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Knock-in mice harboring a Ca(2+) desensitizing mutation in cardiac troponin C develop early onset dilated cardiomyopathy.

PubMed

McConnell, Bradley K; Singh, Sonal; Fan, Qiying; Hernandez, Adriana; Portillo, Jesus P; Reiser, Peter J; Tikunova, Svetlana B

2015-01-01

The physiological consequences of aberrant Ca(2+) binding and exchange with cardiac myofilaments are not clearly understood. In order to examine the effect of decreasing Ca(2+) sensitivity of cTnC on cardiac function, we generated knock-in mice carrying a D73N mutation (not known to be associated with heart disease in human patients) in cTnC. The D73N mutation was engineered into the regulatory N-domain of cTnC in order to reduce Ca(2+) sensitivity of reconstituted thin filaments by increasing the rate of Ca(2+) dissociation. In addition, the D73N mutation drastically blunted the extent of Ca(2+) desensitization of reconstituted thin filaments induced by cTnI pseudo-phosphorylation. Compared to wild-type mice, heterozygous knock-in mice carrying the D73N mutation exhibited a substantially decreased Ca(2+) sensitivity of force development in skinned ventricular trabeculae. Kaplan-Meier survival analysis revealed that median survival time for knock-in mice was 12 weeks. Echocardiographic analysis revealed that knock-in mice exhibited increased left ventricular dimensions with thinner walls. Echocardiographic analysis also revealed that measures of systolic function, such as ejection fraction (EF) and fractional shortening (FS), were dramatically reduced in knock-in mice. In addition, knock-in mice displayed electrophysiological abnormalities, namely prolonged QRS and QT intervals. Furthermore, ventricular myocytes isolated from knock-in mice did not respond to β-adrenergic stimulation. Thus, knock-in mice developed pathological features similar to those observed in human patients with dilated cardiomyopathy (DCM). In conclusion, our results suggest that decreasing Ca(2+) sensitivity of the regulatory N-domain of cTnC is sufficient to trigger the development of DCM.
Human thrombomodulin knock-in mice reveal differential effects of human thrombomodulin on thrombosis and atherosclerosis.

PubMed

Raife, Thomas J; Dwyre, Denis M; Stevens, Jeff W; Erger, Rochelle A; Leo, Lorie; Wilson, Katina M; Fernández, Jose A; Wilder, Jennifer; Kim, Hyung-Suk; Griffin, John H; Maeda, Nobuyo; Lentz, Steven R

2011-11-01

We sought to develop a murine model to examine the antithrombotic and antiinflammatory functions of human thrombomodulin in vivo. Knock-in mice that express human thrombomodulin from the murine thrombomodulin gene locus were generated. Compared with wild-type mice, human thrombomodulin knock-in mice exhibited decreased protein C activation in the aorta (P<0.01) and lung (P<0.001). Activation of endogenous protein C following infusion of thrombin was decreased by 90% in knock-in mice compared with wild-type mice (P<0.05). Carotid artery thrombosis induced by photochemical injury occurred more rapidly in knock-in mice (12±3 minutes) than in wild-type mice (31±6 minutes; P<0.05). No differences in serum cytokine levels were detected between knock-in and wild-type mice after injection of endotoxin. When crossed with apolipoprotein E-deficient mice and fed a Western diet, knock-in mice had a further decrease in protein C activation but did not exhibit increased atherosclerosis. Expression of human thrombomodulin in place of murine thrombomodulin produces viable mice with a prothrombotic phenotype but unaltered responses to systemic inflammatory or atherogenic stimuli. This humanized animal model will be useful for investigating the function of human thrombomodulin under pathophysiological conditions in vivo.
Generation of an allelic series of knock-in mice using recombinase-mediated cassette exchange (RMCE).

PubMed

Roebroek, Anton J M; Van Gool, Bart

2014-01-01

Molecular genetic strategies applying embryonic stem cell (ES cell) technologies to study the function of a gene in mice or to generate a mouse model for a human disease are continuously under development. Next to (conditional) inactivation of genes the application and importance of approaches to generate knock-in mutations are increasing. In this chapter the principle and application of recombinase-mediated cassette exchange (RMCE) are discussed as being a new emerging knock-in strategy, which enables easy generation of a series of different knock-in mutations within one gene. An RMCE protocol, which was used to generate a series of different knock-in mutations in the Lrp1 gene of ES cells, is described in detail as an example of how RMCE can be used to generate highly efficiently an allelic series of differently modified ES cell clones from a parental modified ES cell clone. Subsequently the differently modified ES cell clones can be used to generate an allelic series of mutant knock-in mice.
Electromyographic evidence in support of a knock-in mouse model of DYT1 Dystonia.

PubMed

DeAndrade, Mark P; Trongnetrpunya, Amy; Yokoi, Fumiaki; Cheetham, Chad C; Peng, Ning; Wyss, J Michael; Ding, Mingzhou; Li, Yuqing

2016-11-01

DYT1 dystonia is an autosomal-dominant movement disorder characterized by abnormal, often repetitive, movements and postures. Its hallmark feature is sustained or intermittent contractions of muscles involving co-contractions of antagonist muscle pairs. The symptoms are relieved with the anticholinergic drug trihexyphenidyl. The primary mutation is a trinucleotide deletion (ΔGAG) in DYT1/TOR1A, which codes for torsinA. Previous studies showed that (1) heterozygous Dyt1 ΔGAG knock-in mice, which have an analogous mutation in the endogenous gene, exhibit motor deficits and altered corticostriatal synaptic plasticity in the brain and (2) these deficits can be rescued by trihexyphenidyl. However, brain imaging studies suggest that the Dyt1 knock-in mouse models nonmanifesting mutation carriers of DYT1 dystonia. The aim of this work was to examine the hallmark features of DYT1 dystonia in the Dyt1 knock-in mice by analyzing muscular activities. Wireless telemetry devices with biopotential channels were implanted to the bicep and the rectus femori muscles in Dyt1 knock-in mice, and muscular activities were recorded before and after trihexyphenidyl administration. (1) Consistent with DYT1 dystonia patients, Dyt1 knock-in mice showed sustained contractions and co-contractions of the antagonistic bicep femoris and rectus femoris. (2) The abnormal muscle contractions were normalized by trihexyphenidyl. The results suggest that the motor deficits in Dyt1 knock-in mice are likely produced by abnormal muscle contractions, and Dyt1 knock-in mice can potentially be used as a manifesting disease model to study pathophysiology and develop novel therapeutics. © 2016 International Parkinson and Movement Disorder Society. © 2016 International Parkinson and Movement Disorder Society.
Alterations in ethanol-induced behaviors and consumption in knock-in mice expressing ethanol-resistant NMDA receptors.

PubMed

den Hartog, Carolina R; Beckley, Jacob T; Smothers, Thetford C; Lench, Daniel H; Holseberg, Zack L; Fedarovich, Hleb; Gilstrap, Meghin J; Homanics, Gregg E; Woodward, John J

2013-01-01

Ethanol's action on the brain likely reflects altered function of key ion channels such as glutamatergic N-methyl-D-aspartate receptors (NMDARs). In this study, we determined how expression of a mutant GluN1 subunit (F639A) that reduces ethanol inhibition of NMDARs affects ethanol-induced behaviors in mice. Mice homozygous for the F639A allele died prematurely while heterozygous knock-in mice grew and bred normally. Ethanol (44 mM; ∼0.2 g/dl) significantly inhibited NMDA-mediated EPSCs in wild-type mice but had little effect on responses in knock-in mice. Knock-in mice had normal expression of GluN1 and GluN2B protein across different brain regions and a small reduction in levels of GluN2A in medial prefrontal cortex. Ethanol (0.75-2.0 g/kg; i.p.) increased locomotor activity in wild-type mice but had no effect on knock-in mice while MK-801 enhanced activity to the same extent in both groups. Ethanol (2.0 g/kg) reduced rotarod performance equally in both groups but knock-in mice recovered faster following a higher dose (2.5 g/kg). In the elevated zero maze, knock-in mice had a blunted anxiolytic response to ethanol (1.25 g/kg) as compared to wild-type animals. No differences were noted between wild-type and knock-in mice for ethanol-induced loss of righting reflex, sleep time, hypothermia or ethanol metabolism. Knock-in mice consumed less ethanol than wild-type mice during daily limited-access sessions but drank more in an intermittent 24 h access paradigm with no change in taste reactivity or conditioned taste aversion. Overall, these data support the hypothesis that NMDA receptors are important in regulating a specific constellation of effects following exposure to ethanol.
Alterations in Ethanol-Induced Behaviors and Consumption in Knock-In Mice Expressing Ethanol-Resistant NMDA Receptors

PubMed Central

den Hartog, Carolina R.; Beckley, Jacob T.; Smothers, Thetford C.; Lench, Daniel H.; Holseberg, Zack L.; Fedarovich, Hleb; Gilstrap, Meghin J.; Homanics, Gregg E.; Woodward, John J.

2013-01-01

Ethanol's action on the brain likely reflects altered function of key ion channels such as glutamatergic N-methyl-D-aspartate receptors (NMDARs). In this study, we determined how expression of a mutant GluN1 subunit (F639A) that reduces ethanol inhibition of NMDARs affects ethanol-induced behaviors in mice. Mice homozygous for the F639A allele died prematurely while heterozygous knock-in mice grew and bred normally. Ethanol (44 mM; ∼0.2 g/dl) significantly inhibited NMDA-mediated EPSCs in wild-type mice but had little effect on responses in knock-in mice. Knock-in mice had normal expression of GluN1 and GluN2B protein across different brain regions and a small reduction in levels of GluN2A in medial prefrontal cortex. Ethanol (0.75–2.0 g/kg; IP) increased locomotor activity in wild-type mice but had no effect on knock-in mice while MK-801 enhanced activity to the same extent in both groups. Ethanol (2.0 g/kg) reduced rotarod performance equally in both groups but knock-in mice recovered faster following a higher dose (2.5 g/kg). In the elevated zero maze, knock-in mice had a blunted anxiolytic response to ethanol (1.25 g/kg) as compared to wild-type animals. No differences were noted between wild-type and knock-in mice for ethanol-induced loss of righting reflex, sleep time, hypothermia or ethanol metabolism. Knock-in mice consumed less ethanol than wild-type mice during daily limited-access sessions but drank more in an intermittent 24 h access paradigm with no change in taste reactivity or conditioned taste aversion. Overall, these data support the hypothesis that NMDA receptors are important in regulating a specific constellation of effects following exposure to ethanol. PMID:24244696
CXCR6 plays a critical role in angiotensin II-induced renal injury and fibrosis.

PubMed

Xia, Yunfeng; Jin, Xiaogao; Yan, Jingyin; Entman, Mark L; Wang, Yanlin

2014-07-01

Recent studies have shown that angiotensin II (Ang II) plays a critical role in the pathogenesis and progression of hypertensive kidney disease. However, the signaling mechanisms are poorly understood. In this study, we investigated the role of CXCR6 in Ang II-induced renal injury and fibrosis. Wild-type and CXCR6-green fluorescent protein (GFP) knockin mice were treated with Ang II via subcutaneous osmotic minipumps at 1500 ng/kg per minute after unilateral nephrectomy for ≤ 4 weeks. Wild-type and CXCR6-GFP knockin mice had virtually identical blood pressure at baseline. Ang II treatment led to an increase in blood pressure that was similar between wild-type and CXCR6-GFP knockin mice. CXCR6-GFP knockin mice were protected from Ang II-induced renal dysfunction, proteinuria, and fibrosis. CXCR6-GFP knockin mice accumulated fewer bone marrow-derived fibroblasts and myofibroblasts and produced less extracellular matrix protein in the kidneys after Ang II treatment. Furthermore, CXCR6-GFP knockin mice exhibited fewer F4/80(+) macrophages and CD3(+) T cells and expressed less proinflammatory cytokines in the kidneys after Ang II treatment. Finally, wild-type mice engrafted with CXCR6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts, macrophages, and T cells in the kidney after Ang II treatment when compared with wild-type mice engrafted with CXCR6(+/+) bone marrow cells. Our results indicate that CXCR6 plays a pivotal role in the development of Ang II-induced renal injury and fibrosis through regulation of macrophage and T-cell infiltration and bone marrow-derived fibroblast accumulation. © 2014 American Heart Association, Inc.
CXCR6 Plays a Critical Role in Angiotensin II-induced Renal Injury and Fibrosis

PubMed Central

Xia, Yunfeng; Jin, Xiaogao; Yan, Jingyin; Entman, Mark L.; Wang, Yanlin

2014-01-01

Objective Recent studies have shown that angiotensin II (Ang II) plays a critical role in the pathogenesis and progression of hypertensive kidney disease. However, the signaling mechanisms are poorly understood. In this study, we investigated the role of CXCR6 in Ang II-induced renal injury and fibrosis. Approach and Results Wild-type and CXCR6-GFP knockin mice were treated with Ang II via subcutaneous osmotic minipumps at 1500 ng/kg/min after unilateral nephrectomy for up to 4 weeks. WT and CXCR6-GFP knockin mice had virtually identical blood pressure at baseline. Ang II treatment led to an increase in blood pressure that was similar between WT and CXCR6-GFP knockin mice. CXCR6-GFP knockin mice were protected from Ang II-induced renal dysfunction, proteinuria, and fibrosis. CXCR6-GFP knockin mice accumulated fewer bone marrow-derived fibroblasts and myofibroblasts and produced less extracellular matrix protein in the kidneys following Ang II treatment. Furthermore, CXCR6-GFP knockin mice exhibited fewer F4/80+ macrophages and CD3+ T cells and expressed less proinflammatory cytokines in the kidneys after Ang II treatment. Finally, wild-type mice engrafted with CXCR6−/− bone marrow cells displayed fewer bone marrow-derived fibroblasts, macrophages, and T cells in the kidney after Ang II treatment compared with wild-type mice engrafted with CXCR6+/+ bone marrow cells. Conclusions Our results indicate that CXCR6 plays a pivotal role in the development of Ang II-induced renal injury and fibrosis through regulation of macrophage and T cell infiltration and bone marrow-derived fibroblast accumulation. PMID:24855055
Recapitulating X-Linked Juvenile Retinoschisis in Mouse Model by Knock-In Patient-Specific Novel Mutation.

PubMed

Chen, Ding; Xu, Tao; Tu, Mengjun; Xu, Jinlin; Zhou, Chenchen; Cheng, Lulu; Yang, Ruqing; Yang, Tanchu; Zheng, Weiwei; He, Xiubin; Deng, Ruzhi; Ge, Xianglian; Li, Jin; Song, Zongming; Zhao, Junzhao; Gu, Feng

2017-01-01

X-linked juvenile retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding retinoschisin (RS1), which leads to a significant proportion of visual impairment and blindness. To develop personalized genome editing based gene therapy, knock-in animal disease models that have the exact mutation identified in the patients is extremely crucial, and that the way which genome editing in knock-in animals could be easily transferred to the patients. Here we recruited a family diagnosed with XLRS and identified the causative mutation ( RS1 , p.Y65X), then a knock-in mouse model harboring this disease-causative mutation was generated via TALEN (transcription activator-like effector nucleases). We found that the b-wave amplitude of the ERG of the RS1 -KI mice was significantly decreased. Moreover, we observed that the structure of retina in RS1 -KI mice has become disordered, including the disarray of inner nuclear layer and outer nuclear layer, chaos of outer plexiform layer, decreased inner segments of photoreceptor and the loss of outer segments. The novel knock-in mice ( RS1 -KI) harboring patient-specific mutation will be valuable for development of treatment via genome editing mediated gene correction.
Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy

PubMed Central

Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

2014-01-01

The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knockin mice expressing non-phosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knockin mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knockin mice compared to their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knockin mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knockin and wild type mice, indicating that mTORC1 was still activated in the knockin mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knockin mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth. PMID:25229342
GSK3β isoform-selective regulation of depression, memory and hippocampal cell proliferation

PubMed Central

Pardo, Marta; Abrial, Erika; Jope, Richard S.; Beurel, Eleonore

2016-01-01

Abnormally active glycogen synthase kinase-3 (GSK3) contributes to pathological processes in multiple psychiatric and neurological disorders. Modeled in mice, this includes increasing susceptibility to dysregulation of mood-relevant behaviors, impairing performance in several cognitive tasks, and impairing adult hippocampal neural precursor cell (NPC) proliferation. These deficits are all evident in GSK3α/β knockin mice, in which serine-to-alanine mutations block the inhibitory serine phosphorylation regulation of both GSK3 isoforms, leaving GSK3 hyperactive. It was unknown if both GSK3 isoforms perform redundant actions in these processes, or if hyperactivity of one GSK3 isoform has a predominant effect. To test this, we examined GSK3α or GSK3β knockin mice in which only one isoform was mutated to a hyperactive form. Only GSK3β, not GSK3α, knockin mice displayed heightened vulnerability to the learned helplessness model of depression-like behavior. Three cognitive measures impaired in GSK3α/β knockin mice demonstrated differential regulation by GSK3 isoforms. Novel object recognition was impaired in GSK3β, not GSK3α, knockin mice, whereas temporal order memory was not impaired in GSK3α or GSK3β knockin mice, and coordinate spatial processing was impaired in both GSK3α and GSK3β knockin mice. Adult hippocampal NPC proliferation was severely impaired in GSK3β knockin mice, but not impaired in GSK3α knockin mice. Increased activity of GSK3β, in the absence of over-expression or disease pathology, is sufficient to impair mood regulation, novel object recognition, and hippocampal NPC proliferation, whereas hyperactive GSK3α individually does not impair these processes. These results demonstrate that hyperactivity of the two GSK3 isoforms execute non-redundant effects on these processes. PMID:26749572
GSK3β isoform-selective regulation of depression, memory and hippocampal cell proliferation.

PubMed

Pardo, M; Abrial, E; Jope, R S; Beurel, E

2016-03-01

Abnormally active glycogen synthase kinase-3 (GSK3) contributes to pathological processes in multiple psychiatric and neurological disorders. Modeled in mice, this includes increasing susceptibility to dysregulation of mood-relevant behaviors, impairing performance in several cognitive tasks and impairing adult hippocampal neural precursor cell (NPC) proliferation. These deficits are all evident in GSK3α/β knockin mice, in which serine-to-alanine mutations block the inhibitory serine phosphorylation regulation of both GSK3 isoforms, leaving GSK3 hyperactive. It was unknown if both GSK3 isoforms perform redundant actions in these processes, or if hyperactivity of one GSK3 isoform has a predominant effect. To test this, we examined GSK3α or GSK3β knockin mice in which only one isoform was mutated to a hyperactive form. Only GSK3β, not GSK3α, knockin mice displayed heightened vulnerability to the learned helplessness model of depression-like behavior. Three cognitive measures impaired in GSK3α/β knockin mice showed differential regulation by GSK3 isoforms. Novel object recognition was impaired in GSK3β, not in GSK3α, knockin mice, whereas temporal order memory was not impaired in GSK3α or GSK3β knockin mice, and co-ordinate spatial processing was impaired in both GSK3α and GSK3β knockin mice. Adult hippocampal NPC proliferation was severely impaired in GSK3β knockin mice, but not impaired in GSK3α knockin mice. Increased activity of GSK3β, in the absence of overexpression or disease pathology, is sufficient to impair mood regulation, novel object recognition and hippocampal NPC proliferation, whereas hyperactive GSK3α individually does not impair these processes. These results show that hyperactivity of the two GSK3 isoforms execute non-redundant effects on these processes. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.
Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity

PubMed Central

Jaworski, Maike; Marsland, Ben J; Gehrig, Jasmine; Held, Werner; Favre, Stéphanie; Luther, Sanjiv A; Perroud, Mai; Golshayan, Déla; Gaide, Olivier; Thome, Margot

2014-01-01

The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies. PMID:25319413
Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice.

PubMed

Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

2017-05-15

Robust reproductive engineering techniques are required for the efficient and rapid production of genetically modified mice. We have reported the efficient production of genome-edited mice using reproductive engineering techniques, such as ultra-superovulation, in vitro fertilization (IVF) and vitrification/warming of zygotes. We usually use vitrified/warmed fertilized oocytes created by IVF for microinjection because of work efficiency and flexible scheduling. Here, we investigated whether the culture time of zygotes before microinjection influences the efficiency of producing knock-in mice. Knock-in mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and single-stranded oligodeoxynucleotide (ssODN) or PITCh (Precise Integration into Target Chromosome) system, a method of integrating a donor vector assisted by microhomology-mediated end-joining. The cryopreserved fertilized oocytes were warmed, cultured for several hours and microinjected at different timings. Microinjection was performed with Cas9 protein, guide RNA(s), and an ssODN or PITCh donor plasmid for the ssODN knock-in and the PITCh knock-in, respectively. Different production efficiencies of knock-in mice were observed by changing the timing of microinjection. Our study provides useful information for the CRISPR-Cas9-based generation of knock-in mice. © 2017. Published by The Company of Biologists Ltd.
Role of blood ribosomal protein S19 in coagulum resorption: a study using Gln137Glu-ribosomal protein S19 gene knock-in mouse.

PubMed

Chen, Jun; Fujino, Rika; Zhao, Rui; Semba, Umeko; Araki, Kimi; Yamamoto, Tetsuro

2014-11-01

Sera of human, guinea pig or mouse contain a strong monocyte chemoattractant capacity that is attributed to the ribosomal protein S19 (RP S19) oligomers generated during blood coagulation. In contrast, sera prepared from Gln137Glu-RP S19 gene knock-in mice contained negligible chemoattractant capacity. When coagula that had been pre-formed from the blood of both the wild type and knock-in mice were intraperitoneally inserted into host mice, after 3 days of recovery, the knock-in mouse coagula remained larger than the wild type mouse coagula. The wild type mouse coagula were covered by multiple macrophage layers at the surface and were infiltrated inside by macrophages. Knock-in mouse coagula exhibited less macrophage involvement. When coagula of knock-in mice and coagula of knock-in mice containing C5a/RP S19, an artificial substitute of the RP S19 oligomers, were intraperitoneally inserted as pairs, the C5a/RP S19 containing coagulum was more rapidly absorbed, concomitant with increased macrophage involvement. Finally, when the knock-in mouse and wild type mouse coagula pairs were inserted into mice in which macrophages had been depleted using clodronate liposome, the size difference of recovered coagula was reversed. These results indicate the importance of the RP S19 oligomer-induced macrophage recruitment in coagulum resorption. © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.
Generation and analysis of knock-in mice carrying pseudohypoaldosteronism type II-causing mutations in the cullin 3 gene.

PubMed

Araki, Yuya; Rai, Tatemitsu; Sohara, Eisei; Mori, Takayasu; Inoue, Yuichi; Isobe, Kiyoshi; Kikuchi, Eriko; Ohta, Akihito; Sasaki, Sei; Uchida, Shinichi

2015-10-21

Pseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four different genes: with-no-lysine kinases (WNK) 1 and 4, Kelch-like family member 3 (KLHL3), and cullin 3 (Cul3). Cul3 and KLHL3 form an E3 ligase complex that ubiquitinates and reduces the expression level of WNK4. PHAII-causing mutations in WNK4 and KLHL3 impair WNK4 ubiquitination. However, the molecular pathogenesis of PHAII caused by Cul3 mutations is unclear. In cultured cells and human leukocytes, PHAII-causing Cul3 mutations result in the skipping of exon 9, producing mutant Cul3 protein lacking 57 amino acids. However, whether this phenomenon occurs in the kidneys and is responsible for the pathogenesis of PHAII in vivo is unknown. We generated knock-in mice carrying a mutation in the C-terminus of intron 8 of Cul3, c.1207-1G>A, which corresponds to a PHAII-causing mutation in the human Cul3 gene. Heterozygous Cul3(G(-1)A/+) knock-in mice did not exhibit PHAII phenotypes, and the skipping of exon 9 was not evident in their kidneys. However, the level of Cul3 mRNA expression in the kidneys of heterozygous knock-in mice was approximately half that of wild-type mice. Furthermore, homozygous knock-in mice were nonviable. It suggested that the mutant allele behaved like a knockout allele and did not produce Cul3 mRNA lacking exon 9. A reduction in Cul3 expression alone was not sufficient to develop PHAII in the knock-in mice. Our findings highlighted the pathogenic role of mutant Cul3 protein and provided insight to explain why PHAII-causing mutations in Cul3 cause kidney-predominant PHAII phenotypes. © 2015. Published by The Company of Biologists Ltd.
Transgenic and gene knockout mice in gastric cancer research

PubMed Central

Jiang, Yannan; Yu, Yingyan

2017-01-01

Mouse models are useful tool for carcinogenic study. They will greatly enrich the understanding of pathogenesis and molecular mechanisms for gastric cancer. However, only few of mice could develop gastric cancer spontaneously. With the development and improvement of gene transfer technology, investigators created a variety of transgenic and knockout/knockin mouse models of gastric cancer, such as INS-GAS mice and gastrin knockout mice. Combined with helicobacter infection and carcinogens treatment, these transgenic/knockout/knockin mice developed precancerous or cancerous lesions, which are proper for gene function study or experimental therapy. Here we review the progression of genetically engineered mouse models on gastric cancer research, and emphasize the effects of chemical carcinogens or infectious factors on carcinogenesis of genetically modified mouse. We also emphasize the histological examination on mouse stomach. We expect to provide researchers with some inspirations on this field. PMID:27713138
[Synapse maturation and autism: learning from neuroligin model mice].

PubMed

Tabuchi, Katsuhiko; Chang, WenHsin; Hang, WenHsin; Asgar, Nur Farehan; Asgar, Nur Farehan Mohamed; Pramanik, Gopal

2014-02-01

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, communication, and restricted and repetitive behavior. Synaptic defects have been implicated in autism; nevertheless, the cause is still largely unknown. A mutation that substitutes cysteine for arginine at residue 451 of Neuroligin-3 (R451C) is the first monogenic mutation identified in idiopathic autism patients. To study the relationship between this mutation and autism, we generated knock-in mice that recapitulated this mutation. The knock-in mice were born and grew up normally without showing any major physical phenotypes, but showed a deficit in social interaction. We studied synaptic function in the layer II/III pyramidal neurons in the somatosensory cortex and found inhibitory synaptic transmission was enhanced in the knock-in mice. The administration of GABA blocker rescued social interaction, suggesting that this caused autistic behavior in these mice. We also found, by Morris water maze test, that spatial learning and memory were significantly enhanced in the knock-in mice. Electrophysiology in the CA1 region of the hippocampus revealed that LTP, the NMDA/AMPA ratio, and NR2B function were enhanced, indicating that synaptic maturation was impaired in the knock-in mice. This may cause the deficit in social behavior and extraordinary memory ability occasionally seen in autistic patients.
Vapb/Amyotrophic lateral sclerosis 8 knock-in mice display slowly progressive motor behavior defects accompanying ER stress and autophagic response.

PubMed

Larroquette, Frédérique; Seto, Lesley; Gaub, Perrine L; Kamal, Brishna; Wallis, Deeann; Larivière, Roxanne; Vallée, Joanne; Robitaille, Richard; Tsuda, Hiroshi

2015-11-15

Missense mutations (P56S) in Vapb are associated with autosomal dominant motor neuron diseases: amyotrophic lateral sclerosis and lower motor neuron disease. Although transgenic mice overexpressing the mutant vesicle-associated membrane protein-associated protein B (VAPB) protein with neuron-specific promoters have provided some insight into the toxic properties of the mutant proteins, their role in pathogenesis remains unclear. To identify pathological defects in animals expressing the P56S mutant VAPB protein at physiological levels in the appropriate tissues, we have generated Vapb knock-in mice replacing wild-type Vapb gene with P56S mutant Vapb gene and analyzed the resulting pathological phenotypes. Heterozygous P56S Vapb knock-in mice show mild age-dependent defects in motor behaviors as characteristic features of the disease. The homozygous P56S Vapb knock-in mice show more severe defects compared with heterozygous mice reflecting the dominant and dose-dependent effects of P56S mutation. Significantly, the knock-in mice demonstrate accumulation of P56S VAPB protein and ubiquitinated proteins in cytoplasmic inclusions, selectively in motor neurons. The mutant mice demonstrate induction of ER stress and autophagic response in motor neurons before obvious onset of behavioral defects, suggesting that these cellular biological defects might contribute to the initiation of the disease. The P56S Vapb knock-in mice could be a valuable tool to gain a better understanding of the mechanisms by which the disease arises. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Reduction in open field activity in the absence of memory deficits in the AppNL-G-F knock-in mouse model of Alzheimer's disease.

PubMed

Whyte, Lauren S; Hemsley, Kim M; Lau, Adeline A; Hassiotis, Sofia; Saito, Takashi; Saido, Takaomi C; Hopwood, John J; Sargeant, Timothy J

2018-01-15

The recent development of knock-in mouse models of Alzheimer's disease provides distinct advantages over traditional transgenic mouse models that rely on over-expression of amyloid precursor protein. Two such knock-in models that have recently been widely adopted by Alzheimer's researchers are the App NL-F and App NL-G-F mice. This study aimed to further characterise the behavioural phenotype and amyloid plaque distribution of App NL-G-F/NL-G-F (C57BL/6J background) mice at six-months of age. An attempt to replicate a previous study that observed deficits in working memory in the Y-maze, showed no difference between App NL-G-F/NL-G-F and wild-type mice. Further assessment of these mice using the novel object recognition test and Morris water maze also revealed no differences between App NL-G-F/NL-G-F and wild-type mice. Despite a lack of demonstrated cognitive deficits, we report a reduction in locomotor/exploratory activity in an open field. Histological examination of App NL-G-F/NL-G-F mice showed widespread distribution of amyloid plaques at this age. We conclude that whilst at six-months of age, memory deficits are not sufficiently robust to be replicated in varying environments, amyloid plaque burden is significant in App NL-G-F/NL-G-F knock-in brain. Copyright © 2017 Elsevier B.V. All rights reserved.

Mutations M287L and Q266I in the Glycine Receptor α1 Subunit Change Sensitivity to Volatile Anesthetics in Oocytes and Neurons, but Not the Minimal Alveolar Concentration in Knockin Mice

PubMed Central

Borghese, Cecilia M.; Xiong, Wei; Oh, S. Irene; Ho, Angel; Mihic, S. John; Zhang, Li; Lovinger, David M.; Homanics, Gregg E.; Eger, Edmond I; Harris, R. Adron

2012-01-01

Background Volatile anesthetics (VAs) alter the function of key central nervous system proteins but it is not clear which, if any, of these targets mediates the immobility produced by VAs in the face of noxious stimulation. A leading candidate is the glycine receptor, a ligand-gated ion channel important for spinal physiology. VAs variously enhance such function, and blockade of spinal GlyRs with strychnine affects the minimal alveolar concentration (an anesthetic EC50) in proportion to the degree of enhancement. Methods We produced single amino acid mutations into the glycine receptorα1 subunit that increased (M287L, third transmembrane region) or decreased (Q266I, second transmembrane region) sensitivity to isoflurane in recombinant receptors, and introduced such receptors into mice. The resulting knockin mice presented impaired glycinergic transmission, but heterozygous animals survived to adulthood, and we determined the effect of isoflurane on glycine-evoked responses of brain stem neurons from the knockin mice, and the minimal alveolar concentration for isoflurane and other VAs in the immature and mature knockin mice. Results Studies of glycine-evoked currents in brain stem neurons from knock-in mice confirmed the changes seen with recombinant receptors. No increases in the minimal alveolar concentration were found in knockin mice, but the minimal alveolar concentration for isoflurane and enflurane (but not halothane) decreased in 2-week-old Q266I mice. This change is opposite to the one expected for a mutation that decreases the sensitivity to volatile anesthetics. Conclusion Taken together, these results indicate that glycine receptors containing the α1 subunit are not likely to be crucial for the action of isoflurane and other VAs. PMID:22885675
LatY136F knock-in mouse model for human IgG4-related disease.

PubMed

Yamada, Kazunori; Zuka, Masahiko; Ito, Kiyoaki; Mizuguchi, Keishi; Kakuchi, Yasushi; Onoe, Tamehito; Suzuki, Yasunori; Yamagishi, Masakazu; Izui, Shozo; Malissen, Marie; Malissen, Bernard; Kawano, Mitsuhiro

2018-01-01

The adaptor protein Linker for activation of T cell (LAT) is a key signaling hub used by the T cell antigen receptor. Mutant mice expressing loss-of-function mutations affecting LAT and including a mutation in which tyrosine 136 is replaced by a phenylalanine (LatY136F) develop lymphoproliferative disorder involving T helper type 2 effector cells capable of triggering a massive polyclonal B cell activation that leads to hypergammaglobulinemia G1 and E and to non-resolving inflammation and autoimmunity. The purpose of this study was to evaluate whether the phenotypes of LatY136F knock-in mice resemble the immunohistopathological features of immunoglobulin G4-related disease (IgG4-RD). LatY136F knock-in mice were sacrificed at 4-20 weeks of age, and pancreas, kidney, salivary gland and lung were obtained. All organs were stained with hematoxylin-eosin and with Azan for estimation of collagen in fibrosis, and the severity scores of inflammation and fibrosis were evaluated. Immunostainings were performed to analyze the types of infiltrating cells. In addition, the effects of corticosteroid treatment on the development of tissue lesions and serum levels of IgG1 were assessed. Tissue lesions characterized by inflammatory mononuclear cell infiltration and fibrosis were detected in pancreas, kidney, and salivary gland starting from 6 weeks of age. Immunostainings showed pronounced infiltration of plasma cells, CD4-positive T cells, and macrophages. Infiltrating plasma cells predominantly expressed IgG1. The extent of inflammation in pancreas and salivary glands was markedly reduced by corticosteroid treatment. LatY136F knock-in mice displayed increased production of Th2-type IgG1 (a homologue of human IgG4) and developed multiple organ tissue lesions reminiscent of those seen in patients with IgG4-RD. Moreover, the development of these tissue lesions was highly sensitive to corticosteroid treatment like in IgG4-RD. For these reasons we consider the LatY136F knock-in mouse strain to represent a promising model for human IgG4-RD.
Translating human genetics into mouse: the impact of ultra-rapid in vivo genome editing.

PubMed

Aida, Tomomi; Imahashi, Risa; Tanaka, Kohichi

2014-01-01

Gene-targeted mutant animals, such as knockout or knockin mice, have dramatically improved our understanding of the functions of genes in vivo and the genetic diversity that characterizes health and disease. However, the generation of targeted mice relies on gene targeting in embryonic stem (ES) cells, which is a time-consuming, laborious, and expensive process. The recent groundbreaking development of several genome editing technologies has enabled the targeted alteration of almost any sequence in any cell or organism. These technologies have now been applied to mouse zygotes (in vivo genome editing), thereby providing new avenues for simple, convenient, and ultra-rapid production of knockout or knockin mice without the need for ES cells. Here, we review recent achievements in the production of gene-targeted mice by in vivo genome editing. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.
The kick-in system: a novel rapid knock-in strategy.

PubMed

Tomonoh, Yuko; Deshimaru, Masanobu; Araki, Kimi; Miyazaki, Yasuhiro; Arasaki, Tomoko; Tanaka, Yasuyoshi; Kitamura, Haruna; Mori, Fumiaki; Wakabayashi, Koichi; Yamashita, Sayaka; Saito, Ryo; Itoh, Masayuki; Uchida, Taku; Yamada, Junko; Migita, Keisuke; Ueno, Shinya; Kitaura, Hiroki; Kakita, Akiyoshi; Lossin, Christoph; Takano, Yukio; Hirose, Shinichi

2014-01-01

Knock-in mouse models have contributed tremendously to our understanding of human disorders. However, generation of knock-in animals requires a significant investment of time and effort. We addressed this problem by developing a novel knock-in system that circumvents several traditional challenges by establishing stem cells with acceptor elements enveloping a particular genomic target. Once established, these acceptor embryonic stem (ES) cells are efficient at directionally incorporating mutated target DNA using modified Cre/lox technology. This is advantageous, because knock-ins are not restricted to one a priori selected variation. Rather, it is possible to generate several mutant animal lines harboring desired alterations in the targeted area. Acceptor ES cell generation is the rate-limiting step, lasting approximately 2 months. Subsequent manipulations toward animal production require an additional 8 weeks, but this delimits the full period from conception of the genetic alteration to its animal incorporation. We call this system a "kick-in" to emphasize its unique characteristics of speed and convenience. To demonstrate the functionality of the kick-in methodology, we generated two mouse lines with separate mutant versions of the voltage-dependent potassium channel Kv7.2 (Kcnq2): p.Tyr284Cys (Y284C) and p.Ala306Thr (A306T); both variations have been associated with benign familial neonatal epilepsy. Adult mice homozygous for Y284C, heretofore unexamined in animals, presented with spontaneous seizures, whereas A306T homozygotes died early. Heterozygous mice of both lines showed increased sensitivity to pentylenetetrazole, possibly due to a reduction in M-current in CA1 hippocampal pyramidal neurons. Our observations for the A306T animals match those obtained with traditional knock-in technology, demonstrating that the kick-in system can readily generate mice bearing various mutations, making it a suitable feeder technology toward streamlined phenotyping.
Generation of Knock-in Mouse by Genome Editing.

PubMed

Fujii, Wataru

2017-01-01

Knock-in mice are useful for evaluating endogenous gene expressions and functions in vivo. Instead of the conventional gene-targeting method using embryonic stem cells, an exogenous DNA sequence can be inserted into the target locus in the zygote using genome editing technology. In this chapter, I describe the generation of epitope-tagged mice using engineered endonuclease and single-stranded oligodeoxynucleotide through the mouse zygote as an example of how to generate a knock-in mouse by genome editing.
Tissue thrombin is associated with the pathogenesis of dilated cardiomyopathy.

PubMed

Ito, Keiichi; Hongo, Kenichi; Date, Taro; Ikegami, Masahiro; Hano, Hiroshi; Owada, Mamiko; Morimoto, Satoshi; Kashiwagi, Yusuke; Katoh, Daisuke; Yoshino, Takuya; Yoshii, Akira; Kimura, Haruka; Nagoshi, Tomohisa; Kajimura, Ichige; Kusakari, Yoichiro; Akaike, Toru; Minamisawa, Susumu; Ogawa, Kazuo; Minai, Kosuke; Ogawa, Takayuki; Kawai, Makoto; Yajima, Junji; Matsuo, Seiichiro; Yamane, Teiichi; Taniguchi, Ikuo; Morimoto, Sachio; Yoshimura, Michihiro

2017-02-01

Thrombin is a serine protease known to be the final product of the coagulation cascade. However, thrombin plays other physiological roles in processes such as gastric contractions and vessel wound healing, and a state of coagulability is increased in patients with dilated cardiomyopathy (DCM). In this study, we investigate the role of thrombin in the pathogenesis of DCM. The purpose of this study is to clarify the role of thrombin in the pathogenesis of DCM and investigate the possibility of treatment against DCM by thrombin inhibition. We investigated the expression of thrombin in the left ventricles of five patients with DCM who underwent the Batista operation and four patients without heart disease. Furthermore, we investigated the involvement of thrombin in the development of DCM using knock-in mice with a deletion mutation of cardiac troponin T that causes human DCM (∆K210 knock-in mouse) (B6;129-Tnnt2 tm2Mmto ) and assessed the effects of a direct thrombin inhibitor, dabigatran on ∆K210 knock-in mice using echocardiographic examinations, the Kaplan-Meier method and Western blotting. The immunohistochemical analysis showed a strong thrombin expression in the DCM patients compared to the patients without heart disease. In immunohistochemical analysis, a strong thrombin expression was observed in the heart tissues analysis in the ∆K210 knock-in mice. Dabigatran administration significantly improved fractional shortening according to the echocardiographic examination and the survival outcomes in ∆K210 knock-in mice. Tissue thrombin is involved in the pathogenesis of DCM and thrombin inhibition can be beneficial for the treatment of DCM. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
Optimization of the production of knock-in alleles by CRISPR/Cas9 microinjection into the mouse zygote.

PubMed

Raveux, Aurélien; Vandormael-Pournin, Sandrine; Cohen-Tannoudji, Michel

2017-02-17

Microinjection of the CRISPR/Cas9 system in zygotes is an efficient and comparatively fast method to generate genetically modified mice. So far, only few knock-in mice have been generated using this approach, and because no systematic study has been performed, parameters controlling the efficacy of CRISPR/Cas9-mediated targeted insertion are not fully established. Here, we evaluated the effect of several parameters on knock-in efficiency changing only one variable at a time. We found that knock-in efficiency was dependent on injected Cas9 mRNA and single-guide RNA concentrations and that cytoplasmic injection resulted in more genotypic complexity compared to pronuclear injection. Our results also indicated that injection into the pronucleus compared to the cytoplasm is preferable to generate knock-in alleles with an oligonucleotide or a circular plasmid. Finally, we showed that Cas9D10A nickase variant was less efficient than wild-type Cas9 for generating knock-in alleles and caused a higher rate of mosaicism. Thus, our study provides valuable information that will help to improve the future production of precise genetic modifications in mice.
A Progressive Translational Mouse Model of Human VCP Disease: The VCP R155H/+ Mouse

PubMed Central

Nalbandian, Angèle; Llewellyn, Katrina J.; Badadani, Mallikarjun; Yin, Hong Z.; Nguyen, Christopher; Katheria, Veeral; Watts, Giles; Mukherjee, Jogeshwar; Vesa, Jouni; Caiozzo, Vincent; Mozaffar, Tahseen; Weiss, John H.; Kimonis, Virginia E.

2012-01-01

Introduction Mutations in the valosin containing protein (VCP) gene cause hereditary Inclusion Body Myopathy (hIBM) associated with Paget disease of bone (PDB), and frontotemporal dementia (FTD). More recently they have been linked to 2% of familial ALS cases. A knock-in mouse model offers the opportunity to study VCP-associated pathogenesis. Methods The VCPR155H/+ knock-in mouse model was assessed for muscle strength, immunohistochemical, Western, apoptosis, autophagy and MicroPET/CT imaging analyses. Results VCPR155H/+ mice developed significant progressive muscle weakness, and the quadriceps and brain developed progressive cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-II staining. MicroCT analyses revealed Paget-like lesions at the ends of long bones. Spinal cord demonstrated neurodegenerative changes, ubiquitin, and TDP-43 pathology of motor neurons. Discussion VCPR155H/+ knock-in mice represent an excellent pre-clinical model for understanding VCP-associated disease mechanisms and future treatments. PMID:23169451
Characterization of Two Mutations, M287L and Q266I, in the α1 Glycine Receptor Subunit That Modify Sensitivity to Alcohols

PubMed Central

Borghese, Cecilia M.; Blednov, Yuri A.; Quan, Yu; Iyer, Sangeetha V.; Xiong, Wei; Mihic, S. John; Zhang, Li; Lovinger, David M.; Trudell, James R.; Homanics, Gregg E.

2012-01-01

Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels. Ethanol potentiates glycine activation of the GlyR, and putative binding sites for alcohol are located in the transmembrane (TM) domains between and within subunits. To alter alcohol sensitivity of GlyR, we introduced two mutations in the GlyR α1 subunit, M287L (TM3) and Q266I (TM2). After expression in Xenopus laevis oocytes, both mutants showed a reduction in glycine sensitivity and glycine-induced maximal currents. Activation by taurine, another endogenous agonist, was almost abolished in the M287L GlyR. The ethanol potentiation of glycine currents was reduced in the M287L GlyR and eliminated in Q266I. Physiological levels of zinc (100 nM) potentiate glycine responses in wild-type GlyR and also enhance the ethanol potentiation of glycine responses. Although zinc potentiation of glycine responses was unchanged in both mutants, zinc enhancement of ethanol potentiation of glycine responses was absent in M287L GlyRs. The Q266I mutation decreased conductance but increased mean open time (effects not seen in M287L). Two lines of knockin mice bearing these mutations were developed. Survival of homozygous knockin mice was impaired, probably as a consequence of impaired glycinergic transmission. Glycine showed a decreased capacity for displacing strychnine binding in heterozygous knockin mice. Electrophysiology in isolated neurons of brain stem showed decreased glycine-mediated currents and decreased ethanol potentiation in homozygous knockin mice. Molecular models of the wild-type and mutant GlyRs show a smaller water-filled cavity within the TM domains of the Q266I α1 subunit. The behavioral characterization of these knockin mice is presented in a companion article (J Pharmacol Exp Ther 340:317–329, 2012). PMID:22037201
Effects of short-term Western diet on cerebral oxidative stress and diabetes related factors in APP x PS1 knock-in mice.

PubMed

Studzinski, Christa M; Li, Feng; Bruce-Keller, Annadora J; Fernandez-Kim, Sun Ok; Zhang, Le; Weidner, Adam M; Markesbery, William R; Murphy, M Paul; Keller, Jeffrey N

2009-02-01

A chronic high fat Western diet (WD) promotes a variety of morbidity factors although experimental evidence for short-term WD mediating brain dysfunction remains to be elucidated. The amyloid precursor protein and presenilin-1 (APP x PS1) knock-in mouse model has been demonstrated to recapitulate some key features of Alzheimer's disease pathology, including amyloid-beta (Abeta) pathogenesis. In this study, we placed 1-month-old APP x PS1 mice and non-transgenic littermates on a WD for 4 weeks. The WD resulted in a significant elevation in protein oxidation and lipid peroxidation in the brain of APP x PS1 mice relative to non-transgenic littermates, which occurred in the absence of increased Abeta levels. Altered adipokine levels were also observed in APP x PS1 mice placed on a short-term WD, relative to non-transgenic littermates. Taken together, these data indicate that short-term WD is sufficient to selectively promote cerebral oxidative stress and metabolic disturbances in APP x PS1 knock-in mice, with increased oxidative stress preceding alterations in Abeta. These data have important implications for understanding how WD may potentially contribute to brain dysfunction and the development of neurodegenerative disorders such as Alzheimer's disease.
Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization.

PubMed

Ozawa, Akihiko; Brunori, Gloria; Mercatelli, Daniela; Wu, Jinhua; Cippitelli, Andrea; Zou, Bende; Xie, Xinmin Simon; Williams, Melissa; Zaveri, Nurulain T; Low, Sarah; Scherrer, Grégory; Kieffer, Brigitte L; Toll, Lawrence

2015-08-19

The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [(35)S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with μ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native NOP receptor. These knock-in mice have NOP receptors that function both in vitro and in vivo and have provided a detailed characterization of NOP receptors in brain, spinal cord, and DRG neurons. They appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. Copyright © 2015 the authors 0270-6474/15/3511683-12$15.00/0.
Human IL-3/GM-CSF knock-in mice support human alveolar macrophage development and human immune responses in the lung

PubMed Central

Willinger, Tim; Rongvaux, Anthony; Takizawa, Hitoshi; Yancopoulos, George D.; Valenzuela, David M.; Murphy, Andrew J.; Auerbach, Wojtek; Eynon, Elizabeth E.; Stevens, Sean; Manz, Markus G.; Flavell, Richard A.

2011-01-01

Mice with a functional human immune system have the potential to allow in vivo studies of human infectious diseases and to enable vaccine testing. To this end, mice need to fully support the development of human immune cells, allow infection with human pathogens, and be capable of mounting effective human immune responses. A major limitation of humanized mice is the poor development and function of human myeloid cells and the absence of human immune responses at mucosal surfaces, such as the lung. To overcome this, we generated human IL-3/GM-CSF knock-in (hIL-3/GM-CSF KI) mice. These mice faithfully expressed human GM-CSF and IL-3 and developed pulmonary alveolar proteinosis because of elimination of mouse GM-CSF. We demonstrate that hIL-3/GM-CSF KI mice engrafted with human CD34+ hematopoietic cells had improved human myeloid cell reconstitution in the lung. In particular, hIL-3/GM-CSF KI mice supported the development of human alveolar macrophages that partially rescued the pulmonary alveolar proteinosis syndrome. Moreover, human alveolar macrophages mounted correlates of a human innate immune response against influenza virus. The hIL-3/GM-CSF KI mice represent a unique mouse model that permits the study of human mucosal immune responses to lung pathogens. PMID:21262803
Profoundly different prion diseases in knock-in mice carrying single PrP codon substitutions associated with human diseases.

PubMed

Jackson, Walker S; Borkowski, Andrew W; Watson, Nicki E; King, Oliver D; Faas, Henryk; Jasanoff, Alan; Lindquist, Susan

2013-09-03

In man, mutations in different regions of the prion protein (PrP) are associated with infectious neurodegenerative diseases that have remarkably different clinical signs and neuropathological lesions. To explore the roots of this phenomenon, we created a knock-in mouse model carrying the mutation associated with one of these diseases [Creutzfeldt-Jakob disease (CJD)] that was exactly analogous to a previous knock-in model of a different prion disease [fatal familial insomnia (FFI)]. Together with the WT parent, this created an allelic series of three lines, each expressing the same protein with a single amino acid difference, and with all native regulatory elements intact. The previously described FFI mice develop neuronal loss and intense reactive gliosis in the thalamus, as seen in humans with FFI. In contrast, CJD mice had the hallmark features of CJD, spongiosis and proteinase K-resistant PrP aggregates, initially developing in the hippocampus and cerebellum but absent from the thalamus. A molecular transmission barrier protected the mice from any infectious prion agents that might have been present in our mouse facility and allowed us to conclude that the diseases occurred spontaneously. Importantly, both models created agents that caused a transmissible neurodegenerative disease in WT mice. We conclude that single codon differences in a single gene in an otherwise normal genome can cause remarkably different neurodegenerative diseases and are sufficient to create distinct protein-based infectious elements.
Expression of inactive glutathione peroxidase 4 leads to embryonic lethality, and inactivation of the Alox15 gene does not rescue such knock-in mice.

PubMed

Brütsch, Simone Hanna; Wang, Chi Chiu; Li, Lu; Stender, Hannelore; Neziroglu, Nilgün; Richter, Constanze; Kuhn, Hartmut; Borchert, Astrid

2015-02-01

Glutathione peroxidases (Gpx) and lipoxygenases (Alox) are functional counterplayers in the metabolism of hydroperoxy lipids that regulate cellular redox homeostasis. Gpx4 is a moonlighting protein that has been implicated not only as an enzyme in anti-oxidative defense, gene expression regulation, and programmed cell death, but also as a structural protein in spermatogenesis. Homozygous Gpx4 knock-out mice are not viable, but molecular reasons for intrauterine lethality are not completely understood. This study was aimed at investigating whether the lack of catalytic activity or the impaired function as structural protein is the dominant reason for embryonic lethality. We further explored whether the pro-oxidative enzyme mouse 12/15 lipoxygenase (Alox15) plays a major role in embryonic lethality of Gpx4-deficient mice. To achieve these goals, we first created knock-in mice, which express a catalytically inactive Gpx4 mutant (Sec46Ala). As homozygous Gpx4-knock-out mice Sec46Ala-Gpx4(+/+) knock-in animals are not viable but undergo intrauterine resorption between embryonic day 6 and 7 (E6-7). In contrast, heterozygous knock-in mice (Sec46Ala-Gpx4(-/+)) are viable, fertile and do not show major phenotypic alterations. Interestingly, homozygous Alox15 deficiency did not rescue the U46A-Gpx4(+/+) mice from embryonic lethality. In fact, when heterozygous U46A-Gpx4(-/+) mice were stepwise crossed into an Alox15-deficent background, no viable U46A-Gpx4(+/+)+Alox15(-/-) individuals were obtained. However, we were able to identify U46A-Gpx4(+/+)+Alox15(-/-) embryos in the state of resorption around E7. These data suggest that the lack of catalytic activity is the major reason for the embryonic lethality of Gpx4(-/-) mice and that systemic inactivation of the Alox15 gene does not rescue homozygous knock-in mice expressing catalytically silent Gpx4.
Generation of Oxtr cDNA(HA)-Ires-Cre Mice for Gene Expression in an Oxytocin Receptor Specific Manner.

PubMed

Hidema, Shizu; Fukuda, Tomokazu; Hiraoka, Yuichi; Mizukami, Hiroaki; Hayashi, Ryotaro; Otsuka, Ayano; Suzuki, Shingo; Miyazaki, Shinji; Nishimori, Katsuhiko

2016-05-01

The neurohypophysial hormone oxytocin (OXT) and its receptor (OXTR) have critical roles in the regulation of pro-social behaviors, including social recognition, pair bonding, parental behavior, and stress-related responses. Supporting this hypothesis, a portion of patients suffering from autism spectrum disorder have mutations, such as single nucleotide polymorphisms, or epigenetic modifications in their OXTR gene. We previously reported that OXTR-deficient mice exhibit pervasive social deficits, indicating the critical role of OXTR in social behaviors. In the present study, we generated Oxtr cDNA(HA)-Ires-Cre knock-in mice, expressing both OXTR and Cre recombinase under the control of the endogenous Oxtr promoter. Knock-in cassette of Oxtr cDNA(HA)-Ires-Cre consisted of Oxtr cDNA tagged with the hemagglutinin epitope at the 3' end (Oxtr cDNA(HA)), internal ribosomal entry site (Ires), and Cre. Cre was expressed in the uterus, mammary gland, kidney, and brain of Oxtr cDNA(HA)-Ires-Cre knock-in mice. Furthermore, the distribution of Cre in the brain was similar to that observed in Oxtr-Venus fluorescent protein expressing mice (Oxtr-Venus), another animal model previously generated by our group. Social behavior of Oxtr cDNA(HA)-Ires-Cre knock-in mice was similar to that of wild-type animals. We demonstrated that this construct is expressed in OXTR-expressing neurons specifically after an infection with the recombinant adeno-associated virus carrying the flip-excision switch vector. Using this system, we showed the transport of the wheat-germ agglutinin tracing molecule from the OXTR-expressing neurons to the innervated neurons in knock-in mice. This study might contribute to the monosynaptic analysis of neuronal circuits and to the optogenetic analysis of neurons expressing OXTR. © 2015 Wiley Periodicals, Inc.
Suppression of the hypothalamic-pituitary-gonadal axis by TAK-385 (relugolix), a novel, investigational, orally active, small molecule gonadotropin-releasing hormone (GnRH) antagonist: studies in human GnRH receptor knock-in mice.

PubMed

Nakata, Daisuke; Masaki, Tsuneo; Tanaka, Akira; Yoshimatsu, Mie; Akinaga, Yumiko; Asada, Mari; Sasada, Reiko; Takeyama, Michiyasu; Miwa, Kazuhiro; Watanabe, Tatsuya; Kusaka, Masami

2014-01-15

TAK-385 (relugolix) is a novel, non-peptide, orally active gonadotropin-releasing hormone (GnRH) antagonist, which builds on previous work with non-peptide GnRH antagonist TAK-013. TAK-385 possesses higher affinity and more potent antagonistic activity for human and monkey GnRH receptors compared with TAK-013. Both TAK-385 and TAK-013 have low affinity for the rat GnRH receptor, making them difficult to evaluate in rodent models. Here we report the human GnRH receptor knock-in mouse as a humanized model to investigate pharmacological properties of these compounds on gonadal function. Twice-daily oral administration of TAK-013 (10mg/kg) for 4 weeks decreased the weights of testes and ventral prostate in male knock-in mice but not in male wild-type mice, demonstrating the validity of this model to evaluate antagonists for the human GnRH receptor. The same dose of TAK-385 also reduced the prostate weight to castrate levels in male knock-in mice. In female knock-in mice, twice-daily oral administration of TAK-385 (100mg/kg) induced constant diestrous phases within the first week, decreased the uterus weight to ovariectomized levels and downregulated GnRH receptor mRNA in the pituitary after 4 weeks. Gonadal function of TAK-385-treated knock-in mice began to recover after 5 days and almost completely recovered within 14 days after drug withdrawal in both sexes. Our findings demonstrate that TAK-385 acts as an antagonist for human GnRH receptor in vivo and daily oral administration potently, continuously and reversibly suppresses the hypothalamic-pituitary-gonadal axis. TAK-385 may provide useful therapeutic interventions in hormone-dependent diseases including endometriosis, uterine fibroids and prostate cancer. Copyright © 2013 Elsevier B.V. All rights reserved.
A Novel Mgp-Cre Knock-In Mouse Reveals an Anticalcification/Antistiffness Candidate Gene in the Trabecular Meshwork and Peripapillary Scleral Region.

PubMed

Borrás, Teresa; Smith, Matthew H; Buie, LaKisha K

2015-04-01

Soft tissue calcification is a pathological condition. Matrix Gla (MGP) is a potent mineralization inhibitor secreted by cartilage chondrocytes and arteries' vascular smooth muscle cells. Mgp knock-out mice die at 6 weeks due to massive arterial calcification. Arterial calcification results in arterial stiffness and higher systolic blood pressure. Intriguingly, MGP was highly abundant in trabecular meshwork (TM). Because tissue stiffness is relevant to glaucoma, we investigated which additional eye tissues use Mgp's function using knock-in mice. An Mgp-Cre-recombinase coding sequence (Cre) knock-in mouse, containing Mgp DNA plus an internal ribosomal entry site (IRES)-Cre-cassette was generated by homologous recombination. Founders were crossed with Cre-mediated reporter mouse R26R-lacZ. Their offspring expresses lacZ where Mgp is transcribed. Eyes from MgpCre/+;R26RlacZ/+ (Mgp-lacZ knock-in) and controls, 1 to 8 months were assayed for β-gal enzyme histochemistry. As expected, Mgp-lacZ knock-in's TM was intensely blue. In addition, this mouse revealed high specific expression in the sclera, particularly in the peripapillary scleral region (ppSC). Ciliary muscle and sclera above the TM were also positive. Scleral staining was located immediately underneath the choroid (chondrocyte layer), began midsclera and was remarkably high in the ppSC. Cornea, iris, lens, ciliary body, and retina were negative. All mice exhibited similar staining patterns. All controls were negative. Matrix Gla's restricted expression to glaucoma-associated tissues from anterior and posterior segments suggests its involvement in the development of the disease. Matrix Gla's anticalcification/antistiffness properties in the vascular tissue, together with its high TM and ppCS expression, place this gene as a strong candidate for TM's softness and sclera's stiffness regulation in glaucoma.
Erythroblast differentiation at spleen in Q137E mutant ribosomal protein S19 gene knock-in C57BL/6J mice.

PubMed

Yamanegi, Koji; Yamada, Naoko; Nakasho, Keiji; Nishiura, Hiroshi

2018-01-01

We recently found that erythroblast-like cells derived from human leukaemia K562 cells express C5a receptor (C5aR) and produce its antagonistic and agonistic ligand ribosomal protein S19 (RP S19) polymer, which is cross-linked between K122 and Q137 by tissue transglutaminases. RP S19 polymer binds to the reciprocal C5aRs on erythroblast-like cells and macrophage-like cells derived from human monocytic THP-1 cells and promotes differentiation into reticulocyte-like cells through enucleation in vitro. To examine the roles of RP S19 polymer in mouse erythropoiesis, we prepared Q137E mutant RP S19 gene knock-in C57BL/6J mice. In contrast to wild-type mice, erythroblast numbers at the preliminary stage (CD71 high /TER119 low ) in spleen based on transferrin receptor (CD71) and glycophorin A (TER119) values and erythrocyte numbers in orbital artery bloods were not largely changed in knock-in mice. Conversely, erythroblast numbers at the early stage (CD71 high /TER119 high ) were significantly decreased in spleen by knock-in mice. The reduction of early erythroblast numbers in spleen was enhanced by the phenylhydrazine-induced pernicious anemia model knock-in mice and was rescued by a functional analogue of RP S19 dimer S-tagged C5a/RP S19. These data indicated that RP S19 polymer plays the roles in the early erythroblast differentiation of C57BL/6J mouse spleen. Copyright © 2017 Elsevier GmbH. All rights reserved.
Initial elevations in glutamate and dopamine neurotransmission decline with age, as does exploratory behavior, in LRRK2 G2019S knock-in mice

PubMed Central

Kuhlmann, Naila; Kadgien, Chelsie A; Tatarnikov, Igor; Fox, Jesse; Khinda, Jaskaran; Mitchell, Emma; Bergeron, Sabrina; Melrose, Heather

2017-01-01

LRRK2 mutations produce end-stage Parkinson’s disease (PD) with reduced nigrostriatal dopamine, whereas, asymptomatic carriers have increased dopamine turnover and altered brain connectivity. LRRK2 pathophysiology remains unclear, but reduced dopamine and mitochondrial abnormalities occur in aged G2019S mutant knock-in (GKI) mice. Conversely, cultured GKI neurons exhibit increased synaptic transmission. We assessed behavior and synaptic glutamate and dopamine function across a range of ages. Young GKI mice exhibit more vertical exploration, elevated glutamate and dopamine transmission, and aberrant D2-receptor responses. These phenomena decline with age, but are stable in littermates. In young GKI mice, dopamine transients are slower, independent of dopamine transporter (DAT), increasing the lifetime of extracellular dopamine. Slowing of dopamine transients is observed with age in littermates, suggesting premature ageing of dopamine synapses in GKI mice. Thus, GKI mice exhibit early, but declining, synaptic and behavioral phenotypes, making them amenable to investigation of early pathophysiological, and later parkinsonian-like, alterations. This model will prove valuable in efforts to develop neuroprotection for PD. PMID:28930069
Initial elevations in glutamate and dopamine neurotransmission decline with age, as does exploratory behavior, in LRRK2 G2019S knock-in mice.

PubMed

Volta, Mattia; Beccano-Kelly, Dayne A; Paschall, Sarah A; Cataldi, Stefano; MacIsaac, Sarah E; Kuhlmann, Naila; Kadgien, Chelsie A; Tatarnikov, Igor; Fox, Jesse; Khinda, Jaskaran; Mitchell, Emma; Bergeron, Sabrina; Melrose, Heather; Farrer, Matthew J; Milnerwood, Austen J

2017-09-20

LRRK2 mutations produce end-stage Parkinson's disease (PD) with reduced nigrostriatal dopamine, whereas, asymptomatic carriers have increased dopamine turnover and altered brain connectivity. LRRK2 pathophysiology remains unclear, but reduced dopamine and mitochondrial abnormalities occur in aged G2019S mutant knock-in (GKI) mice. Conversely, cultured GKI neurons exhibit increased synaptic transmission. We assessed behavior and synaptic glutamate and dopamine function across a range of ages. Young GKI mice exhibit more vertical exploration, elevated glutamate and dopamine transmission, and aberrant D2-receptor responses. These phenomena decline with age, but are stable in littermates. In young GKI mice, dopamine transients are slower, independent of dopamine transporter (DAT), increasing the lifetime of extracellular dopamine. Slowing of dopamine transients is observed with age in littermates, suggesting premature ageing of dopamine synapses in GKI mice. Thus, GKI mice exhibit early, but declining, synaptic and behavioral phenotypes, making them amenable to investigation of early pathophysiological, and later parkinsonian-like, alterations. This model will prove valuable in efforts to develop neuroprotection for PD.

Retigabine, a Kv7.2/Kv7.3-Channel Opener, Attenuates Drug-Induced Seizures in Knock-In Mice Harboring Kcnq2 Mutations.

PubMed

Ihara, Yukiko; Tomonoh, Yuko; Deshimaru, Masanobu; Zhang, Bo; Uchida, Taku; Ishii, Atsushi; Hirose, Shinichi

2016-01-01

The hetero-tetrameric voltage-gated potassium channel Kv7.2/Kv7.3, which is encoded by KCNQ2 and KCNQ3, plays an important role in limiting network excitability in the neonatal brain. Kv7.2/Kv7.3 dysfunction resulting from KCNQ2 mutations predominantly causes self-limited or benign epilepsy in neonates, but also causes early onset epileptic encephalopathy. Retigabine (RTG), a Kv7.2/ Kv7.3-channel opener, seems to be a rational antiepileptic drug for epilepsies caused by KCNQ2 mutations. We therefore evaluated the effects of RTG on seizures in two strains of knock-in mice harboring different Kcnq2 mutations, in comparison to the effects of phenobarbital (PB), which is the first-line antiepileptic drug for seizures in neonates. The subjects were heterozygous knock-in mice (Kcnq2Y284C/+ and Kcnq2A306T/+) bearing the Y284C or A306T Kcnq2 mutation, respectively, and their wild-type (WT) littermates, at 63-100 days of age. Seizures induced by intraperitoneal injection of kainic acid (KA, 12mg/kg) were recorded using a video-electroencephalography (EEG) monitoring system. Effects of RTG on KA-induced seizures of both strains of knock-in mice were assessed using seizure scores from a modified Racine's scale and compared with those of PB. The number and total duration of spike bursts on EEG and behaviors monitored by video recording were also used to evaluate the effects of RTG and PB. Both Kcnq2Y284C/+ and Kcnq2A306T/+ mice showed significantly more KA-induced seizures than WT mice. RTG significantly attenuated KA-induced seizure activities in both Kcnq2Y284C/+ and Kcnq2A306T/+ mice, and more markedly than PB. This is the first reported evidence of RTG ameliorating KA-induced seizures in knock-in mice bearing mutations of Kcnq2, with more marked effects than those observed with PB. RTG or other Kv7.2-channel openers may be considered as first-line antiepileptic treatments for epilepsies resulting from KCNQ2 mutations.
Pou4f2-GFP knock-in mouse line: A model for studying retinal ganglion cell development.

PubMed

Zheng, Dongwang; Yang, Xiaoyan; Sheng, Donglai; Yu, Dongliang; Liang, Guoqing; Guo, Luming; Xu, Mei; Hu, Xu; He, Daqiang; Yang, Yang; Wang, Yuying

2016-10-01

Pou4f2 acts as a key node in the comprehensive and step-wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2-green fluorescent protein (GFP) fusion protein expressed in RGCs. Co-localization of POU4F2 and GFP in the retina and brain of Pou4f2-GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild-type mice, the expression patterns of POU4F2 and POU4F1 and the co-expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2-GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2-GFP/GFP homozygote and wild-type mice. These results demonstrated that the development of RGCs in Pou4f2-GFP/GFP homozygote mice was the same as in wild-type mice. Thus, the present Pou4f2-GFP knock-in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs. © 2016 Wiley Periodicals, Inc.
Pro416Arg cherubism mutation in Sh3bp2 knock-in mice affects osteoblasts and alters bone mineral and matrix properties

PubMed Central

Wang, Chiachien J.; Chen, I-Ping; Koczon-Jaremko, Boguslawa; Boskey, Adele L.; Ueki, Yasuyoshi; Kuhn, Liisa; Reichenberger, Ernst J.

2010-01-01

Cherubism is an autosomal dominant disorder in children characterized by unwarranted symmetrical bone resorption of the jaws with fibrous tissue deposition. Mutations causing cherubism have been identified in the adaptor protein SH3BP2. Knock-in mice with a Pro416Arg mutation in Sh3bp2 exhibit a generalized osteoporotic bone phenotype. In this study, we examined the effects of this “cherubism” mutation on spectroscopic indices of “bone quality” and on osteoblast differentiation. Fourier-transform infrared imaging (FTIRI) analysis of femurs from wild-type and Sh3bp2 knock-in mice showed decreased mineral content, decreased mineral crystallinity/crystal size, and increased collagen maturity in homozygous mutants. To assess osteoblast maturation in vivo, knock-in mice were crossed with transgenic mice over-expressing GFP driven by 3.6-kb or 2.3-kb Col1a1 promoter fragments. Reduced numbers of mature osteoblasts were observed in homozygous mice. Neonatal calvarial cultures, which were enriched for osteoblasts by depletion of hematopoietic cells (negative selection for Ter119- and CD45-positive cells) were investigated for osteoblast-specific gene expression and differentiation, which demonstrated that differentiation and mineralization in homozygous osteoblast cultures was impaired. Co-cultures with calvarial osteoblasts and bone marrow macrophages showed that mutant osteoblasts appear to increase osteoclastogenesis resulting in increased bone resorption on bone chips. In summary, the Sh3bp2 mutation in cherubism mice alters bone quality, reduces osteoblast function, and may contribute to excessive bone resorption by osteoclasts. Our data, together with previous osteoclast studies, demonstrate a critical role of Sh3bp2 in bone remodeling and osteoblast differentiation. PMID:20117257
Mice with mutant Inf2 show impaired podocyte and slit diaphragm integrity in response to protamine-induced kidney injury.

PubMed

Subramanian, Balajikarthick; Sun, Hua; Yan, Paul; Charoonratana, Victoria T; Higgs, Henry N; Wang, Fang; Lai, Ka-Man V; Valenzuela, David M; Brown, Elizabeth J; Schlöndorff, Johannes S; Pollak, Martin R

2016-08-01

Mutations in the INF2 (inverted formin 2) gene, encoding a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause human focal segmental glomerulosclerosis (FSGS). INF2 interacts directly with certain other mammalian diaphanous formin proteins (mDia) that function as RhoA effector molecules. FSGS-causing INF2 mutations impair these interactions and disrupt the ability of INF2 to regulate Rho/Dia-mediated actin dynamics in vitro. However, the precise mechanisms by which INF2 regulates and INF2 mutations impair glomerular structure and function remain unknown. Here, we characterize an Inf2 R218Q point-mutant (knockin) mouse to help answer these questions. Knockin mice have no significant renal pathology or proteinuria at baseline despite diminished INF2 protein levels. INF2 mutant podocytes do show impaired reversal of protamine sulfate-induced foot process effacement by heparin sulfate perfusion. This is associated with persistent podocyte cytoplasmic aggregation, nephrin phosphorylation, and nephrin and podocin mislocalization, as well as impaired recovery of mDia membrane localization. These changes were partially mimicked in podocyte outgrowth cultures, in which podocytes from knockin mice show altered cellular protrusions compared to those from wild-type mice. Thus, in mice, normal INF2 function is not required for glomerular development but normal INF2 is required for regulation of the actin-based behaviors necessary for response to and/or recovery from injury. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
Reducing the Levels of Akt Activation by PDK1 Knock-in Mutation Protects Neuronal Cultures against Synthetic Amyloid-Beta Peptides.

PubMed

Yang, Shaobin; Pascual-Guiral, Sònia; Ponce, Rebeca; Giménez-Llort, Lydia; Baltrons, María A; Arancio, Ottavio; Palacio, Jose R; Clos, Victoria M; Yuste, Victor J; Bayascas, Jose R

2017-01-01

The Akt kinase has been widely assumed for years as a key downstream effector of the PI3K signaling pathway in promoting neuronal survival. This notion was however challenged by the finding that neuronal survival responses were still preserved in mice with reduced Akt activity. Moreover, here we show that the Akt signaling is elevated in the aged brain of two different mice models of Alzheimer Disease. We manipulate the rate of Akt stimulation by employing knock-in mice expressing a mutant form of PDK1 (phosphoinositide-dependent protein kinase 1) with reduced, but not abolished, ability to activate Akt. We found increased membrane localization and activity of the TACE/ADAM17 α-secretase in the brain of the PDK1 mutant mice with concomitant TNFR1 processing, which provided neurons with resistance against TNFα-induced neurotoxicity. Opposite to the Alzheimer Disease transgenic mice, the PDK1 knock-in mice exhibited an age-dependent attenuation of the unfolding protein response, which protected the mutant neurons against endoplasmic reticulum stressors. Moreover, these two mechanisms cooperatively provide the mutant neurons with resistance against amyloid-beta oligomers, and might singularly also contribute to protect these mice against amyloid-beta pathology.
Immune tolerance negatively regulates B cells in knock-in mice expressing broadly neutralizing HIV antibody 4E10.

PubMed

Doyle-Cooper, Colleen; Hudson, Krystalyn E; Cooper, Anthony B; Ota, Takayuki; Skog, Patrick; Dawson, Phillip E; Zwick, Michael B; Schief, William R; Burton, Dennis R; Nemazee, David

2013-09-15

A major goal of HIV research is to develop vaccines reproducibly eliciting broadly neutralizing Abs (bNAbs); however, this has proved to be challenging. One suggested explanation for this difficulty is that epitopes seen by bNAbs mimic self, leading to immune tolerance. We generated knock-in mice expressing bNAb 4E10, which recognizes the membrane proximal external region of gp41. Unlike b12 knock-in mice, described in the companion article (Ota et al. 2013. J. Immunol. 191: 3179-3185), 4E10HL mice were found to undergo profound negative selection of B cells, indicating that 4E10 is, to a physiologically significant extent, autoreactive. Negative selection occurred by various mechanisms, including receptor editing, clonal deletion, and receptor downregulation. Despite significant deletion, small amounts of IgM and IgG anti-gp41 were found in the sera of 4E10HL mice. On a Rag1⁻/⁻ background, 4E10HL mice had virtually no serum Ig of any kind. These results are consistent with a model in which B cells with 4E10 specificity are counterselected, raising the question of how 4E10 was generated in the patient from whom it was isolated. This represents the second example of a membrane proximal external region-directed bNAb that is apparently autoreactive in a physiological setting. The relative conservation in HIV of the 4E10 epitope might reflect the fact that it is under less intense immunological selection as a result of B cell self-tolerance. The safety and desirability of targeting this epitope by a vaccine is discussed in light of the newly described bNAb 10E8.
[Pathomechanism and therapeutic strategy of Fukuyama congenital muscular dystrophy and related disorders].

PubMed

Toda, Tatsushi

2009-11-01

Hypoglycosylation and reduced laminin-binding activity of alpha-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. We previously identified the genes for Fukuyama congenital muscular dystrophy (FCMD) and muscle-eye-brain disease (MEB). FCMD, caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. Knock-in mice carrying the founder retrotransposal insertion exhibited hypoglycosylated alpha-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact alpha-dystroglycan, and solid-phase assays determined laminin binding levels to be approximately 50% of normal. In contrast, intact alpha-dystroglycan is undetectable in the dystrophic Large mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact alpha-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. Transfer of fukutin into knock-in mice restored glycosylation of alpha-dystroglycan. Transfer of LARGE produced laminin-binding forms of alpha-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse. These data suggest that even partial restoration of alpha-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.
Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis

PubMed Central

Smagris, Eriks; BasuRay, Soumik; Li, John; Huang, Yongcheng; Lai, Ka-man V; Gromada, Jesper; Cohen, Jonathan C; Hobbs, Helen H

2015-01-01

A sequence polymorphism (rs738409, I148M) in patatin-like phospholipid domain containing protein 3 (PNPLA3) is strongly associated with nonalcoholic fatty liver disease (NAFLD), but the mechanistic basis for this association remains enigmatic. Neither ablation nor overexpression of wild-type PNPLA3 affects liver fat content in mice, whereas hepatic overexpression of the human 148M transgene causes steatosis. To determine whether the 148M allele causes fat accumulation in the liver when expressed at physiological levels, we introduced a methionine codon at position 148 of the mouse Pnpla3 gene. Knockin mice had normal levels of hepatic fat on a chow diet, but when challenged with a high-sucrose diet their liver fat levels increased 2 to 3-fold compared to wild-type littermates without any associated changes in glucose homeostasis. The increased liver fat in the knockin mice was accompanied by a 40-fold increase in PNPLA3 on hepatic lipid droplets, with no increase in hepatic PNPLA3 messenger RNA (mRNA). Similar results were obtained when the catalytic dyad of PNPLA3 was inactivated by substituting the catalytic serine with alanine (S47A). Conclusion: These data provide the first direct evidence that physiological expression of PNPLA3 148M variant causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of TG in the liver. (Hepatology 2015;61:108–118) PMID:24917523
Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant CaV2.1 channels

PubMed Central

Watase, Kei; Barrett, Curtis F.; Miyazaki, Taisuke; Ishiguro, Taro; Ishikawa, Kinya; Hu, Yuanxin; Unno, Toshinori; Sun, Yaling; Kasai, Sayumi; Watanabe, Masahiko; Gomez, Christopher M.; Mizusawa, Hidehiro; Tsien, Richard W.; Zoghbi, Huda Y.

2008-01-01

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (CaV) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of CaV2.1 channel or through a gain-of-function mechanism associated with accumulation of the expanded polyglutamine protein. We generated three strains of knockin (KI) mice carrying normal, expanded, or hyperexpanded CAG repeat tracts in the Cacna1a locus. The mice expressing hyperexpanded polyglutamine (Sca684Q) developed progressive motor impairment and aggregation of mutant CaV2.1 channels. Electrophysiological analysis of cerebellar Purkinje cells revealed similar Ca2+ channel current density among the three KI models. Neither voltage sensitivity of activation nor inactivation was altered in the Sca684Q neurons, suggesting that expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channels. The pathogenesis of SCA6 is apparently linked to an age-dependent process accompanied by accumulation of mutant CaV2.1 channels. PMID:18687887
Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage.

PubMed

Li, Juan; Prins, Daniel; Park, Hyun Jung; Grinfeld, Jacob; Gonzalez-Arias, Carlos; Loughran, Stephen; Dovey, Oliver M; Klampfl, Thorsten; Bennett, Cavan; Hamilton, Tina L; Pask, Dean C; Sneade, Rachel; Williams, Matthew; Aungier, Juliet; Ghevaert, Cedric; Vassiliou, George S; Kent, David G; Green, Anthony R

2018-02-08

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALR del/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALR del/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALR del/+ HSCs were more proliferative in vitro, but neither CALR del/+ nor CALR del/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF. © 2018 by The American Society of Hematology.
Autophagy and UPR in alpha-crystallin mutant knock-in mouse models of hereditary cataracts.

PubMed

Andley, Usha P; Goldman, Joshua W

2016-01-01

Knock-in mice provide useful models of congenital and age-related cataracts caused by α-crystallin mutations. R49C αA-crystallin and R120G αB-crystallin mutations are linked with hereditary cataracts. Knock-in αA-R49C+/- heterozygotes develop cataracts by 1-2months, whereas homozygote mice have cataracts at birth. The R49C mutation drastically reduces lens protein water solubility and causes cell death in knock-in mouse lenses. Mutant crystallin cannot function as a chaperone, which leads to protein aggregation and lens opacity. Protein aggregation disrupts the lens fiber cell structure and normal development and causes cell death in epithelial and fiber cells. We determined what aspects of the wild-type phenotype are age-dependently altered in the mutant lens. Wild-type, heterozygote (αA-R49C+/-), and homozygote (αA-R49C+/+) mouse lenses were assessed pre- and postnatally for lens morphology (electron microscopy, immunohistochemistry), and autophagy or unfolded protein response markers (immunoblotting). Morphology was altered by embryonic day 17 in R49C+/+ lenses; R49C+/- lens morphology was unaffected at this stage. Active autophagy in the lens epithelium of mutant lenses was indicated by the presence of autophagosomes using electron microscopy. Protein p62 levels, which are degraded specifically by autophagy, increased in αA-R49C mutant versus wild-type lenses, suggesting autophagy inhibition in the mutant lenses. The unfolded protein response marker XBP-1 was upregulated in adult lenses of αB-R120G+/+ mice, suggesting its role in lens opacification. Mutated crystallins alter lens morphology, autophagy, and stress responses. Therapeutic modulation of autophagic pathways may improve protein degradation in cataractous lenses and reduce lens opacity. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease. Copyright © 2015 Elsevier B.V. All rights reserved.
Generation and characterization of Lhx9 – GFPCreERT2 knock-in mouse line

PubMed Central

Xie, Xiaoling; Deng, Min; Gan, Lin

2014-01-01

Summary LHX9 is a LIM-homeodomain transcription factor essential for the development of gonads, spinal cord interneurons, and thalamic neurons to name a few. We recently reported the expression of LHX9 in retinal amacrine cells during development. In this study, we generated an Lhx9 - GFPCreERT2 (GCE) knock-in mouse line by knocking-in a GCE cassette at the Lhx9 locus, thus inactivating endogenous Lhx9. Lhx9GCE/+ mice were viable, fertile, and displayed no overt phenotypical characteristics. Lhx9GCE/GCE mice were all phenotypically female, smaller in size, viable, but infertile. The specificity and efficacy of the Lhx9-GCE mouse line was verified by crossing it to a Rosa26 - tdTomato reporter mouse line, which reveals the Cre recombinase activities in retinal amacrine cells, developing limbs, testis, hippocampal neurons, thalamic neurons, and cerebellar neurons. Taken together, the Lhx9-GCE mouse line could serve as a beneficial tool for lineage tracing and gene manipulation experiments. PMID:25112520
Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities.

PubMed

Igoucheva, Olga; Alexeev, Vitali; Halabi, Carmen M; Adams, Sheila M; Stoilov, Ivan; Sasaki, Takako; Arita, Machiko; Donahue, Adele; Mecham, Robert P; Birk, David E; Chu, Mon-Li

2015-08-28

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Transformation by HrasG12V is Consistently Associated with Mutant Allele Copy Gains and is Reversed by Farnesyl Transferase Inhibition

PubMed Central

Chen, Xu; Makarewicz, Jacek M.; Knauf, Jeffrey A.; Johnson, Linda K.; Fagin, James A.

2014-01-01

RAS-driven malignancies remain a major therapeutic challenge. The two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-o-tetradecanoylphorbol-13-acetate (TPA) model of mouse skin carcinogenesis has been used to study mechanisms of epithelial tumor development by oncogenic Hras. We used mice with a HrasG12V knock-in allele to elucidate the early events after Hras activation, and to evaluate the therapeutic effectiveness of farnesyltransferase (FTI) inhibition. Treatment of Caggs-Cre/FR-HrasG12V mice with TPA alone was sufficient to trigger papilloma development with shorter latency and a ~10-fold greater tumor burden than DMBA/TPA-treated WT controls. HrasG12V allele copy number was increased in all papillomas induced by TPA. DMBA/TPA treatment of HrasG12V knock-in mice induced an even greater incidence of papillomas, which either harbored HrasG12V amplification, or developed a HrasQ61L mutation in the second allele. Laser-capture microdissection of normal skin, hyperplastic skin and papillomas showed that amplification occurred only at the papilloma stage. HRAS mutant allelic imbalance was also observed in human cancer cell lines, consistent with a requirement for augmented oncogenic HRAS signaling for tumor development. The FTI SCH66336 blocks HRAS farnesylation and delocalizes it from the plasma membrane. NRAS and KRAS are not affected as they are alternatively prenylated. When tested in lines harboring HRAS, NRAS or KRAS mutations, SCH66336 delocalized, inhibited signaling and preferentially inhibited growth only of HRAS-mutant lines. Treatment with SCH66336 also induced near-complete regression of papillomas of TPA-treated HrasG12V knock-in mice. These data suggest that farnesyl transferase inhibitors should be reevaluated as targeted agents for human HRAS-driven cancers, such as those of bladder, thyroid and other epithelial lineages. PMID:24240680
Magnolol-mediated regulation of plasma triglyceride through affecting lipoprotein lipase activity in apolipoprotein A5 knock-in mice.

PubMed

Chang, Chun-Kai; Lin, Xiu-Ru; Lin, Yen-Lin; Fang, Woei-Horng; Lin, Shu-Wha; Chang, Sui-Yuan; Kao, Jau-Tsuen

2018-01-01

Hyperlipidemia is a risk factor of arteriosclerosis, stroke, and other coronary heart disease, which has been shown to correlate with single nucleotide polymorphisms of genes essential for lipid metabolism, such as lipoprotein lipase (LPL) and apolipoprotein A5 (APOA5). In this study, the effect of magnolol, the main active component extracted from Magnolia officinalis, on LPL activity was investigated. A dose-dependent up-regulation of LPL activity, possibly through increasing LPL mRNA transcription, was observed in mouse 3T3-L1 pre-adipocytes cultured in the presence of magnolol for 6 days. Subsequently, a transgenic knock-in mice carrying APOA5 c.553G>T variant was established and then fed with corn oil with or without magnolol for four days. The baseline plasma triglyceride levels in transgenic knock-in mice were higher than those in wild-type mice, with the highest increase occurred in homozygous transgenic mice (106 mg/dL vs 51 mg/dL, p<0.01). After the induction of hyperglyceridemia along with the administration of magnolol, the plasma triglyceride level in heterozygous transgenic mice was significantly reduced by half. In summary, magnolol could effectively lower the plasma triglyceride levels in APOA5 c.553G>T variant carrier mice and facilitate the triglyceride metabolism in postprandial hypertriglyceridemia.
Magnolol-mediated regulation of plasma triglyceride through affecting lipoprotein lipase activity in apolipoprotein A5 knock-in mice

PubMed Central

Lin, Yen-Lin; Fang, Woei-Horng; Lin, Shu-Wha; Kao, Jau-Tsuen

2018-01-01

Hyperlipidemia is a risk factor of arteriosclerosis, stroke, and other coronary heart disease, which has been shown to correlate with single nucleotide polymorphisms of genes essential for lipid metabolism, such as lipoprotein lipase (LPL) and apolipoprotein A5 (APOA5). In this study, the effect of magnolol, the main active component extracted from Magnolia officinalis, on LPL activity was investigated. A dose-dependent up-regulation of LPL activity, possibly through increasing LPL mRNA transcription, was observed in mouse 3T3-L1 pre-adipocytes cultured in the presence of magnolol for 6 days. Subsequently, a transgenic knock-in mice carrying APOA5 c.553G>T variant was established and then fed with corn oil with or without magnolol for four days. The baseline plasma triglyceride levels in transgenic knock-in mice were higher than those in wild-type mice, with the highest increase occurred in homozygous transgenic mice (106 mg/dL vs 51 mg/dL, p<0.01). After the induction of hyperglyceridemia along with the administration of magnolol, the plasma triglyceride level in heterozygous transgenic mice was significantly reduced by half. In summary, magnolol could effectively lower the plasma triglyceride levels in APOA5 c.553G>T variant carrier mice and facilitate the triglyceride metabolism in postprandial hypertriglyceridemia. PMID:29425239
Systematic Localization and Identification of SUMOylation Substrates in Knock-In Mice Expressing Affinity-Tagged SUMO1.

PubMed

Tirard, Marilyn; Brose, Nils

2016-01-01

Protein SUMOylation is a posttranslational protein modification that is emerging as a key regulatory process in neurobiology. To date, however, SUMOylation in vivo has only been studied cursorily. Knock-in mice expressing His6-HA-SUMO1 from the Sumo1 locus allow for the highly specific localization and identification of endogenous SUMO1 substrates under physiological and pathophysiological conditions. By making use of the HA-tag and using wild-type mice for highly stringent negative control samples, SUMO1 targets can be specifically localized in and purified from cultured mouse nerve cells and mouse tissues.
Knock-in human GDF5 proregion L373R mutation as a mouse model for proximal symphalangism.

PubMed

Zhang, Xinxin; Xing, Xuesha; Liu, Xing; Hu, Yu; Qu, Shengqiang; Wang, Heyi; Luo, Yang

2017-12-26

Proximal symphalangism (SYM1) is an autosomal dominant disorder, mainly characterized by bony fusions of the proximal phalanges of the hands and feet. GDF5 and NOG were identified to be responsible for SYM1. We have previously reported on a p.Leu373Arg mutation in the GDF5 proregion present in a Chinese family with SYM1. Here, we investigated the effects of the GDF-L373R mutation. The variant caused proteolysis efficiency of GDF5 increased in ATDC5 cells. The variant also caused upregulation of SMAD1/5/8 phosphorylation and increased expression of target genes SMURF1 , along with COL2A1 and SOX9 which are factors associated with chondrosis. Furthermore, we developed a human-relevant SYM1 mouse model by making a Gdf5 L367R (the orthologous position for L373R in humans) knock-in mouse. Gdf5 L367R/+ and Gdf5 L367R/L367R mice displayed stiffness and adhesions across the proximal phalanx joint which were in complete accord with SYM1. It was also confirmed the joint formation and development was abnormal in Gdf5 L367R/+ and Gdf5 L367R/L367R mice, including the failure to develop the primary ossification center and be hypertrophic chondrocytes during embryonic development. This knock-in mouse model offers a tool for assessing the pathogenesis of SYM1 and the function of the GDF5 proregion.
Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis.

PubMed

Smagris, Eriks; BasuRay, Soumik; Li, John; Huang, Yongcheng; Lai, Ka-man V; Gromada, Jesper; Cohen, Jonathan C; Hobbs, Helen H

2015-01-01

A sequence polymorphism (rs738409, I148M) in patatin-like phospholipid domain containing protein 3 (PNPLA3) is strongly associated with nonalcoholic fatty liver disease (NAFLD), but the mechanistic basis for this association remains enigmatic. Neither ablation nor overexpression of wild-type PNPLA3 affects liver fat content in mice, whereas hepatic overexpression of the human 148M transgene causes steatosis. To determine whether the 148M allele causes fat accumulation in the liver when expressed at physiological levels, we introduced a methionine codon at position 148 of the mouse Pnpla3 gene. Knockin mice had normal levels of hepatic fat on a chow diet, but when challenged with a high-sucrose diet their liver fat levels increased 2 to 3-fold compared to wild-type littermates without any associated changes in glucose homeostasis. The increased liver fat in the knockin mice was accompanied by a 40-fold increase in PNPLA3 on hepatic lipid droplets, with no increase in hepatic PNPLA3 messenger RNA (mRNA). Similar results were obtained when the catalytic dyad of PNPLA3 was inactivated by substituting the catalytic serine with alanine (S47A). These data provide the first direct evidence that physiological expression of PNPLA3 148M variant causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of TG in the liver. © 2014 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.
In Vivo Investigation of Escitalopram’s Allosteric Site on the Serotonin Transporter

PubMed Central

Murray, Karen E.; Ressler, Kerry J.; Owens, Michael J.

2015-01-01

Escitalopram is a commonly prescribed antidepressant of the selective serotonin reuptake inhibitor class. Clinical evidence and mapping of the serotonin transporter (SERT) identified that escitalopram, in addition to its binding to a primary uptake-blocking site, is capable of binding to the SERT via an allosteric site that is hypothesized to alter escitalopram’s kinetics at the SERT. The studies reported here examined the in vivo role of the SERT allosteric site in escitalopram action. A knockin mouse model that possesses an allosteric-null SERT was developed. Autoradiographic studies indicated that the knockin protein was expressed at a lower density than endogenous mouse SERT (approximately 10–30% of endogenous mouse SERT), but the knockin mice are a viable tool to study the allosteric site. Microdialysis studies in the ventral hippocampus found no measurable decrease in extracellular serotonin response after local escitalopram challenge in mice without the allosteric site compared to mice with the site (p = 0.297). In marble burying assays there was a modest effect of the absence of the allosteric site, with a larger systemic dose of escitalopram (10-fold) necessary for the same effect as in mice with intact SERT (p = 0.023). However, there was no effect of the allosteric site in the tail suspension test. Together these data suggest that there may be a regional specificity in the role of the allosteric site. The lack of a robust effect overall suggests that the role of the allosteric site for escitalopram on the SERT may not produce meaningful in vivo effects. PMID:26621784

Loss of ethanol conditioned taste aversion and motor stimulation in knockin mice with ethanol-insensitive α2-containing GABA(A) receptors.

PubMed

Blednov, Y A; Borghese, C M; McCracken, M L; Benavidez, J M; Geil, C R; Osterndorff-Kahanek, E; Werner, D F; Iyer, S; Swihart, A; Harrison, N L; Homanics, G E; Harris, R A

2011-01-01

GABA type A receptors (GABA(A)-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABA(A)-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705-714, 2004; Pharmacol Biochem Behav 90:95-104, 2008; J Psychiatr Res 42:184-191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism.
Loss of Ethanol Conditioned Taste Aversion and Motor Stimulation in Knockin Mice with Ethanol-Insensitive α2-Containing GABAA Receptors

PubMed Central

Borghese, C. M.; McCracken, M. L.; Benavidez, J. M.; Geil, C. R.; Osterndorff-Kahanek, E.; Werner, D. F.; Iyer, S.; Swihart, A.; Harrison, N. L.; Homanics, G. E.; Harris, R. A.

2011-01-01

GABA type A receptors (GABAA-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABAA-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705–714, 2004; Pharmacol Biochem Behav 90:95–104, 2008; J Psychiatr Res 42:184–191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism. PMID:20876231
Interferon β induces clearance of mutant ataxin 7 and improves locomotion in SCA7 knock-in mice.

PubMed

Chort, Alice; Alves, Sandro; Marinello, Martina; Dufresnois, Béatrice; Dornbierer, Jean-Gabriel; Tesson, Christelle; Latouche, Morwena; Baker, Darren P; Barkats, Martine; El Hachimi, Khalid H; Ruberg, Merle; Janer, Alexandre; Stevanin, Giovanni; Brice, Alexis; Sittler, Annie

2013-06-01

We showed previously, in a cell model of spinocerebellar ataxia 7, that interferon beta induces the expression of PML protein and the formation of PML protein nuclear bodies that degrade mutant ataxin 7, suggesting that the cytokine, used to treat multiple sclerosis, might have therapeutic value in spinocerebellar ataxia 7. We now show that interferon beta also induces PML-dependent clearance of ataxin 7 in a preclinical model, SCA7(266Q/5Q) knock-in mice, and improves motor function. Interestingly, the presence of mutant ataxin 7 in the mice induces itself the expression of endogenous interferon beta and its receptor. Immunohistological studies in brains from two patients with spinocerebellar ataxia 7 confirmed that these modifications are also caused by the disease in humans. Interferon beta, administered intraperitoneally three times a week in the knock-in mice, was internalized with its receptor in Purkinje and other cells and translocated to the nucleus. The treatment induced PML protein expression and the formation of PML protein nuclear bodies and decreased mutant ataxin 7 in neuronal intranuclear inclusions, the hallmark of the disease. No reactive gliosis or other signs of toxicity were observed in the brain or internal organs. The performance of the SCA7(266Q/5Q) knock-in mice was significantly improved on two behavioural tests sensitive to cerebellar function: the Locotronic® Test of locomotor function and the Beam Walking Test of balance, motor coordination and fine movements, which are affected in patients with spinocerebellar ataxia 7. In addition to motor dysfunction, SCA7(266Q/5Q) mice present abnormalities in the retina as in patients: ataxin 7-positive neuronal intranuclear inclusions that were reduced by interferon beta treatment. Finally, since neuronal death does not occur in the cerebellum of SCA7(266Q/5Q) mice, we showed in primary cell cultures expressing mutant ataxin 7 that interferon beta treatment improves Purkinje cell survival.
Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety.

PubMed

Labarca, C; Schwarz, J; Deshpande, P; Schwarz, S; Nowak, M W; Fonck, C; Nashmi, R; Kofuji, P; Dang, H; Shi, W; Fidan, M; Khakh, B S; Chen, Z; Bowers, B J; Boulter, J; Wehner, J M; Lester, H A

2001-02-27

Knock-in mice were generated that harbored a leucine-to-serine mutation in the alpha4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.
Alcohol-induced tolerance and physical dependence in mice with ethanol insensitive α1 GABAA receptors

PubMed Central

Werner, David F.; Swihart, Andrew R.; Ferguson, Carolyn; Lariviere, William R.; Harrison, Neil L.; Homanics, Gregg E.

2009-01-01

Background Although many people consume alcohol (ethanol), it remains unknown why some become addicted. Elucidating the molecular mechanisms of tolerance and physical dependence (withdrawal) may provide insight into alcohol addiction. While the exact molecular mechanisms of ethanol action are unclear, γ-aminobutyric acid type A receptors (GABAA-Rs) have been extensively implicated in ethanol action. The α1 GABAA-R subunit is associated with tolerance and physical dependence, but its exact role remains unknown. In this report, we tested the hypothesis that α1-GABAA-Rs mediate in part these effects of ethanol. Methods Ethanol-induced behavioral responses related to tolerance and physical dependence were investigated in knockin mice that have ethanol-insensitive α1 GABAA-Rs and wildtype controls. Acute functional tolerance (AFT) was assessed using the stationary dowel and loss of righting reflex assays. Chronic tolerance was assessed on the loss of righting reflex, fixed speed rotarod, hypothermia, and radiant tail flick assays following ten consecutive days of ethanol exposure. Withdrawal-related hyperexcitability was assessed by handling-induced convulsions following 3 cycles of ethanol vapor exposure/withdrawal. Immunoblots were used to assess α1 protein levels. Results Compared to controls, knockin mice displayed decreased AFT and chronic tolerance to ethanol-induced motor ataxia, and also displayed heightened ethanol-withdrawal hyperexcitability. No differences between wildtype and knockin mice were seen in other ethanol-induced behavioral measures. Following chronic exposure to ethanol, control mice displayed reductions in α1 protein levels, but knockins did not. Conclusions We conclude that α1-GABAA-Rs play a role in tolerance to ethanol-induced motor ataxia and withdrawal-related hyperexcitability. However, other aspects of behavioral tolerance and physical dependence do not rely on α1-containing GABAA-Rs. PMID:19032579
Establishment of Mouse Model of MYH9 Disorders: Heterozygous R702C Mutation Provokes Macrothrombocytopenia with Leukocyte Inclusion Bodies, Renal Glomerulosclerosis and Hearing Disability

PubMed Central

Suzuki, Nobuaki; Kunishima, Shinji; Ikejiri, Makoto; Maruyama, Shoichi; Sone, Michihiko; Takagi, Akira; Ikawa, Masahito; Okabe, Masaru; Kojima, Tetsuhito; Saito, Hidehiko; Naoe, Tomoki; Matsushita, Tadashi

2013-01-01

Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation. We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/− mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/− mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May–Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm. In other organs, R702C+/− mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response. These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation. PMID:23976996
Establishment of mouse model of MYH9 disorders: heterozygous R702C mutation provokes macrothrombocytopenia with leukocyte inclusion bodies, renal glomerulosclerosis and hearing disability.

PubMed

Suzuki, Nobuaki; Kunishima, Shinji; Ikejiri, Makoto; Maruyama, Shoichi; Sone, Michihiko; Takagi, Akira; Ikawa, Masahito; Okabe, Masaru; Kojima, Tetsuhito; Saito, Hidehiko; Naoe, Tomoki; Matsushita, Tadashi

2013-01-01

Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation. We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/- mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/- mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May-Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm. In other organs, R702C+/- mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response. These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation.
Generation and characterization of PDGFRα-GFPCreERT2 knock-In mouse line.

PubMed

Miwa, Hiroyuki; Era, Takumi

2015-05-01

Platelet-derived growth factor (PDGF) and its receptor play an important role in embryogenesis. PDGF receptor α (PDGFRα) is expressed specifically in the embryonic day 7.5 (E7.5) mesoderm and in the E9.5 neural crest among other tissues. PDGFRα-expressing cells and their descendants are involved in the formation of various tissues. To trace PDGFRα-expressing cells in vivo, we generated a knock-in mouse line that expressed a fusion protein of green fluorescent protein (GFP), Cre recombinase (Cre), and mutated estrogen receptor ligand-binding domain (ERT2) under the control of the PDGFRα promoter. In these mice, Cre activity in PDGFRα-expressing cells could be induced by tamoxifen treatment. Taken together, our results suggest that the knock-in mouse line generated here could be useful for studying PDGFRα-expressing cells and their descendants in vivo at various stages of development. © 2015 Wiley Periodicals, Inc.
Spatial reversal learning defect coincides with hypersynchronous telencephalic BOLD functional connectivity in APPNL-F/NL-F knock-in mice.

PubMed

Shah, Disha; Latif-Hernandez, Amira; De Strooper, Bart; Saito, Takashi; Saido, Takaomi; Verhoye, Marleen; D'Hooge, Rudi; Van der Linden, Annemie

2018-04-19

Amyloid pathology occurs early in Alzheimer's disease (AD), and has therefore been the focus of numerous studies. Transgenic mouse models have been instrumental to study amyloidosis, but observations might have been confounded by APP-overexpression artifacts. The current study investigated early functional defects in an APP knock-in mouse model, which allows assessing the effects of pathological amyloid-beta (Aβ) without interference of APP-artifacts. Female APP NL/NL knock-in mice of 3 and 7 months old were compared to age-matched APP NL-F/NL-F mice with increased Aβ42/40 ratio and initial Aβ-plaque deposition around 6 months of age. Spatial learning was examined using a Morris water maze protocol consisting of acquisition and reversal trials interleaved with reference memory tests. Functional connectivity (FC) of brain networks was assessed using resting-state functional MRI (rsfMRI). The Morris water maze data revealed that 3 months old APP NL-F/NL-F mice were unable to reach the same reference memory proficiency as APP NL/NL mice after reversal training. This cognitive defect in 3-month-old APP NL-F/NL-F mice coincided with hypersynchronous FC of the hippocampal, cingulate, caudate-putamen, and default-mode-like networks. The occurrence of these defects in APP NL-F/NL-F mice demonstrates that cognitive flexibility and synchronicity of telencephalic activity are specifically altered by early Aβ pathology without changes in APP neurochemistry.
Characterization of a knock-in mouse model of the homozygous p.V37I variant in Gjb2.

PubMed

Chen, Ying; Hu, Lingxiang; Wang, Xueling; Sun, Changling; Lin, Xin; Li, Lei; Mei, Ling; Huang, Zhiwu; Yang, Tao; Wu, Hao

2016-09-13

The homozygous p.V37I variant in GJB2 is prevalent in East and Southeast Asians and may lead to mild-to-moderate hearing loss with reduced penetrance. To investigate the pathogenic mechanism underlying this variant, we generated a knock-in mouse model of homozygous p.V37I by an embryonic stem cell gene targeting method. Auditory brainstem response test showed that the knock-in mice developed progressive, mild-to-moderate hearing loss over the first 4-9 months. Overall no significant developmental and morphological abnormality was observed in the knock-in mouse cochlea, while confocal immunostaining and electron microscopic scanning revealed minor loss of the outer hair cells. Gene expression microarray analysis identified 105 up-regulated and 43 down-regulated genes in P5 knock-in mouse cochleae (P < 0.05 adjusted by the Benjamini &Hochberg method), among which four top candidate genes with the highest fold-changes or implication to deafness Fcer1g, Nnmt and Lars2 and Cuedc1 were verified by quantitative real-time PCR. Our study demonstrated that the homozygous p.V37I knock-in mouse modeled the hearing phenotype of the human patients and can serve as a useful animal model for further studies. The differentially expressed genes identified in this study may shed new insights into the understanding of the pathogenic mechanism and the phenotypic modification of homozygous p.V37I.
The caspase-3/p120 RasGAP stress-sensing module reduces liver cancer incidence but does not affect overall survival in gamma-irradiated and carcinogen-treated mice.

PubMed

Vanli, Güliz; Sempoux, Christine; Widmann, Christian

2017-06-01

Activation of oncogenes is the initial step in cellular transformation. Oncogenes favor aberrant proliferation, which, at least initially, induces cellular stress. This oncogenic stress can act as a safeguard mechanism against further transformation by inducing senescence or apoptosis. Yet, the few premalignant cells that tolerate and escape these senescent or apoptotic responses are those that will ultimately generate tumors. The caspase-3/p120 RasGAP module is a stress-sensing device that promotes survival under mild stress conditions. A point mutation in RasGAP that prevents its cleavage by caspase-3 inactivates the pro-survival capacity of the device. When the mice homozygous for this mutation (D455A knock-in mice) are patho-physiologically challenged, they experience much stronger cellular damage than their wild-type counterparts and the affected organs rapidly lose their functionality. We reasoned that the caspase-3/p120 RasGAP module could help premalignant cells to cope with oncogenic stress and hence favor the development of tumors. Using gamma-irradiation and N-ethyl-N-nitrosourea (ENU) as tumor initiators, we assessed the survival advantage that the caspase-3/p120 RasGAP module could provide to premalignant cells. No difference in overall mortality between wild-type and D455A knock-in mice were observed. However, the number of ENU-induced liver tumors in the knock-in mice was higher than in control mice. These results indicate that the caspase-3/p120 RasGAP stress-sensing module impacts on carcinogen-induced liver cancer incidence but not sufficiently so as to affect overall survival. Hence, gamma irradiation and ENU-induced tumorigenesis processes do not critically rely on a survival mechanism that contributes to the maintenance of organ homeostasis in stressed healthy tissues. © 2017 Wiley Periodicals, Inc.
RNase H2 catalytic core Aicardi-Goutières syndrome–related mutant invokes cGAS–STING innate immune-sensing pathway in mice

PubMed Central

Pokatayev, Vladislav; Hasin, Naushaba; Chon, Hyongi; Cerritelli, Susana M.; Sakhuja, Kiran; Ward, Jerrold M.; Morris, H. Douglas; Yan, Nan

2016-01-01

The neuroinflammatory autoimmune disease Aicardi-Goutières syndrome (AGS) develops from mutations in genes encoding several nucleotide-processing proteins, including RNase H2. Defective RNase H2 may induce accumulation of self-nucleic acid species that trigger chronic type I interferon and inflammatory responses, leading to AGS pathology. We created a knock-in mouse model with an RNase H2 AGS mutation in a highly conserved residue of the catalytic subunit, Rnaseh2aG37S/G37S (G37S), to understand disease pathology. G37S homozygotes are perinatal lethal, in contrast to the early embryonic lethality previously reported for Rnaseh2b- or Rnaseh2c-null mice. Importantly, we found that the G37S mutation led to increased expression of interferon-stimulated genes dependent on the cGAS–STING signaling pathway. Ablation of STING in the G37S mice results in partial rescue of the perinatal lethality, with viable mice exhibiting white spotting on their ventral surface. We believe that the G37S knock-in mouse provides an excellent animal model for studying RNASEH2-associated autoimmune diseases. PMID:26880576
75 FR 66104 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-27

... receptor gene exhibit impaired growth and resistance to thyroid hormone. Proc Natl Acad Sci U S A. 2000 Nov... overactivated. These mice have a knock-in dominantly negative mutant thyroid hormone receptor [beta] gene (TR... mutation in the thyroid hormone receptor beta gene spontaneously develop thyroid carcinoma: a mouse model...
Genetic Contributors to Intergenerational CAG Repeat Instability in Huntington’s Disease Knock-In Mice

PubMed Central

Neto, João Luís; Lee, Jong-Min; Afridi, Ali; Gillis, Tammy; Guide, Jolene R.; Dempsey, Stephani; Lager, Brenda; Alonso, Isabel; Wheeler, Vanessa C.; Pinto, Ricardo Mouro

2017-01-01

Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in exon 1 of the HTT gene. Longer repeat sizes are associated with increased disease penetrance and earlier ages of onset. Intergenerationally unstable transmissions are common in HD families, partly underlying the genetic anticipation seen in this disorder. HD CAG knock-in mouse models also exhibit a propensity for intergenerational repeat size changes. In this work, we examine intergenerational instability of the CAG repeat in over 20,000 transmissions in the largest HD knock-in mouse model breeding datasets reported to date. We confirmed previous observations that parental sex drives the relative ratio of expansions and contractions. The large datasets further allowed us to distinguish effects of paternal CAG repeat length on the magnitude and frequency of expansions and contractions, as well as the identification of large repeat size jumps in the knock-in models. Distinct degrees of intergenerational instability were observed between knock-in mice of six background strains, indicating the occurrence of trans-acting genetic modifiers. We also found that lines harboring a neomycin resistance cassette upstream of Htt showed reduced expansion frequency, indicative of a contributing role for sequences in cis, with the expanded repeat as modifiers of intergenerational instability. These results provide a basis for further understanding of the mechanisms underlying intergenerational repeat instability. PMID:27913616
Genetic Contributors to Intergenerational CAG Repeat Instability in Huntington's Disease Knock-In Mice.

PubMed

Neto, João Luís; Lee, Jong-Min; Afridi, Ali; Gillis, Tammy; Guide, Jolene R; Dempsey, Stephani; Lager, Brenda; Alonso, Isabel; Wheeler, Vanessa C; Pinto, Ricardo Mouro

2017-02-01

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in exon 1 of the HTT gene. Longer repeat sizes are associated with increased disease penetrance and earlier ages of onset. Intergenerationally unstable transmissions are common in HD families, partly underlying the genetic anticipation seen in this disorder. HD CAG knock-in mouse models also exhibit a propensity for intergenerational repeat size changes. In this work, we examine intergenerational instability of the CAG repeat in over 20,000 transmissions in the largest HD knock-in mouse model breeding datasets reported to date. We confirmed previous observations that parental sex drives the relative ratio of expansions and contractions. The large datasets further allowed us to distinguish effects of paternal CAG repeat length on the magnitude and frequency of expansions and contractions, as well as the identification of large repeat size jumps in the knock-in models. Distinct degrees of intergenerational instability were observed between knock-in mice of six background strains, indicating the occurrence of trans-acting genetic modifiers. We also found that lines harboring a neomycin resistance cassette upstream of Htt showed reduced expansion frequency, indicative of a contributing role for sequences in cis, with the expanded repeat as modifiers of intergenerational instability. These results provide a basis for further understanding of the mechanisms underlying intergenerational repeat instability. Copyright © 2017 by the Genetics Society of America.
Identification of rat Rosa26 locus enables generation of knock-in rat lines ubiquitously expressing tdTomato.

PubMed

Kobayashi, Toshihiro; Kato-Itoh, Megumi; Yamaguchi, Tomoyuki; Tamura, Chihiro; Sanbo, Makoto; Hirabayashi, Masumi; Nakauchi, Hiromitsu

2012-11-01

Recent discovery of a method for derivation and culture of germline-competent rat pluripotent stem cells (PSCs) enables generation of transgenic rats or knock-out rats via genetic modification of such PSCs. This opens the way to use rats, as is routine in mice, for analyses of gene functions or physiological features. In mouse or human, one widely used technique to express a gene of interest stably and ubiquitously is to insert that gene into the Rosa26 locus via gene targeting of PSCs. Rosa26 knock-in mice conditionally expressing a reporter or a toxin gene have contributed to tracing or ablation of specific cell lineages. We successfully identified a rat orthologue of the mouse Rosa26 locus. Insertion of tdTomato, a variant of red fluorescent protein, into the Rosa26 locus of PSCs of various rat strains allows ubiquitous expression of tdTomato. Through germline transmission of one Rosa26-tdTomato knock-in embryonic stem cell line, we also obtained tdTomato knock-in rats. These expressed tdTomato ubiquitously throughout their bodies, which indicates that the rat Rosa26 locus conserves functions of its orthologues in mouse and human. The new tools described here (targeting vectors, knock-in PSCs, and rats) should be useful for a variety of research using rats.
A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8.

PubMed

Bouhy, Delphine; Juneja, Manisha; Katona, Istvan; Holmgren, Anne; Asselbergh, Bob; De Winter, Vicky; Hochepied, Tino; Goossens, Steven; Haigh, Jody J; Libert, Claude; Ceuterick-de Groote, Chantal; Irobi, Joy; Weis, Joachim; Timmerman, Vincent

2018-01-01

Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot-Marie-Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces impairments in autophagy that may aggravate the phenotype.
B cells from knock-in mice expressing broadly neutralizing HIV antibody b12 carry an innocuous B cell receptor responsive to HIV vaccine candidates.

PubMed

Ota, Takayuki; Doyle-Cooper, Colleen; Cooper, Anthony B; Doores, Katherine J; Aoki-Ota, Miyo; Le, Khoa; Schief, William R; Wyatt, Richard T; Burton, Dennis R; Nemazee, David

2013-09-15

Broadly neutralizing Abs against HIV protect from infection, but their routine elicitation by vaccination has not been achieved. To generate small animal models to test vaccine candidates, we have generated targeted transgenic ("knock-in") mice expressing, in the physiological Ig H and L chain loci, two well-studied broadly neutralizing Abs: 4E10, which interacts with the membrane proximal external region of gp41, and b12, which binds to the CD4 binding site on gp120. 4E10HL mice are described in the companion article (Doyle-Cooper et al., J. Immunol. 191: 3186-3191). In this article, we describe b12 mice. B cells in b12HL mice, in contrast to the case in 4E10 mice, were abundant and essentially monoclonal, retaining the b12 specificity. In cell culture, b12HL B cells responded avidly to HIV envelope gp140 trimers and to BCR ligands. Upon transfer to wild-type recipients, b12HL B cells responded robustly to vaccination with gp140 trimers. Vaccinated b12H mice, although generating abundant precursors and Abs with affinity for Env, were unable to rapidly generate neutralizing Abs, highlighting the importance of developing Ag forms that better focus responses to neutralizing epitopes. The b12HL and b12H mice should be useful in optimizing HIV vaccine candidates to elicit a neutralizing response while avoiding nonprotective specificities.
Sqstm1-GFP knock-in mice reveal dynamic actions of Sqstm1 during autophagy and under stress conditions in living cells.

PubMed

Eino, Atsushi; Kageyama, Shun; Uemura, Takefumi; Annoh, Hiromichi; Saito, Tetsuya; Narita, Ichiei; Waguri, Satoshi; Komatsu, Masaaki

2015-12-01

Sqstm1 serves as a signaling hub and receptor for selective autophagy. Consequently, dysregulation of Sqstm1 causes imbalances in signaling pathways and disrupts proteostasis, thereby contributing to the development of human diseases. Environmental stresses influence the level of Sqstm1 by altering its expression and/or autophagic degradation, and also changes the localization of Sqstm1, making it difficult to elucidate the actions and roles of this protein. In this study, we developed knock-in mice expressing Sqstm1 fused to GFP (Sqstm1-GFP(KI/+)). Using these Sqstm1-GFP(KI/+) mice, we revealed for the first time the dynamics of endogenous Sqstm1 in living cells. Sqstm1-GFP was translocated to a restricted area of LC3-positive structures, which primarily correspond to the inside of autophagosomes, and then degraded. Moreover, exposure to arsenite induced expression of Sqstm1-GFP, followed by accumulation of the fusion protein in large aggregates that were degraded by autophagy. Furthermore, suppression of autophagy in Sqstm1-GFP(KI/+) mouse livers caused accumulation of Sqstm1-GFP and formation of GFP-positive aggregate structures, leading to severe hepatic failure. These results indicate that Sqstm1-GFP(KI/+) mice are a useful tool for analyzing Sqstm1 in living cells and intact animals. © 2015. Published by The Company of Biologists Ltd.
Generation and analysis of the thiazide-sensitive Na+ -Cl- cotransporter (Ncc/Slc12a3) Ser707X knockin mouse as a model of Gitelman syndrome.

PubMed

Yang, Sung-Sen; Lo, Yi-Fen; Yu, I-Shing; Lin, Shu-Wha; Chang, Tai-Hsiang; Hsu, Yu-Juei; Chao, Tai-Kuang; Sytwu, Huey-Kang; Uchida, Shinichi; Sasaki, Sei; Lin, Shih-Hua

2010-12-01

Gitelman syndrome (GS) is characterized by salt-losing hypotension, hypomagnesemia, hypokalemic metabolic alkalosis, and hypocalciuria. To better model human GS caused by a specific mutation in the thiazide-sensitive Na(+) -Cl(-) cotransporter (NCC) gene SLC12A3, we generated a nonsense Ncc Ser707X knockin mouse corresponding to human p.Ser710X (c.2135C>A), a recurrent mutation with severe phenotypes in Chinese GS patients. Compared with wild-type or heterozygous littermates, homozygous (Hom) knockin mice fully recapitulated the phenotype of human GS. The markedly reduced Ncc mRNA and virtually absent Ncc protein expression in kidneys of Hom mice was primarily due to nonsense-mediated mRNA decay (NMD) surveillance mechanisms. Expression of epithelial Na(+) channel (Enac), Ca(2+) channels (Trpv5 and Trpv6), and K(+) channels (Romk1 and maxi-K) were significantly increased. Late distal convoluted tubules (DCT) volume was increased and DCT cell ultrastructure appeared intact. High K(+) intake could not correct hypokalemia but caused a further increase in maxi-K but not Romk1 expression. Renal tissue from a patient with GS also showed the enhanced TRPV5 and ROMK1 expression in distal tubules. We suggest that the upregulation of TRPV5/6 and of ROMK1 and Maxi-K may contribute to hypocalciuria and hypokalemia in Ncc Ser707X knockin mice and human GS, respectively. © 2010 Wiley-Liss, Inc.

Foxp2 Regulates Identities and Projection Patterns of Thalamic Nuclei During Development.

PubMed

Ebisu, Haruka; Iwai-Takekoshi, Lena; Fujita-Jimbo, Eriko; Momoi, Takashi; Kawasaki, Hiroshi

2017-07-01

The molecular mechanisms underlying the formation of the thalamus during development have been investigated intensively. Although transcription factors distinguishing the thalamic primordium from adjacent brain structures have been uncovered, those involved in patterning inside the thalamus are largely unclear. Here, we show that Foxp2, a member of the forkhead transcription factor family, regulates thalamic patterning during development. We found a graded expression pattern of Foxp2 in the thalamic primordium of the mouse embryo. The expression levels of Foxp2 were high in the posterior region and low in the anterior region of the thalamic primordium. In Foxp2 (R552H) knockin mice, which have a missense loss-of-function mutation in the forkhead domain of Foxp2, thalamic nuclei of the posterior region of the thalamus were shrunken, while those of the intermediate region were expanded. Consistently, Foxp2 (R552H) knockin mice showed changes in thalamocortical projection patterns. Our results uncovered important roles of Foxp2 in thalamic patterning and thalamocortical projections during development. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice.

PubMed

Li, Kun; Wohlford-Lenane, Christine L; Channappanavar, Rudragouda; Park, Jung-Eun; Earnest, James T; Bair, Thomas B; Bates, Amber M; Brogden, Kim A; Flaherty, Heather A; Gallagher, Tom; Meyerholz, David K; Perlman, Stanley; McCray, Paul B

2017-04-11

The Middle East respiratory syndrome (MERS) emerged in Saudi Arabia in 2012, caused by a zoonotically transmitted coronavirus (CoV). Over 1,900 cases have been reported to date, with ∼36% fatality rate. Lack of autopsies from MERS cases has hindered understanding of MERS-CoV pathogenesis. A small animal model that develops progressive pulmonary manifestations when infected with MERS-CoV would advance the field. As mice are restricted to infection at the level of DPP4, the MERS-CoV receptor, we generated mice with humanized exons 10-12 of the mouse Dpp4 locus. Upon inoculation with MERS-CoV, human DPP4 knockin (KI) mice supported virus replication in the lungs, but developed no illness. After 30 serial passages through the lungs of KI mice, a mouse-adapted virus emerged (MERS MA ) that grew in lungs to over 100 times higher titers than the starting virus. A plaque-purified MERS MA clone caused weight loss and fatal infection. Virus antigen was observed in airway epithelia, pneumocytes, and macrophages. Pathologic findings included diffuse alveolar damage with pulmonary edema and hyaline membrane formation associated with accumulation of activated inflammatory monocyte-macrophages and neutrophils in the lungs. Relative to the parental MERS-CoV, MERS MA viruses contained 13-22 mutations, including several within the spike (S) glycoprotein gene. S-protein mutations sensitized viruses to entry-activating serine proteases and conferred more rapid entry kinetics. Recombinant MERS MA bearing mutant S proteins were more virulent than the parental virus in hDPP4 KI mice. The hDPP4 KI mouse and the MERS MA provide tools to investigate disease causes and develop new therapies.
Studies of protein aggregation in A53T α-synuclein transgenic, Tg2576 transgenic, and P246L presenilin-1 knock-in cross bred mice.

PubMed

Emmer, Kristel L; Covy, Jason P; Giasson, Benoit I

2012-01-24

Synucleinopathies are a group of neurodegenerative disorders, including Parkinson disease, associated with neuronal amyloid inclusions comprised of the presynaptic protein α-synuclein (α-syn); however the biological events that initiate and lead to the formation of these inclusions are still poorly understood. There is mounting evidence that intracellular α-syn aggregation may proceed via a seeding mechanism and could spread between neurons through a prion-like mechanism that may involve other amyloidogenic proteins. Several lines of evidence suggest that Aβ peptides and/or extracellular Aβ deposits may directly or indirectly promote intracellular α-syn aggregation. To assess the effects of Aβ peptides and extracellular Aβ deposits on α-syn aggregate formation, transgenic mice (line M83) expressing A53T human α-syn that are sensitive to developing α-syn pathological inclusions were cross bred to Tg2576 transgenic mice that generated elevated levels of Aβ peptides and develop abundant Aβ plaques. In addition these mice were bred to mice with the P264L presenilin-1 knock-in mutation that further promotes Aβ plaque formation. These mice demonstrated the expected formation of Aβ plaques; however despite the accumulation of hyperphosphorylated α-syn dystrophic neurites within or surrounding Aβ plaques, no additional α-syn pathologies were observed. These studies show that Aβ amyloid deposits can cause the local aggregation of α-syn, but these did not lead to more extensive α-syn pathology. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
Calcilytic Ameliorates Abnormalities of Mutant Calcium-Sensing Receptor (CaSR) Knock-In Mice Mimicking Autosomal Dominant Hypocalcemia (ADH).

PubMed

Dong, Bingzi; Endo, Itsuro; Ohnishi, Yukiyo; Kondo, Takeshi; Hasegawa, Tomoka; Amizuka, Norio; Kiyonari, Hiroshi; Shioi, Go; Abe, Masahiro; Fukumoto, Seiji; Matsumoto, Toshio

2015-11-01

Activating mutations of calcium-sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D3 or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT-305/MK-5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT-305/MK-5442 suppressed the hypersensitivity to extracellular Ca(2+) of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock-in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT-305/MK-5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1-34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock-in mice exhibited low bone turnover due to the deficiency of PTH, and JTT-305/MK-5442 as well as PTH(1-34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a novel therapeutic agent for ADH. © 2015 American Society for Bone and Mineral Research.
Comprehensive behavioral and molecular characterization of a new knock-in mouse model of Huntington's disease: zQ175.

PubMed

Menalled, Liliana B; Kudwa, Andrea E; Miller, Sam; Fitzpatrick, Jon; Watson-Johnson, Judy; Keating, Nicole; Ruiz, Melinda; Mushlin, Richard; Alosio, William; McConnell, Kristi; Connor, David; Murphy, Carol; Oakeshott, Steve; Kwan, Mei; Beltran, Jose; Ghavami, Afshin; Brunner, Dani; Park, Larry C; Ramboz, Sylvie; Howland, David

2012-01-01

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive and psychiatric manifestations. Since the mutation responsible for the disease was identified as an unstable expansion of CAG repeats in the gene encoding the huntingtin protein in 1993, numerous mouse models of HD have been generated to study disease pathogenesis and evaluate potential therapeutic approaches. Of these, knock-in models best mimic the human condition from a genetic perspective since they express the mutation in the appropriate genetic and protein context. Behaviorally, however, while some abnormal phenotypes have been detected in knock-in mouse models, a model with an earlier and more robust phenotype than the existing models is required. We describe here for the first time a new mouse line, the zQ175 knock-in mouse, derived from a spontaneous expansion of the CAG copy number in our CAG 140 knock-in colony [1]. Given the inverse relationship typically observed between age of HD onset and length of CAG repeat, since this new mouse line carries a significantly higher CAG repeat length it was expected to be more significantly impaired than the parent line. Using a battery of behavioral tests we evaluated both heterozygous and homozygous zQ175 mice. Homozygous mice showed motor and grip strength abnormalities with an early onset (8 and 4 weeks of age, respectively), which were followed by deficits in rotarod and climbing activity at 30 weeks of age and by cognitive deficits at around 1 year of age. Of particular interest for translational work, we also found clear behavioral deficits in heterozygous mice from around 4.5 months of age, especially in the dark phase of the diurnal cycle. Decreased body weight was observed in both heterozygotes and homozygotes, along with significantly reduced survival in the homozygotes. In addition, we detected an early and significant decrease of striatal gene markers from 12 weeks of age. These data suggest that the zQ175 knock-in line could be a suitable model for the evaluation of therapeutic approaches and early events in the pathogenesis of HD.
Generation and characterization of Atoh1-Cre knock-in mouse line

PubMed Central

Yang, Hua; Xie, Xiaoling; Deng, Min; Chen, Xiaowei; Gan, Lin

2010-01-01

Summary Atoh1 encodes a basic helix-loop-helix (bHLH) transcription factor required for the development of the inner ear sensory epithelia, the dorsal spinal cord, brainstem, cerebellum, and intestinal secretory cells. In this study to create a genetic tool for the research on gene function in the ear sensory organs, we generated an Atoh1-Cre knock-in mouse line by replacing the entire Atoh1 coding sequences with the Cre coding sequences. Atoh1Cre/+mice were viable, fertile, and displayed no visible defects whereas the Atoh1Cre/Cremice died perinatally. The spatiotemporal activities of Cre recombinase were examined by crossing Atoh1-Cre mice with the R26R-lacZ conditional reporter mice. Atoh1-Cre activities were detected in the developing inner ear, the hindbrain, the spinal cord, and the intestine. In the inner ear, Atoh1-Cre activities were confined to the sensory organs in which lacZ expression is detected in nearly all of the hair cells and in many supporting cells. Thus, Atoh1-Cre mouse line serves as a useful tool for the functional study of genes in the inner ear. In addition, our results demonstrate that Atoh1 is expressed in the common progenitors destined for both hair and supporting cells. PMID:20533400
Ultrasonic vocalization impairment of Foxp2 (R552H) knockin mice related to speech-language disorder and abnormality of Purkinje cells

PubMed Central

Fujita, Eriko; Tanabe, Yuko; Shiota, Akira; Ueda, Masatsugu; Suwa, Kiyotaka; Momoi, Mariko Y.; Momoi, Takashi

2008-01-01

Previous studies have demonstrated that mutation in the forkhead domain of the forkhead box P2 (FOXP2) protein (R553H) causes speech-language disorders. To further analyze FOXP2 function in speech learning, we generated a knockin (KI) mouse for Foxp2 (R552H) [Foxp2 (R552H)-KI], corresponding to the human FOXP2 (R553H) mutation, by homologous recombination. Homozygous Foxp2 (R552H)-KI mice showed reduced weight, immature development of the cerebellum with incompletely folded folia, Purkinje cells with poor dendritic arbors and less synaptophysin immunoreactivity, and achieved crisis stage for survival 3 weeks after birth. At postnatal day 10, these mice also showed severe ultrasonic vocalization (USV) and motor impairment, whereas the heterozygous Foxp2 (R552H)-KI mice exhibited modest impairments. Similar to the wild-type protein, Foxp2 (R552H) localized in the nuclei of the Purkinje cells and the thalamus, striatum, cortex, and hippocampus (CA1) neurons of the homozygous Foxp2 (R552H)-KI mice (postnatal day 10), and some of the neurons showed nuclear aggregates of Foxp2 (R552H). In addition to the immature development of the cerebellum, Foxp2 (R552H) nuclear aggregates may further compromise the function of the Purkinje cells and cerebral neurons of the homozygous mice, resulting in their death. In contrast, heterozygous Foxp2 (R552H)-KI mice, which showed modest impairment of USVs with different USV qualities and which did not exhibit nuclear aggregates, should provide insights into the common molecular mechanisms between the mouse USV and human speech learning and the relationship between the USV and motor neural systems. PMID:18287060
Absence of Vsx1 expression in the normal and damaged mouse cornea

PubMed Central

Chow, Robert L.

2011-01-01

Purpose To examine the expression of visual system homeobox 1 (Vsx1) in the mouse cornea and its potential role in the corneal wound response pathway. Methods Expression of Vsx1 was examined by quantitative reverse-transcription PCR (qRT–PCR) in corneal tissue from developing and adult mice and from mice that had undergone alkali-burn corneal wounding. Immunolabeling and Vsx1 knock-in reporter gene expression in wild type and Vsx1 null-mice were used to confirm the qRT–PCR data. Results Using qRT–PCR, Vsx1 expression was not detected in either the postnatal or adult mouse cornea or in corneas following wounding. This qRT–PCR data was supported by the absence of specific Vsx1 immunolabeling and Vsx1 knock-in reporter expression in untreated and wounded corneas. Conclusions In mice, Vsx1 mRNA, protein or reporter gene expression is not detected in the normal or damaged cornea. These results make it uncertain what role VSX1/Vsx1 plays in corneal biology. Future experiments examining the pathogenicity of VSX1 mutations associated with corneal dystrophy are required to rule out species differences and possible non-cell autonomous roles for VSX1 in the cornea. PMID:21437200
Contribution of Dysferlin Deficiency to Skeletal Muscle Pathology in Asymptomatic and Severe Dystroglycanopathy Models: Generation of a New Model for Fukuyama Congenital Muscular Dystrophy

PubMed Central

Kanagawa, Motoi; Lu, Zhongpeng; Ito, Chiyomi; Matsuda, Chie; Miyake, Katsuya; Toda, Tatsushi

2014-01-01

Defects in dystroglycan glycosylation are associated with a group of muscular dystrophies, termed dystroglycanopathies, that include Fukuyama congenital muscular dystrophy (FCMD). It is widely believed that abnormal glycosylation of dystroglycan leads to disease-causing membrane fragility. We previously generated knock-in mice carrying a founder retrotransposal insertion in fukutin, the gene responsible for FCMD, but these mice did not develop muscular dystrophy, which hindered exploring therapeutic strategies. We hypothesized that dysferlin functions may contribute to muscle cell viability in the knock-in mice; however, pathological interactions between glycosylation abnormalities and dysferlin defects remain unexplored. To investigate contributions of dysferlin deficiency to the pathology of dystroglycanopathy, we have crossed dysferlin-deficient dysferlin sjl/sjl mice to the fukutin-knock-in fukutin Hp/− and Large-deficient Large myd/myd mice, which are phenotypically distinct models of dystroglycanopathy. The fukutin Hp/− mice do not show a dystrophic phenotype; however, (dysferlin sjl/sjl: fukutin Hp/−) mice showed a deteriorated phenotype compared with (dysferlin sjl/sjl: fukutin Hp/+) mice. These data indicate that the absence of functional dysferlin in the asymptomatic fukutin Hp/− mice triggers disease manifestation and aggravates the dystrophic phenotype. A series of pathological analyses using double mutant mice for Large and dysferlin indicate that the protective effects of dysferlin appear diminished when the dystrophic pathology is severe and also may depend on the amount of dysferlin proteins. Together, our results show that dysferlin exerts protective effects on the fukutin Hp/− FCMD mouse model, and the (dysferlin sjl/sjl: fukutin Hp/−) mice will be useful as a novel model for a recently proposed antisense oligonucleotide therapy for FCMD. PMID:25198651
Production of knock-in mice in a single generation from embryonic stem cells.

PubMed

Ukai, Hideki; Kiyonari, Hiroshi; Ueda, Hiroki R

2017-12-01

The system-level identification and analysis of molecular networks in mammals can be accelerated by 'next-generation' genetics, defined as genetics that does not require crossing of multiple generations of animals in order to achieve the desired genetic makeup. We have established a highly efficient procedure for producing knock-in (KI) mice within a single generation, by optimizing the genome-editing protocol for KI embryonic stem (ES) cells and the protocol for the generation of fully ES-cell-derived mice (ES mice). Using this protocol, the production of chimeric mice is eliminated, and, therefore, there is no requirement for the crossing of chimeric mice to produce mice that carry the KI gene in all cells of the body. Our procedure thus shortens the time required to produce KI ES mice from about a year to ∼3 months. Various kinds of KI ES mice can be produced with a minimized amount of work, facilitating the elucidation of organism-level phenomena using a systems biology approach. In this report, we describe the basic technologies and protocols for this procedure, and discuss the current challenges for next-generation mammalian genetics in organism-level systems biology studies.
Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out

PubMed Central

Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

2012-01-01

Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. PMID:22391119
Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance.

PubMed

Akman, Hasan O; Dorado, Beatriz; López, Luis C; García-Cazorla, Angeles; Vilà, Maya R; Tanabe, Lauren M; Dauer, William T; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

2008-08-15

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2(-/-)) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2(-/-) animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype.
Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance

PubMed Central

Akman, Hasan O.; Dorado, Beatriz; López, Luis C.; García-Cazorla, Ángeles; Vilà, Maya R.; Tanabe, Lauren M.; Dauer, William T.; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

2008-01-01

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2−/−) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2−/− animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype. PMID:18467430
Three gene-targeted mouse models of RNA splicing factor RP show late-onset RPE and retinal degeneration.

PubMed

Graziotto, John J; Farkas, Michael H; Bujakowska, Kinga; Deramaudt, Bertrand M; Zhang, Qi; Nandrot, Emeline F; Inglehearn, Chris F; Bhattacharya, Shomi S; Pierce, Eric A

2011-01-01

Mutations in genes that produce proteins involved in mRNA splicing, including pre-mRNA processing factors 3, 8, and 31 (PRPF3, 8, and 31), RP9, and SNRNP200 are common causes of the late-onset inherited blinding disorder retinitis pigmentosa (RP). It is not known how mutations in these ubiquitously expressed genes lead to retina-specific disease. To investigate the pathogenesis of the RNA splicing factor forms of RP, the authors generated and characterized the retinal phenotypes of Prpf3-T494M, Prpf8-H2309P knockin mice. The retinal ultrastructure of Prpf31-knockout mice was also investigated. The knockin mice have single codon alterations in their endogenous Prpf3 and Prpf8 genes that mimic the most common disease causing mutations in human PRPF3 and PRPF8. The Prpf31-knockout mice mimic the null alleles that result from the majority of mutations identified in PRPF31 patients. The retinal phenotypes of the gene targeted mice were evaluated by electroretinography (ERG), light, and electron microscopy. The RPE cells of heterozygous Prpf3(+/T494M) and Prpf8(+/H2309P) knockin mice exhibited loss of the basal infoldings and vacuolization, with accumulation of amorphous deposits between the RPE and Bruch[b]'s membrane at age two years. These changes were more severe in the homozygous mice, and were associated with decreased rod function in the Prpf3-T494M mice. Similar degenerative changes in the RPE were detected in Prpf31(±) mice at one year of age. The finding of similar degenerative changes in RPE cells of all three mouse models suggests that the RPE may be the primary cell type affected in the RNA splicing factor forms of RP. The relatively late-onset phenotype observed in these mice is consistent with the typical adult onset of disease in patients with RP.
Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes.

PubMed

Chu, Van Trung; Weber, Timm; Graf, Robin; Sommermann, Thomas; Petsch, Kerstin; Sack, Ulrike; Volchkov, Pavel; Rajewsky, Klaus; Kühn, Ralf

2016-01-16

The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes. We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9. Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.
Characterization of Variant Creutzfeldt-Jakob Disease Prions in Prion Protein-humanized Mice Carrying Distinct Codon 129 Genotypes*

PubMed Central

Takeuchi, Atsuko; Kobayashi, Atsushi; Ironside, James W.; Mohri, Shirou; Kitamoto, Tetsuyuki

2013-01-01

To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype. PMID:23792955
Characterization of variant Creutzfeldt-Jakob disease prions in prion protein-humanized mice carrying distinct codon 129 genotypes.

PubMed

Takeuchi, Atsuko; Kobayashi, Atsushi; Ironside, James W; Mohri, Shirou; Kitamoto, Tetsuyuki

2013-07-26

To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype.
Variable Bone Fragility Associated With an Amish COL1A2 Variant and a Knock-in Mouse Model

PubMed Central

Daley, Ethan; Streeten, Elizabeth A; Sorkin, John D; Kuznetsova, Natalia; Shapses, Sue A; Carleton, Stephanie M; Shuldiner, Alan R; Marini, Joan C; Phillips, Charlotte L; Goldstein, Steven A; Leikin, Sergey; McBride, Daniel J

2010-01-01

Osteogenesis imperfecta (OI) is a heritable form of bone fragility typically associated with a dominant COL1A1 or COL1A2 mutation. Variable phenotype for OI patients with identical collagen mutations is well established, but phenotype variability is described using the qualitative Sillence classification. Patterning a new OI mouse model on a specific collagen mutation therefore has been hindered by the absence of an appropriate kindred with extensive quantitative phenotype data. We benefited from the large sibships of the Old Order Amish (OOA) to define a wide range of OI phenotypes in 64 individuals with the identical COL1A2 mutation. Stratification of carrier spine (L1–4) areal bone mineral density (aBMD) Z-scores demonstrated that 73% had moderate to severe disease (less than −2), 23% had mild disease (−1 to −2), and 4% were in the unaffected range (greater than −1). A line of knock-in mice was patterned on the OOA mutation. Bone phenotype was evaluated in four F1 lines of knock-in mice that each shared approximately 50% of their genetic background. Consistent with the human pedigree, these mice had reduced body mass, aBMD, and bone strength. Whole-bone fracture susceptibility was influenced by individual genomic factors that were reflected in size, shape, and possibly bone metabolic regulation. The results indicate that the G610C OI (Amish) knock-in mouse is a novel translational model to identify modifying genes that influence phenotype and for testing potential therapies for OI. © 2010 American Society for Bone and Mineral Research PMID:19594296
Brain catecholamine depletion and motor impairment in a Th knock-in mouse with type B tyrosine hydroxylase deficiency.

PubMed

Korner, Germaine; Noain, Daniela; Ying, Ming; Hole, Magnus; Flydal, Marte I; Scherer, Tanja; Allegri, Gabriella; Rassi, Anahita; Fingerhut, Ralph; Becu-Villalobos, Damasia; Pillai, Samyuktha; Wueest, Stephan; Konrad, Daniel; Lauber-Biason, Anna; Baumann, Christian R; Bindoff, Laurence A; Martinez, Aurora; Thöny, Beat

2015-10-01

Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate-limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
A CTRP5 gene S163R mutation knock-in mouse model for late-onset retinal degeneration.

PubMed

Chavali, Venkata R M; Khan, Naheed W; Cukras, Catherine A; Bartsch, Dirk-Uwe; Jablonski, Monica M; Ayyagari, Radha

2011-05-15

Late-onset retinal macular degeneration (L-ORD) is an autosomal dominant inherited disorder caused by a single missense mutation (S163R) in the CTRP5/C1QTNF5 protein. Early phenotypic features of L-ORD include: dark adaptation abnormalities, nyctalopia, and drusen deposits in the peripheral macular region. Apart from posterior segment abnormalities, these patients also develop abnormally long anterior lens zonules. In the sixth decade of life the rod and cone function declines, accompanied by electroretinogram (ERG) abnormalities. Some patients also develop choroidal neovascularization and glaucoma. In order to understand the disease pathology and mechanisms involved in retinal dystrophy, we generated a knock-in (Ctrp5(+/-)) mouse model carrying the disease-associated mutation in the mouse Ctrp5/C1QTNF5 gene. These mice develop slower rod-b wave recovery consistent with early dark adaptation abnormalities, accumulation of hyperautofluorescence spots, retinal pigment epithelium abnormalities, drusen, Bruch's membrane abnormalities, loss of photoreceptors, and retinal vascular leakage. The Ctrp5(+/-) mice, which have most of the pathological features of age-related macular degeneration, are unique and may serve as a valuable model both to understand the molecular pathology of late-onset retinal degeneration and to evaluate therapies.

A CTRP5 gene S163R mutation knock-in mouse model for late-onset retinal degeneration

PubMed Central

Chavali, Venkata R.M.; Khan, Naheed W.; Cukras, Catherine A.; Bartsch, Dirk-Uwe; Jablonski, Monica M.; Ayyagari, Radha

2011-01-01

Late-onset retinal macular degeneration (L-ORD) is an autosomal dominant inherited disorder caused by a single missense mutation (S163R) in the CTRP5/C1QTNF5 protein. Early phenotypic features of L-ORD include: dark adaptation abnormalities, nyctalopia, and drusen deposits in the peripheral macular region. Apart from posterior segment abnormalities, these patients also develop abnormally long anterior lens zonules. In the sixth decade of life the rod and cone function declines, accompanied by electroretinogram (ERG) abnormalities. Some patients also develop choroidal neovascularization and glaucoma. In order to understand the disease pathology and mechanisms involved in retinal dystrophy, we generated a knock-in (Ctrp5+/−) mouse model carrying the disease-associated mutation in the mouse Ctrp5/C1QTNF5 gene. These mice develop slower rod-b wave recovery consistent with early dark adaptation abnormalities, accumulation of hyperautofluorescence spots, retinal pigment epithelium abnormalities, drusen, Bruch's membrane abnormalities, loss of photoreceptors, and retinal vascular leakage. The Ctrp5+/−mice, which have most of the pathological features of age-related macular degeneration, are unique and may serve as a valuable model both to understand the molecular pathology of late-onset retinal degeneration and to evaluate therapies. PMID:21349921
Novel In Vivo Model for Combinatorial Fluorescence Labeling in Mouse Prostate

PubMed Central

Fang, Xiaolan; Gyabaah, Kenneth; Nickkholgh, Bita; Cline, J. Mark; Balaji, K.C.

2015-01-01

BACKGROUND The epithelial layer of prostate glands contains several types of cells, including luminal and basal cells. Yet there is paucity of animal models to study the cellular origin of normal or neoplastic development in the prostate to facilitate the treatment of heterogenous prostate diseases by targeting individual cell lineages. METHODS We developed a mouse model that expresses different types of fluorescent proteins (XFPs) specifically in prostatic cells. Using an in vivo stochastic fluorescent protein combinatorial strategy, XFP signals were expressed specifically in prostate of Protein Kinase D1 (PKD1) knock-out, K-RasG12D knock-in, and Phosphatase and tensin homolog (PTEN) and PKD1 double knock-out mice under the control of PB-Cre promoter. RESULTS In vivo XFP signals were observed in prostate of PKD1 knock-out, K-RasG12D knock-in, and PTEN PKD1 double knock-out mice, which developed normal, hyperplastic, and neoplastic prostate, respectively. The patchy expression pattern of XFPs in neoplasia tissue indicated the clonal origin of cancer cells in the prostate. CONCLUSIONS The transgenic mouse models demonstrate combinatorial fluorescent protein expression in normal and cancerous prostatic tissues. This novel prostate-specific fluorescent labeled mouse model, which we named Prorainbow, could be useful in studying benign and malignant pathology of prostate. PMID:25753731
Novel In Vivo model for combinatorial fluorescence labeling in mouse prostate.

PubMed

Fang, Xiaolan; Gyabaah, Kenneth; Nickkholgh, Bita; Cline, J Mark; Balaji, K C

2015-06-15

The epithelial layer of prostate glands contains several types of cells, including luminal and basal cells. Yet there is paucity of animal models to study the cellular origin of normal or neoplastic development in the prostate to facilitate the treatment of heterogenous prostate diseases by targeting individual cell lineages. We developed a mouse model that expresses different types of fluorescent proteins (XFPs) specifically in prostatic cells. Using an in vivo stochastic fluorescent protein combinatorial strategy, XFP signals were expressed specifically in prostate of Protein Kinase D1 (PKD1) knock-out, K-Ras(G) (12) (D) knock-in, and Phosphatase and tensin homolog (PTEN) and PKD1 double knock-out mice under the control of PB-Cre promoter. In vivo XFP signals were observed in prostate of PKD1 knock-out, K-Ras(G) (12) (D) knock-in, and PTEN PKD1 double knock-out mice, which developed normal, hyperplastic, and neoplastic prostate, respectively. The patchy expression pattern of XFPs in neoplasia tissue indicated the clonal origin of cancer cells in the prostate. The transgenic mouse models demonstrate combinatorial fluorescent protein expression in normal and cancerous prostatic tissues. This novel prostate-specific fluorescent labeled mouse model, which we named Prorainbow, could be useful in studying benign and malignant pathology of prostate. © 2015 Wiley Periodicals, Inc.
An Intracellular Threonine of Amyloid-β Precursor Protein Mediates Synaptic Plasticity Deficits and Memory Loss

PubMed Central

Lombino, Franco; Biundo, Fabrizio; Tamayev, Robert; Arancio, Ottavio; D’Adamio, Luciano

2013-01-01

Mutations in Amyloid-ß Precursor Protein (APP) and BRI2/ITM2b genes cause Familial Alzheimer and Danish Dementias (FAD/FDD), respectively. APP processing by BACE1, which is inhibited by BRI2, yields sAPPß and ß-CTF. ß-CTF is cleaved by gamma-secretase to produce Aß. A knock-in mouse model of FDD, called FDDKI, shows deficits in memory and synaptic plasticity, which can be attributed to sAPPß/ß-CTF but not Aß. We have investigated further the pathogenic function of ß-CTF focusing on Thr668 of ß-CTF because phosphorylation of Thr668 is increased in AD cases. We created a knock-in mouse bearing a Thr668Ala mutation (APPTA mice) that prevents phosphorylation at this site. This mutation prevents the development of memory and synaptic plasticity deficits in FDDKI mice. These data are consistent with a role for the carboxyl-terminal APP domain in the pathogenesis of dementia and suggest that averting the noxious role of Thr668 is a viable therapeutic strategy for human dementias. PMID:23451158
Modeling familial British and Danish dementia.

PubMed

Garringer, Holly J; Murrell, Jill; D'Adamio, Luciano; Ghetti, Bernardino; Vidal, Ruben

2010-03-01

Familial British dementia (FBD) and familial Danish dementia (FDD) are two autosomal dominant neurodegenerative diseases caused by mutations in the BRI ( 2 ) gene. FBD and FDD are characterized by widespread cerebral amyloid angiopathy (CAA), parenchymal amyloid deposition, and neurofibrillary tangles. Transgenic mice expressing wild-type and mutant forms of the BRI(2) protein, Bri ( 2 ) knock-in mutant mice, and Bri ( 2 ) gene knock-out mice have been developed. Transgenic mice expressing a human FDD-mutated form of the BRI ( 2 ) gene have partially reproduced the neuropathological lesions observed in FDD. These mice develop extensive CAA, parenchymal amyloid deposition, and neuroinflammation in the central nervous system. These animal models allow the study of the molecular mechanism(s) underlying the neuronal dysfunction in these diseases and allow the development of potential therapeutic approaches for these and related neurodegenerative conditions. In this review, a comprehensive account of the advances in the development of animal models for FBD and FDD and of their relevance to the study of Alzheimer disease is presented.
Hmga2 promotes the development of myelofibrosis in Jak2V617F knockin mice by enhancing TGF-β1 and Cxcl12 pathways.

PubMed

Dutta, Avik; Hutchison, Robert E; Mohi, Golam

2017-08-17

Myelofibrosis (MF) is a devastating blood disorder. The JAK2V617F mutation has been detected in ∼50% cases of MF. Elevated expression of high-mobility group AT hook 2 (HMGA2) has also been frequently observed in patients with MF. Interestingly, upregulation of HMGA2 expression has been found in association with the JAK2V617F mutation in significant cases of MF. However, the contribution of HMGA2 in the pathogenesis of MF remains elusive. To determine the effects of concurrent expression of HMGA2 and JAK2V617F mutation in hematopoiesis, we transduced bone marrow cells from Jak2 V617F knockin mice with lentivirus expressing Hmga2 and performed bone marrow transplantation. Expression of Hmga2 enhanced megakaryopoiesis, increased extramedullary hematopoiesis, and accelerated the development of MF in mice expressing Jak2 V617F Mechanistically, the data show that expression of Hmga2 enhances the activation of transforming growth factor-β1 (TGF-β1) and Cxcl12 pathways in mice expressing Jak2 V617F In addition, expression of Hmga2 causes upregulation of Fzd2, Ifi27l2a, and TGF-β receptor 2. Forced expression of Cxcl12, Fzd2, or Ifi27l2a increases megakaryocytic differentiation and proliferation in the bone marrow of Jak2 V617F mice, whereas TGF-β1 or Cxcl12 stimulation induces collagen deposition in the bone marrow mesenchymal stromal cells. Together, these findings demonstrate that expression of Hmga2 cooperates with Jak2 V617F in the pathogenesis of MF. © 2017 by The American Society of Hematology.
Characterization of Behavioral, Neuropathological, Brain Metabolic and Key Molecular Changes in zQ175 Knock-In Mouse Model of Huntington's Disease.

PubMed

Peng, Qi; Wu, Bin; Jiang, Mali; Jin, Jing; Hou, Zhipeng; Zheng, Jennifer; Zhang, Jiangyang; Duan, Wenzhen

2016-01-01

Huntington's disease (HD) is caused by an expansion of the trinucleotide poly (CAG) tract located in exon 1 of the huntingtin (Htt) gene leading to progressive neurodegeneration in selected brain regions, and associated functional impairments in motor, cognitive, and psychiatric domains. Since the discovery of the gene mutation that causes the disease, mouse models have been developed by different strategies. Recently, a new model, the zQ175 knock-in (KI) line, was developed in an attempt to have the Htt gene in a context and causing a phenotype that more closely mimics HD in humans. The behavioral phenotype was characterized across the independent laboratories and important features reminiscent of human HD are observed in zQ175 mice. In the current study, we characterized the zQ175 model housed in an academic laboratory under reversed dark-light cycle, including motor function, in vivo longitudinal structural MRI imaging for brain volume, MRS for striatal metabolites, neuropathology, as well as a panel of key disease marker proteins in the striatum at different ages. Our results suggest that homozygous zQ175 mice exhibited significant brain atrophy before the motor deficits and brain metabolite changes. Altered striatal medium spiny neuronal marker, postsynaptic marker protein and complement component C1qC also characterized zQ175 mice. Our results confirmed that the zQ175 KI model is valuable in understanding of HD-like pathophysiology and evaluation of potential therapeutics. Our data also provide suggestions to select appropriate outcome measurements in preclinical studies using the zQ175 mice.
Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out.

PubMed

Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

2012-05-01

Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. Copyright © 2012 Elsevier B.V. All rights reserved.
Thiazide-sensitive Na+ -Cl- cotransporter (NCC) gene inactivation results in increased duodenal Ca2+ absorption, enhanced osteoblast differentiation and elevated bone mineral density.

PubMed

Hsu, Yu-Juei; Yang, Sung-Sen; Cheng, Chih-Jen; Liu, Shu-Ting; Huang, Shih-Ming; Chau, Tom; Chu, Pauling; Salter, Donald M; Lee, Herng-Sheng; Lin, Shih-Hua

2015-01-01

Inactivation of the thiazide-sensitive sodium chloride cotransporter (NCC) due to genetic mutations in Gitelman's syndrome (GS) or pharmacological inhibition with thiazide diuretics causes hypocalciuria and increased bone mineral density (BMD) with unclear extrarenal calcium (Ca(2+) ) regulation. We investigated intestinal Ca(2+) absorption and bone Ca(2+) metabolism in nonsense Ncc Ser707X (S707X) homozygous knockin mice (Ncc(S707X/S707X) mice). Compared to wild-type and heterozygous knockin littermates, Ncc(S707X/S707X) mice had increased intestinal absorption of (45) Ca(2+) and expression of the active Ca(2+) transport machinery (transient receptor potential vanilloid 6, calbindin-D9K , and plasma membrane Ca(2+) ATPase isoform 1b). Ncc(S707X/S707X) mice had also significantly increased Ca(2+) content accompanied by greater mineral apposition rate (MAR) in their femurs and higher trabecular bone volume, cortical bone thickness, and BMD determined by μCT. Their osteoblast differentiation markers, such as bone alkaline phosphatase, procollagen I, osteocalcin, and osterix, were also significantly increased while osteoclast activity was unaffected. Analysis of marrow-derived bone cells, either treated with thiazide or directly cultured from Ncc S707X knockin mice, showed that the differentiation of osteoblasts was associated with increased phosphorylation of mechanical stress-induced focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). In conclusion, NCC inhibition stimulates duodenal Ca(2+) absorption as well as osteoblast differentiation and bone Ca(2+) storage, possibly through a FAK/ERK dependent mechanism. © 2014 American Society for Bone and Mineral Research.
Generation of Pax6-IRES-EGFP knock-in mouse via the cloning-free CRISPR/Cas9 system to reliably visualize neurodevelopmental dynamics.

PubMed

Inoue, Yukiko U; Morimoto, Yuki; Hoshino, Mikio; Inoue, Takayoshi

2018-07-01

Pax6 encodes a transcription factor that plays pivotal roles in eye development, early brain patterning, neocortical arealization, and so forth. Visualization of Pax6 expression dynamics in these events could offer numerous advantages to neurodevelopmental studies. While CRISPR/Cas9 system has dramatically accelerated one-step generation of knock-out mouse, establishment of gene-cassette knock-in mouse via zygote injection has been considered insufficient due to its low efficiency. Recently, an improved CRISPR/Cas9 system for effective gene-cassette knock-in has been reported, where the native form of guide RNAs (crRNA and tracrRNA) assembled with recombinant Cas9 protein are directly delivered into mouse fertilized eggs. Here we apply this strategy to insert IRES-EGFP-pA cassette into Pax6 locus and achieve efficient targeted insertions of the 1.8 kb reporter gene. In Pax6-IRES-EGFP mouse we have generated, EGFP-positive cells reside in the eyes and cerebellum as endogenous Pax6 expressing cells at postnatal day 2. At the early embryonic stages when the embryos are transparent, EGFP-positive regions can be easily identified without PCR-based genotyping, precisely recapitulating the endogenous Pax6 expression patterns. Remarkably, at E12.5, the graded expression patterns of Pax6 in the developing neocortex now become recognizable in our knock-in mice, serving a sufficiently sensitive and useful tool to precisely visualize neurodevelopmental processes. Copyright © 2018 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.
Morphological and Functional Attenuation of Degeneration of Peripheral Neurons by Mesenchymal Stem Cell-Conditioned Medium in Spinocerebellar Ataxia Type 1-Knock-in Mice.

PubMed

Suto, Nana; Mieda, Tokue; Iizuka, Akira; Nakamura, Kazuhiro; Hirai, Hirokazu

2016-08-01

Spinocerebellar ataxia type 1 (SCA1) is caused by the ataxin-1 protein (ATXN1) with an abnormally expanded polyglutamine tract and is characterized by progressive neurodegeneration. We previously showed that intrathecal injection of mesenchymal stem cells (MSCs) during the nonsymptomatic stage mitigates the degeneration of the peripheral nervous system (PNS) neurons in SCA1-knock-in (SCA1-KI) mice. We tested in this study whether the therapeutic effects of MSCs in SCA1-KI mice could be reproduced with MSC-releasing factor(s). To test the effects of MSC-releasing factor(s), we used MSC-conditioned medium (MSC-CM). MSC-CM was intrathecally and/or intravenously injected into young SCA1-KI mice, and the therapeutic effects were assessed in the PNS at later ages using immunostaining, electrophysiology, and behavioral tests. MSC-CM attenuated the degeneration of axons and myelin of spinal motor neurons. Consequently, the injected SCA1-KI mice exhibited smaller reductions in nerve conduction velocity in spinal motor neurons and reduced motor incoordination than the untreated mice. These results suggest that factors released from MSC mitigate the morphological and functional abnormalities in the PNS that are observed in SCA1-KI mice in a paracrine manner. © 2016 John Wiley & Sons Ltd.
In Vivo Tagging of Lung Epithelial Cells To Define the Early Steps of Tumor Cell Dissemination

DTIC Science & Technology

2015-12-01

was interbred with Nkx2.1-CreERT2 knock-in mouse strain, containing a tamoxifen -inducible lineage specific Cre recombinase, to generate adult lung...KrasG12D(L/-); Lkb1(L/L) mice 7 days after tamoxifen injections show that KrasG12D mice develop isolated tumors one week after the end of tamoxifen ...SA2a-b) (timeframe months 6-10 by Drs Kathuria, Cao, Ramirez, and Kotton). 1. First we will induce tumors by tamoxifen injection of Kras G12D /Lkb1L/L
Studying the Role of Eukaryotic Translation Initiation Factor 4E (eIF4E) Phosphorylation by MNK1/2 Kinases in Prostate Cancer Development and Progression

DTIC Science & Technology

2012-06-01

preclinical mouse model. J Clin Invest 118: 3051–3064. 49. Le Bacquer O, et al. (2007) Elevated sensitivity to diet-induced obesity and insulin resistance in...Elevated sensitivity to diet-induced obesity and insulin resistance in mice lacking 4E-BP1 and 4E-BP2. J Clin Invest 117:387–396. 7. Frederickson RM...that knock-in mice expressing a nonphosphorylatable form of eIF4E are resistant to tumorigenesis in a prostate cancer model. By using a genome-wide
Production of genome-edited pluripotent stem cells and mice by CRISPR/Cas.

PubMed

Horii, Takuro; Hatada, Izuho

2016-01-01

Clustered regularly at interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases, so-called CRISPR/Cas, was recently developed as an epoch-making genome engineering technology. This system only requires Cas9 nuclease and single-guide RNA complementary to a target locus. CRISPR/Cas enables the generation of knockout cells and animals in a single step. This system can also be used to generate multiple mutations and knockin in a single step, which is not possible using other methods. In this review, we provide an overview of genome editing by CRISPR/Cas in pluripotent stem cells and mice.
Specific expression of FOXP2 in cerebellum improves ultrasonic vocalization in heterozygous but not in homozygous Foxp2 (R552H) knock-in pups.

PubMed

Fujita-Jimbo, Eriko; Momoi, Takashi

2014-04-30

The R553H mutation has been found in the FOXP2 gene of patients with speech-language disorder. Foxp2(R552H) knock-in (KI) mice exhibit poor dendritic development of Purkinje cells in the cerebellum and impaired ultrasonic vocalization (USV), which is related to human speech and language; compared with wild-type mice, heterozygous Foxp2(R552H)-KI pups exhibit the reduced number of whistle-type USVs and the increased short-type ones, while homozygous pups exhibit only click-type USVs but no whistle-type or short-type ones. To make clear the relationship between the role of Foxp2 in the cerebellum and whistle-type USVs activity, we prepared transgenic (Tg) mice specifically expressing human FOXP2-myc in cerebellum (Pcp2-FOXP2-myc-Tg mice) by using purkinje cell protein-2 (Pcp2) promoter. FOXP2-myc expression in the cerebellum increased the relative numbers of whistle-type USVs in the heterozygous Foxp2(R552H)-KI pups and recovered their USVs but did not in the homozygous ones. Foxp2 in the cerebellum may pertain to the brain network engaged in whistle-type USVs activities including modification, but not their production. There may be common molecular contribution of Purkinje cells to human FOXP2-mediated speech-language and mouse Foxp2-mediated USVs. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
Early white matter abnormalities, progressive brain pathology and motor deficits in a novel knock-in mouse model of Huntington's disease

PubMed Central

Jin, Jing; Peng, Qi; Hou, Zhipeng; Jiang, Mali; Wang, Xin; Langseth, Abraham J.; Tao, Michael; Barker, Peter B.; Mori, Susumu; Bergles, Dwight E.; Ross, Christopher A.; Detloff, Peter J.; Zhang, Jiangyang; Duan, Wenzhen

2015-01-01

White matter abnormalities have been reported in premanifest Huntington's disease (HD) subjects before overt striatal neuronal loss, but whether the white matter changes represent a necessary step towards further pathology and the underlying mechanism of these changes remains unknown. Here, we characterized a novel knock-in mouse model that expresses mouse HD gene homolog (Hdh) with extended CAG repeat- HdhQ250, which was derived from the selective breeding of HdhQ150 mice. HdhQ250 mice manifest an accelerated and robust phenotype compared with its parent line. HdhQ250 mice exhibit progressive motor deficits, reduction in striatal and cortical volume, accumulation of mutant huntingtin aggregation, decreased levels of DARPP32 and BDNF and altered striatal metabolites. The abnormalities detected in this mouse model are reminiscent of several aspects of human HD. In addition, disturbed myelination was evident in postnatal Day 14 HdhQ250 mouse brain, including reduced levels of myelin regulatory factor and myelin basic protein, and decreased numbers of myelinated axons in the corpus callosum. Thinner myelin sheaths, indicated by increased G-ratio of myelin, were also detected in the corpus callosum of adult HdhQ250 mice. Moreover, proliferation of oligodendrocyte precursor cells is altered by mutant huntingtin both in vitro and in vivo. Our data indicate that this model is suitable for understanding comprehensive pathogenesis of HD in white matter and gray matter as well as developing therapeutics for HD. PMID:25609071
Generation of knock-in mice that express nuclear enhanced green fluorescent protein and tamoxifen-inducible Cre recombinase in the notochord from Foxa2 and T loci.

PubMed

Imuta, Yu; Kiyonari, Hiroshi; Jang, Chuan-Wei; Behringer, Richard R; Sasaki, Hiroshi

2013-03-01

The node and the notochord are important embryonic signaling centers that control embryonic pattern formation. Notochord progenitor cells present in the node and later in the posterior end of the notochord move anteriorly to generate the notochord. To understand the dynamics of cell movement during notochord development and the molecular mechanisms controlling this event, analyses of cell movements using time-lapse imaging and conditional manipulation of gene activities are required. To achieve this goal, we generated two knock-in mouse lines that simultaneously express nuclear enhanced green fluorescent protein (EGFP) and tamoxifen-inducible Cre, CreER(T2) , from two notochord gene loci, Foxa2 and T (Brachury). In Foxa2(nEGFP-CreERT2/+) and T(nEGFP-CreERT2/+) embryos, nuclei of the Foxa2 or T-expressing cells, which include the node, notochord, and endoderm (Foxa2) or wide range of posterior mesoderm (T), were labeled with EGFP at intensities that can be used for live imaging. Cre activity was also induced in cells expressing Foxa2 and T 1 day after tamoxifen administration. These mice are expected to be useful tools for analyzing the mechanisms of notochord development. Copyright © 2013 Wiley Periodicals, Inc.
VE-cadherin Y685F knock-in mouse is sensitive to vascular permeability in recurrent angiogenic organs.

PubMed

Sidibé, Adama; Polena, Helena; Pernet-Gallay, Karin; Razanajatovo, Jeremy; Mannic, Tiphaine; Chaumontel, Nicolas; Bama, Soumalamaya; Maréchal, Irène; Huber, Philippe; Gulino-Debrac, Danielle; Bouillet, Laurence; Vilgrain, Isabelle

2014-08-01

Covalent modifications such as tyrosine phosphorylation are associated with the breakdown of endothelial cell junctions and increased vascular permeability. We previously showed that vascular endothelial (VE)-cadherin was tyrosine phosphorylated in vivo in the mouse reproductive tract and that Y685 was a target site for Src in response to vascular endothelial growth factor in vitro. In the present study, we aimed to understand the implication of VE-cadherin phosphorylation at site Y685 in cyclic angiogenic organs. To achieve this aim, we generated a knock-in mouse carrying a tyrosine-to-phenylalanine point mutation of VE-cadherin Y685 (VE-Y685F). Although homozygous VE-Y685F mice were viable and fertile, the nulliparous knock-in female mice exhibited enlarged uteri with edema. This phenotype was observed in 30% of females between 4 to 14 mo old. Histological examination of longitudinal sections of the VE-Y685F uterus showed an extensive disorganization of myometrium and endometrium with highly edematous uterine glands, numerous areas with sparse cells, and increased accumulation of collagen fibers around blood vessels, indicating a fibrotic state. Analysis of cross section of ovaries showed the appearance of spontaneous cysts, which suggested increased vascular hyperpermeability. Electron microscopy analysis of capillaries in the ovary showed a slight but significant increase in the gap size between two adjacent endothelial cell membranes in the junctions of VE-Y685F mice (wild-type, 11.5 ± 0.3, n = 78; and VE-Y685F, 12.48 ± 0.3, n = 65; P = 0.045), as well as collagen fiber accumulation around capillaries. Miles assay revealed that either basal or vascular endothelial growth factor-stimulated permeability in the skin was increased in VE-Y685F mice. Since edema and fibrotic appearance have been identified as hallmarks of initial increased vascular permeability, we conclude that the site Y685 in VE-cadherin is involved in the physiological regulation of capillary permeability. Furthermore, this knock-in mouse model is of potential interest for further studies of diseases that are associated with abnormal vascular permeability. Copyright © 2014 the American Physiological Society.
Late-onset of spinal neurodegeneration in knock-in mice expressing a mutant BiP.

PubMed

Jin, Hisayo; Mimura, Naoya; Kashio, Makiko; Koseki, Haruhiko; Aoe, Tomohiko

2014-01-01

Most human neurodegenerative diseases are sporadic, and appear later in life. While the underlying mechanisms of the progression of those diseases are still unclear, investigations into the familial forms of comparable diseases suggest that endoplasmic reticulum (ER) stress is involved in the pathogenesis. Binding immunoglobulin protein (BiP) is an ER chaperone that is central to ER function. We produced knock-in mice expressing a mutant BiP that lacked the retrieval sequence in order to evaluate the effect of a functional defect in an ER chaperone in multi-cellular organisms. Here we report that heterozygous mutant BiP mice revealed motor disabilities in aging. We found a degeneration of some motoneurons in the spinal cord accompanied by accumulations of ubiquitinated proteins. The defect in retrieval of BiP by the KDEL receptor leads to impaired activities in quality control and autophagy, suggesting that functional defects in the ER chaperones may contribute to the late onset of neurodegenerative diseases.
Thalamocortical neuron loss and localized astrocytosis in the Cln3Deltaex7/8 knock-in mouse model of Batten disease.

PubMed

Pontikis, Charlie C; Cotman, Susan L; MacDonald, Marcy E; Cooper, Jonathan D

2005-12-01

Juvenile neuronal ceroid lipofuscinosis (JNCL) is the result of mutations in the Cln3 gene. The Cln3 knock-in mouse (Cln3Deltaex7/8) reproduces the most common Cln3 mutation and we have now characterized the CNS of these mice at 12 months of age. With the exception of the thalamus, Cln3Deltaex7/8 homozygotes displayed no significant regional atrophy, but a range of changes in individual laminar thickness that resulted in variable cortical thinning across subfields. Stereological analysis revealed a pronounced loss of neurons within individual laminae of somatosensory cortex of affected mice and the novel finding of a loss of sensory relay thalamic neurons. These affected mice also exhibited profound astrocytic reactions that were most pronounced in the neocortex and thalamus, but diminished in other brain regions. These data provide the first direct evidence for neurodegenerative and reactive changes in the thalamocortical system in JNCL and emphasize the localized nature of these events.

CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice.

PubMed

Oji, Asami; Noda, Taichi; Fujihara, Yoshitaka; Miyata, Haruhiko; Kim, Yeon Joo; Muto, Masanaga; Nozawa, Kaori; Matsumura, Takafumi; Isotani, Ayako; Ikawa, Masahito

2016-08-17

Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene, Dnajb13, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that Dnajb13 is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism.
An anticholinergic reverses motor control and corticostriatal LTD deficits in Dyt1 ΔGAG knock-in mice

PubMed Central

Dang, Mai T.; Yokoi, Fumiaki; Cheetham, Chad C.; Lu, Jun; Vo, Viet; Lovinger, David M.; Li, Yuqing

2011-01-01

DYT1 early-onset generalized torsion dystonia is an inherited movement disorder associated with mutations in DYT1 that codes for torsinA protein. The most common mutation seen in this gene is a trinucleotide deletion of GAG. We previously reported a motor control deficit on a beam-walking task in our Dyt1 ΔGAG knock-in heterozygous mice. In this report we show the reversal of this motor deficit with the anticholinergic trihexyphenidyl (THP), a drug commonly used to treat movement problems in dystonia patients. THP also restored the reduced corticostriatal long-term depression (LTD) observed in these mice. Corticostriatal LTD has long been known to be dependent on D2 receptor activation. In this mouse model, striatal D2 receptors were expressed at lower quantities in comparison to wild-type mice. Furthermore, the mice were also partially resistant to FPL64176, an agonist of L-type calcium channels that have been previously reported to cause severe dystonic-like symptoms in wild-type mice. Our findings collectively suggest that altered communication between cholinergic interneurons and medium spiny neurons is responsible for the LTD deficit and that this synaptic plasticity modification may be involved in the striatal motor control abnormalities in our mouse model of DYT1 dystonia. PMID:21995941
The de-ubiquitinating enzyme ataxin-3 does not modulate disease progression in a knock-in mouse model of Huntington disease.

PubMed

Zeng, Li; Tallaksen-Greene, Sara J; Wang, Bo; Albin, Roger L; Paulson, Henry L

2013-01-01

Ataxin-3 is a deubiquitinating enzyme (DUB) that participates in ubiquitin-dependent protein quality control pathways and, based on studies in model systems, may be neuroprotective against toxic polyglutamine proteins such as the Huntington's disease (HD) protein, huntingtin (htt). HD is one of at least nine polyglutamine neurodegenerative diseases in which disease-causing proteins accumulate in ubiquitin-positive inclusions within neurons. In studies crossing mice null for ataxin-3 to an established HD knock-in mouse model (HdhQ200), we tested whether loss of ataxin-3 alters disease progression, perhaps by impairing the clearance of mutant htt or the ubiquitination of inclusions. While loss of ataxin-3 mildly exacerbated age-dependent motor deficits, it did not alter inclusion formation, ubiquitination of inclusions or levels of mutant or normal htt. Ataxin-3, itself a polyglutamine-containing protein with multiple ubiquitin binding domains, was not observed to localize to htt inclusions. Changes in neurotransmitter receptor binding known to occur in HD knock-in mice also were not altered by the loss of ataxin-3, although we unexpectedly observed increased GABAA receptor binding in the striatum of HdhQ200 mice, which has not previously been noted. Finally, we confirmed that CNS levels of hsp70 are decreased in HD mice as has been reported in other HD mouse models, regardless of the presence or absence of ataxin-3. We conclude that while ataxin-3 may participate in protein quality control pathways, it does not critically regulate the handling of mutant htt or contribute to major features of disease pathogenesis in HD.
Inhaled Anesthetic Responses of Recombinant Receptors and Knockin Mice Harboring α2(S270H/L277A) GABAA Receptor Subunits That Are Resistant to Isoflurane

PubMed Central

Werner, D. F.; Swihart, A.; Rau, V.; Jia, F.; Borghese, C. M.; McCracken, M. L.; Iyer, S.; Fanselow, M. S.; Oh, I.; Sonner, J. M.; Eger, E. I.; Harrison, N. L.; Harris, R. A.

2011-01-01

The mechanism by which the inhaled anesthetic isoflurane produces amnesia and immobility is not understood. Isoflurane modulates GABAA receptors (GABAA-Rs) in a manner that makes them plausible targets. We asked whether GABAA-R α2 subunits contribute to a site of anesthetic action in vivo. Previous studies demonstrated that Ser270 in the second transmembrane domain is involved in the modulation of GABAA-Rs by volatile anesthetics and alcohol, either as a binding site or a critical allosteric residue. We engineered GABAA-Rs with two mutations in the α2 subunit, changing Ser270 to His and Leu277 to Ala. Recombinant receptors with these mutations demonstrated normal affinity for GABA, but substantially reduced responses to isoflurane. We then produced mutant (knockin) mice in which this mutated subunit replaced the wild-type α2 subunit. The adult mutant mice were overtly normal, although there was evidence of enhanced neonatal mortality and fear conditioning. Electrophysiological recordings from dentate granule neurons in brain slices confirmed the decreased actions of isoflurane on mutant receptors contributing to inhibitory synaptic currents. The loss of righting reflex EC50 for isoflurane did not differ between genotypes, but time to regain the righting reflex was increased in N2 generation knockins. This effect was not observed at the N4 generation. Isoflurane produced immobility (as measured by tail clamp) and amnesia (as measured by fear conditioning) in both wild-type and mutant mice, and potencies (EC50) did not differ between the strains for these actions of isoflurane. Thus, immobility or amnesia does not require isoflurane potentiation of the α2 subunit. PMID:20807777
Synaptic function is modulated by LRRK2 and glutamate release is increased in cortical neurons of G2019S LRRK2 knock-in mice.

PubMed

Beccano-Kelly, Dayne A; Kuhlmann, Naila; Tatarnikov, Igor; Volta, Mattia; Munsie, Lise N; Chou, Patrick; Cao, Li-Ping; Han, Heather; Tapia, Lucia; Farrer, Matthew J; Milnerwood, Austen J

2014-01-01

Mutations in Leucine-Rich Repeat Kinase-2 (LRRK2) result in familial Parkinson's disease and the G2019S mutation alone accounts for up to 30% in some ethnicities. Despite this, the function of LRRK2 is largely undetermined although evidence suggests roles in phosphorylation, protein interactions, autophagy and endocytosis. Emerging reports link loss of LRRK2 to altered synaptic transmission, but the effects of the G2019S mutation upon synaptic release in mammalian neurons are unknown. To assess wild type and mutant LRRK2 in established neuronal networks, we conducted immunocytochemical, electrophysiological and biochemical characterization of >3 week old cortical cultures of LRRK2 knock-out, wild-type overexpressing and G2019S knock-in mice. Synaptic release and synapse numbers were grossly normal in LRRK2 knock-out cells, but discretely reduced glutamatergic activity and reduced synaptic protein levels were observed. Conversely, synapse density was modestly but significantly increased in wild-type LRRK2 overexpressing cultures although event frequency was not. In knock-in cultures, glutamate release was markedly elevated, in the absence of any change to synapse density, indicating that physiological levels of G2019S LRRK2 elevate probability of release. Several pre-synaptic regulatory proteins shown by others to interact with LRRK2 were expressed at normal levels in knock-in cultures; however, synapsin 1 phosphorylation was significantly reduced. Thus, perturbations to the pre-synaptic release machinery and elevated synaptic transmission are early neuronal effects of LRRK2 G2019S. Furthermore, the comparison of knock-in and overexpressing cultures suggests that one copy of the G2019S mutation has a more pronounced effect than an ~3-fold increase in LRRK2 protein. Mutant-induced increases in transmission may convey additional stressors to neuronal physiology that may eventually contribute to the pathogenesis of Parkinson's disease.
Foxc2CreERT2 knock-in mice mark stage-specific Foxc2-expressing cells during mouse organogenesis.

PubMed

Amin, Mohammed Badrul; Miura, Naoyuki; Uddin, Mohammad Khaja Mafij; Islam, Mohammod Johirul; Yoshida, Nobuaki; Iseki, Sachiko; Kume, Tsutomu; Trainor, Paul A; Saitsu, Hirotomo; Aoto, Kazushi

2017-01-01

Foxc2, a member of the winged helix transcription factor family, is essential for eye, calvarial bone, cardiovascular and kidney development in mice. Nevertheless, how Foxc2-expressing cells and their descendent cells contribute to the development of these tissues and organs has not been elucidated. Here, we generated a Foxc2 knock-in (Foxc2 CreERT2 ) mouse, in which administration of estrogen receptor antagonist tamoxifen induces nuclear translocation of Cre recombinase in Foxc2-expressing cells. By crossing with ROSA-LacZ reporter mice (Foxc2 CreERT2 ; R26R), the fate of Foxc2 positive (Foxc2 + ) cells was analyzed through LacZ staining at various embryonic stages. We found Foxc2 + cell descendants in the supraoccipital and exoccipital bone in E18.5 embryos, when tamoxifen was administered at embryonic day (E) 8.5. Furthermore, Foxc2 + descendant cranial neural crest cells at E8-10 were restricted to the corneal mesenchyme, while Foxc2 + cell derived cardiac neural crest cells at E6-12 were found in the aorta, pulmonary trunk and valves, and endocardial cushions. Foxc2 + cell descendant contributions to the glomerular podocytes in the kidney were also observed following E6.5 tamoxifen treatment. Our results are consistent with previous reports of Foxc2 expression during early embryogenesis and the Foxc2 CreERT2 mouse provides a tool to investigate spatiotemporal roles of Foxc2 and contributions of Foxc2 + expressing cells during mouse embryogenesis. © 2016 Japanese Teratology Society.
HB-EGF function in cardiac valve development requires interaction with heparan sulfate proteoglycans.

PubMed

Iwamoto, Ryo; Mine, Naoki; Kawaguchi, Taichiro; Minami, Seigo; Saeki, Kazuko; Mekada, Eisuke

2010-07-01

HB-EGF, a member of the EGF family of growth factors, plays an important role in cardiac valve development by suppressing mesenchymal cell proliferation. Here, we show that HB-EGF must interact with heparan sulfate proteoglycans (HSPGs) to properly function in this process. In developing valves, HB-EGF is synthesized in endocardial cells but accumulates in the mesenchyme by interacting with HSPGs. Disrupting the interaction between HB-EGF and HSPGs in an ex vivo model of endocardial cushion explants resulted in increased mesenchymal cell proliferation. Moreover, homozygous knock-in mice (HB(Delta)(hb/)(Delta)(hb)) expressing a mutant HB-EGF that cannot bind to HSPGs developed enlarged cardiac valves with hyperproliferation of mesenchymal cells; this resulted in a phenotype that resembled that of Hbegf-null mice. Interestingly, although Hbegf-null mice had abnormal heart chambers and lung alveoli, HB(Delta)(hb/)(Delta)(hb) mice did not exhibit these defects. These results indicate that interactions with HSPGs are essential for the function of HB-EGF, especially in cardiac valve development, in which HB-EGF suppresses mesenchymal cell proliferation.
Behavioral Characterization of Knockin Mice with Mutations M287L and Q266I in the Glycine Receptor α1 Subunit

PubMed Central

Blednov, Yuri A.; Benavidez, Jill M.; Homanics, Gregg E.

2012-01-01

We used behavioral pharmacology to characterize heterozygous knockin mice with mutations (Q266I or M287L) in the α1 subunit of the glycine receptor (GlyR) (J Pharmacol Exp Ther 340:304–316, 2012). These mutations were designed to reduce (M287L) or eliminate (Q266I) ethanol potentiation of GlyR function. We asked which behavioral effects of ethanol would be reduced more in the Q266I mutant than the M287L and found rotarod ataxia to be the behavior that fulfilled this criterion. Compared with controls, the mutant mice also differed in ethanol consumption, ethanol-stimulated startle response, signs of acute physical dependence, and duration of loss of righting response produced by ethanol, butanol, ketamine, pentobarbital, and flurazepam. Some of these behavioral changes were mimicked in wild-type mice by acute injections of low, subconvulsive doses of strychnine. Both mutants showed increased acoustic startle response and increased sensitivity to strychnine seizures. Thus, in addition to reducing ethanol action on the GlyRs, these mutations reduced glycinergic inhibition, which may also alter sensitivity to GABAergic drugs. PMID:22037202
Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss.

PubMed

Scekic-Zahirovic, Jelena; Sendscheid, Oliver; El Oussini, Hajer; Jambeau, Mélanie; Sun, Ying; Mersmann, Sina; Wagner, Marina; Dieterlé, Stéphane; Sinniger, Jérome; Dirrig-Grosch, Sylvie; Drenner, Kevin; Birling, Marie-Christine; Qiu, Jinsong; Zhou, Yu; Li, Hairi; Fu, Xiang-Dong; Rouaux, Caroline; Shelkovnikova, Tatyana; Witting, Anke; Ludolph, Albert C; Kiefer, Friedemann; Storkebaum, Erik; Lagier-Tourenne, Clotilde; Dupuis, Luc

2016-05-17

FUS is an RNA-binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS-containing aggregates are often associated with concomitant loss of nuclear FUS Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell-specific CRE-mediated expression of wild-type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.
Divergent Regulation of Energy Expenditure and Hepatic Glucose Production by Insulin Receptor in Agouti-Related Protein and POMC Neurons

PubMed Central

Lin, Hua V.; Plum, Leona; Ono, Hiraku; Gutiérrez-Juárez, Roger; Shanabrough, Marya; Borok, Erzsebet; Horvath, Tamas L.; Rossetti, Luciano; Accili, Domenico

2010-01-01

OBJECTIVE The sites of insulin action in the central nervous system that regulate glucose metabolism and energy expenditure are incompletely characterized. We have shown that mice with hypothalamic deficiency (L1) of insulin receptors (InsRs) fail to regulate hepatic glucose production (HGP) in response to insulin. RESEARCH DESIGN AND METHODS To distinguish neurons that mediate insulin's effects on HGP from those that regulate energy homeostasis, we used targeted knock-ins to express InsRs in agouti-related protein (AgRP) or proopiomelanocortin (POMC) neurons of L1 mice. RESULTS Restoration of insulin action in AgRP neurons normalized insulin suppression of HGP. Surprisingly, POMC-specific InsR knock-in increased energy expenditure and locomotor activity, exacerbated insulin resistance and increased HGP, associated with decreased expression of the ATP-sensitive K+ channel (KATP channel) sulfonylurea receptor 1 subunit, and decreased inhibitory synaptic contacts on POMC neurons. CONCLUSIONS The contrasting phenotypes of InsR knock-ins in POMC and AgRP neurons suggest a branched-pathway model of hypothalamic insulin signaling in which InsR signaling in AgRP neurons decreases HGP, whereas InsR activation in POMC neurons promotes HGP and activates the melanocortinergic energy expenditure program. PMID:19933998
Tafenoquine and NPC-1161B require CYP 2D metabolism for anti-malarial activity: implications for the 8-aminoquinoline class of anti-malarial compounds

PubMed Central

2014-01-01

Background Tafenoquine (TQ) is an 8-aminoquinoline (8AQ) that has been tested in several Phase II and Phase III clinical studies and is currently in late stage development as an anti-malarial prophylactic agent. NPC-1161B is a promising 8AQ in late preclinical development. It has recently been reported that the 8AQ drug primaquine requires metabolic activation by CYP 2D6 for efficacy in humans and in mice, highlighting the importance of pharmacogenomics in the target population when administering primaquine. A logical follow-up study was to determine whether CYP 2D activation is required for other compounds in the 8AQ structural class. Methods In the present study, the anti-malarial activities of NPC-1161B and TQ were assessed against luciferase expressing Plasmodium berghei in CYP 2D knock-out mice in comparison with normal C57BL/6 mice (WT) and with humanized/CYP 2D6 knock-in mice by monitoring luminescence with an in vivo imaging system. These experiments were designed to determine the direct effects of CYP 2D metabolic activation on the anti-malarial efficacy of NPC-1161B and TQ. Results NPC-1161B and TQ exhibited no anti-malarial activity in CYP 2D knock-out mice when dosed at their ED100 values (1 mg/kg and 3 mg/kg, respectively) established in WT mice. TQ anti-malarial activity was partially restored in humanized/CYP 2D6 knock-in mice when tested at two times its ED100. Conclusions The results reported here strongly suggest that metabolism of NPC-1161B and TQ by the CYP 2D enzyme class is essential for their anti-malarial activity. Furthermore, these results may provide a possible explanation for therapeutic failures for patients who do not respond to 8AQ treatment for relapsing malaria. Because CYP 2D6 is highly polymorphic, variable expression of this enzyme in humans represents a significant pharmacogenomic liability for 8AQs which require CYP 2D metabolic activation for efficacy, particularly for large-scale prophylaxis and eradication campaigns. PMID:24386891
Tafenoquine and NPC-1161B require CYP 2D metabolism for anti-malarial activity: implications for the 8-aminoquinoline class of anti-malarial compounds.

PubMed

Marcsisin, Sean R; Sousa, Jason C; Reichard, Gregory A; Caridha, Diana; Zeng, Qiang; Roncal, Norma; McNulty, Ronan; Careagabarja, Julio; Sciotti, Richard J; Bennett, Jason W; Zottig, Victor E; Deye, Gregory; Li, Qigui; Read, Lisa; Hickman, Mark; Dhammika Nanayakkara, N P; Walker, Larry A; Smith, Bryan; Melendez, Victor; Pybus, Brandon S

2014-01-03

Tafenoquine (TQ) is an 8-aminoquinoline (8AQ) that has been tested in several Phase II and Phase III clinical studies and is currently in late stage development as an anti-malarial prophylactic agent. NPC-1161B is a promising 8AQ in late preclinical development. It has recently been reported that the 8AQ drug primaquine requires metabolic activation by CYP 2D6 for efficacy in humans and in mice, highlighting the importance of pharmacogenomics in the target population when administering primaquine. A logical follow-up study was to determine whether CYP 2D activation is required for other compounds in the 8AQ structural class. In the present study, the anti-malarial activities of NPC-1161B and TQ were assessed against luciferase expressing Plasmodium berghei in CYP 2D knock-out mice in comparison with normal C57BL/6 mice (WT) and with humanized/CYP 2D6 knock-in mice by monitoring luminescence with an in vivo imaging system. These experiments were designed to determine the direct effects of CYP 2D metabolic activation on the anti-malarial efficacy of NPC-1161B and TQ. NPC-1161B and TQ exhibited no anti-malarial activity in CYP 2D knock-out mice when dosed at their ED100 values (1 mg/kg and 3 mg/kg, respectively) established in WT mice. TQ anti-malarial activity was partially restored in humanized/CYP 2D6 knock-in mice when tested at two times its ED100. The results reported here strongly suggest that metabolism of NPC-1161B and TQ by the CYP 2D enzyme class is essential for their anti-malarial activity. Furthermore, these results may provide a possible explanation for therapeutic failures for patients who do not respond to 8AQ treatment for relapsing malaria. Because CYP 2D6 is highly polymorphic, variable expression of this enzyme in humans represents a significant pharmacogenomic liability for 8AQs which require CYP 2D metabolic activation for efficacy, particularly for large-scale prophylaxis and eradication campaigns.
Long Term Ablation of Protein Kinase A (PKA)-mediated Cardiac Troponin I Phosphorylation Leads to Excitation-Contraction Uncoupling and Diastolic Dysfunction in a Knock-in Mouse Model of Hypertrophic Cardiomyopathy*

PubMed Central

Dweck, David; Sanchez-Gonzalez, Marcos A.; Chang, Audrey N.; Dulce, Raul A.; Badger, Crystal-Dawn; Koutnik, Andrew P.; Ruiz, Edda L.; Griffin, Brittany; Liang, Jingsheng; Kabbaj, Mohamed; Fincham, Frank D.; Hare, Joshua M.; Overton, J. Michael; Pinto, Jose R.

2014-01-01

The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca2+ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca2+-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca2+ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca2+ content. This abnormal Ca2+ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na+-Ca2+ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy. PMID:24973218
In the absence of its cytosolic domain, the CD28 molecule still contributes to T cell activation

PubMed Central

Morin, Stéphanie; Giroux, Valentin; Favre, Cédric; Bechah, Yassina; Auphan-Anezin, Nathalie; Roncagalli, Romain; Mège, Jean-Louis; Olive, Daniel; Malissen, Marie; Nunes, Jacques

2015-01-01

The CD28 costimulatory receptor has a pivotal role in T cell biology as this molecule amplifies T cell receptor (TCR) signals to provide an efficient immune T cell response. There is a large debate about how CD28 mediates these signals. Here, we designed a CD28 gene targeted knock-in mouse strain lacking the cytoplasmic tail of CD28. As is the case in CD28-deficient (CD28 knock-out) mice, regulatory T cell homeostasis and T cell activation are altered in these CD28 knock-in mice. Unexpectedly, the presence of a CD28 molecule deprived of its cytoplasmic tail could partially induce some early activation events in T cells such as signaling events or expression of early activation markers. These results unravel a new mechanism of T cell costimulation by CD28, independent of its cytoplasmic tail. PMID:25725801
An anticholinergic reverses motor control and corticostriatal LTD deficits in Dyt1 ΔGAG knock-in mice.

PubMed

Dang, Mai T; Yokoi, Fumiaki; Cheetham, Chad C; Lu, Jun; Vo, Viet; Lovinger, David M; Li, Yuqing

2012-01-15

DYT1 early-onset generalized torsion dystonia is an inherited movement disorder associated with mutations in DYT1 that codes for torsinA protein. The most common mutation seen in this gene is a trinucleotide deletion of GAG. We previously reported a motor control deficit on a beam-walking task in our Dyt1 ΔGAG knock-in heterozygous mice. In this report we show the reversal of this motor deficit with the anticholinergic trihexyphenidyl (THP), a drug commonly used to treat movement problems in dystonia patients. THP also restored the reduced corticostriatal long-term depression (LTD) observed in these mice. Corticostriatal LTD has long been known to be dependent on D2 receptor activation. In this mouse model, striatal D2 receptors were expressed at lower quantities in comparison to wild-type mice. Furthermore, the mice were also partially resistant to FPL64176, an agonist of L-type calcium channels that have been previously reported to cause severe dystonic-like symptoms in wild-type mice. Our findings collectively suggest that altered communication between cholinergic interneurons and medium spiny neurons is responsible for the LTD deficit and that this synaptic plasticity modification may be involved in the striatal motor control abnormalities in our mouse model of DYT1 dystonia. Copyright © 2011 Elsevier B.V. All rights reserved.
Long-term oral administration of the NMDA receptor antagonist memantine extends life span in spinocerebellar ataxia type 1 knock-in mice.

PubMed

Iizuka, Akira; Nakamura, Kazuhiro; Hirai, Hirokazu

2015-04-10

Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease caused by extension of a CAG repeat in the Sca1gene. Although the mechanisms underlying the symptoms of SCA1 have not been determined, aberrant neuronal activation potentially contributes to the neuronal cell death characteristic of the disease. Here we examined the potential involvement of extrasynaptic N-methyl-d-aspartate receptor (NMDAR) activation in the pathogenesis of SCA1 by administering memantine, a low-affinity noncompetitive NMDAR antagonist, in SCA1 knock-in (KI) mice. In KI mice, the exon in the ataxin 1 gene is replaced with abnormally expanded 154CAG repeats. Memantine was administered orally to the SCA1 KI mice from 4 weeks of age until death. The treatment significantly attenuated body-weight loss and prolonged the life span of SCA1 KI mice. Furthermore, memantine significantly suppressed the loss of Purkinje cells in the cerebellum and motor neurons in the dorsal motor nucleus of the vagus, which are critical for motor function and parasympathetic function, respectively. These findings support the contribution of aberrant activation of extrasynaptic NMDARs to neuronal cell death in SCA1 KI mice and suggest that memantine may also have therapeutic benefits in human SCA1 patients. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Diabetes increases mortality after myocardial infarction by oxidizing CaMKII

PubMed Central

Luo, Min; Guan, Xiaoqun; Luczak, Elizabeth D.; Lang, Di; Kutschke, William; Gao, Zhan; Yang, Jinying; Glynn, Patric; Sossalla, Samuel; Swaminathan, Paari D.; Weiss, Robert M.; Yang, Baoli; Rokita, Adam G.; Maier, Lars S.; Efimov, Igor R.; Hund, Thomas J.; Anderson, Mark E.

2013-01-01

Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca2+/calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction. PMID:23426181
Osteoclasts in the dental microenvironment: a delicate balance controls dental histogenesis.

PubMed

Berdal, A; Castaneda, B; Aïoub, M; Néfussi, J R; Mueller, C; Descroix, V; Lézot, F

2011-01-01

The impact of osteoclast activity on dental development has been previously analyzed but in the context of severe osteopetrosis. The present study sought to investigate the effects of osteoclast hypofunction,present in Msx2 gene knockin mutant mice (Msx2-/-), and hyperfunction, in transgenic mice driving RANK over-expression in osteoclast precursors (RANK(Tg)), on tooth development. In Msx2-/- mice, moderate osteopetrosis was observed, occurring exclusively in the periodontal region. Microradiographical and histological analyses revealed an abnormal dental epithelium histogenesis that gave rise to odontogenic tumor-like structures. This led to impaired tooth eruption, especially of the third mandibular molars. In RANK(Tg) mice, root histogenesis showed site-specific upregulation of dental cell proliferation and differentiation rates. This culminated in roots with a reduced diameter and pulp size albeit of normal length. These two reverse experimental systems will enable the investigation of distinctive dental cell and osteoclast communication in normal growth and tumorigenesis. Copyright © 2011 S. Karger AG, Basel.
Role of kinase-independent and -dependent functions of FAK in endothelial cell survival and barrier function during embryonic development.

PubMed

Zhao, Xiaofeng; Peng, Xu; Sun, Shaogang; Park, Ann Y J; Guan, Jun-Lin

2010-06-14

Focal adhesion kinase (FAK) is essential for vascular development as endothelial cell (EC)-specific knockout of FAK (conditional FAK knockout [CFKO] mice) leads to embryonic lethality. In this study, we report the differential kinase-independent and -dependent functions of FAK in vascular development by creating and analyzing an EC-specific FAK kinase-defective (KD) mutant knockin (conditional FAK knockin [CFKI]) mouse model. CFKI embryos showed apparently normal development through embryonic day (E) 13.5, whereas the majority of CFKO embryos died at the same stage. Expression of KD FAK reversed increased EC apoptosis observed with FAK deletion in embryos and in vitro through suppression of up-regulated p21. However, vessel dilation and defective angiogenesis of CFKO embryos were not rescued in CFKI embryos. ECs without FAK or expressing KD FAK showed increased permeability, abnormal distribution of vascular endothelial cadherin (VE-cadherin), and reduced VE-cadherin Y658 phosphorylation. Together, our data suggest that kinase-independent functions of FAK can support EC survival in vascular development through E13.5 but are insufficient for maintaining EC function to allow for completion of embryogenesis.
Periodontal Defects in the A116T Knock-in Murine Model of Odontohypophosphatasia.

PubMed

Foster, B L; Sheen, C R; Hatch, N E; Liu, J; Cory, E; Narisawa, S; Kiffer-Moreira, T; Sah, R L; Whyte, M P; Somerman, M J; Millán, J L

2015-05-01

Mutations in ALPL result in hypophosphatasia (HPP), a disease causing defective skeletal mineralization. ALPL encodes tissue nonspecific alkaline phosphatase (ALP), an enzyme that promotes mineralization by reducing inorganic pyrophosphate, a mineralization inhibitor. In addition to skeletal defects, HPP causes dental defects, and a mild clinical form of HPP, odontohypophosphatasia, features only a dental phenotype. The Alpl knockout (Alpl (-/-)) mouse phenocopies severe infantile HPP, including profound skeletal and dental defects. However, the severity of disease in Alpl (-/-) mice prevents analysis at advanced ages, including studies to target rescue of dental tissues. We aimed to generate a knock-in mouse model of odontohypophosphatasia with a primarily dental phenotype, based on a mutation (c.346G>A) identified in a human kindred with autosomal dominant odontohypophosphatasia. Biochemical, skeletal, and dental analyses were performed on the resulting Alpl(+/A116T) mice to validate this model. Alpl(+/A116T) mice featured 50% reduction in plasma ALP activity compared with wild-type controls. No differences in litter size, survival, or body weight were observed in Alpl(+/A116T) versus wild-type mice. The postcranial skeleton of Alpl(+/A116T) mice was normal by radiography, with no differences in femur length, cortical/trabecular structure or mineral density, or mechanical properties. Parietal bone trabecular compartment was mildly altered. Alpl(+/A116T) mice featured alterations in the alveolar bone, including radiolucencies and resorptive lesions, osteoid accumulation on the alveolar bone crest, and significant differences in several bone properties measured by micro-computed tomography. Nonsignificant changes in acellular cementum did not appear to affect periodontal attachment or function, although circulating ALP activity was correlated significantly with incisor cementum thickness. The Alpl(+/A116T) mouse is the first model of odontohypophosphatasia, providing insights on dentoalveolar development and function under reduced ALP, bringing attention to direct effects of HPP on alveolar bone, and offering a new model for testing potential dental-targeted therapies in future studies. © International & American Associations for Dental Research 2015.

Gsg1, Trnp1, and Tmem215 Mark Subpopulations of Bipolar Interneurons in the Mouse Retina

PubMed Central

Park, Ko Uoon; Randazzo, Grace; Jones, Kenneth L.; Brzezinski, Joseph A.

2017-01-01

Purpose How retinal bipolar cell interneurons are specified and assigned to specialized subtypes is only partially understood. In part, this is due to a lack of early pan- and subtype-specific bipolar cell markers. To discover these factors, we identified genes that were upregulated in Blimp1 (Prdm1) mutant retinas, which exhibit precocious bipolar cell development. Methods Postnatal day (P)2 retinas from Blimp1 conditional knock-out (CKO) mice and controls were processed for RNA sequencing. Genes that increased at least 45% and were statistically different between conditions were considered candidate bipolar-specific factors. Candidates were further evaluated by RT-PCR, in situ hybridization, and immunohistochemistry. Knock-in Tmem215-LacZ mice were used to better trace retinal expression. Results A comparison between Blimp1 CKO and control RNA-seq datasets revealed approximately 40 significantly upregulated genes. We characterized the expression of three genes that have no known function in the retina, Gsg1 (germ cell associated gene), Trnp1 (TMF-regulated nuclear protein), and Tmem215 (a predicted transmembrane protein). Germ cell associated gene appeared restricted to a small subset of cone bipolars while Trnp1 was seen in all ON type bipolar cells. Using Tmem215-LacZ heterozygous knock-in mice, we observed that β-galactosidase expression started early in bipolar cell development. In adults, Tmem215 was expressed by a subset of ON and OFF cone bipolar cells. Conclusions We have identified Gsg1, Tmem215, and Trnp1 as novel bipolar subtype-specific genes. The spatial and temporal pattern of their expression is consistent with a role in controlling bipolar subtype fate choice, differentiation, or physiology. PMID:28199486
Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay

PubMed Central

Nguyen, Thi A.; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M.; Xu, Xialian; Miller, Bruce L.; Rigo, Frank; Ferguson, Shawn M.; Walther, Tobias C.; Farese, Robert V.

2018-01-01

Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized GrnR493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous GrnR493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in GrnR493X mice and cell lines and in fibroblasts from patients containing the GRNR493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. PMID:29511098
Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ.

PubMed

Aida, Tomomi; Nakade, Shota; Sakuma, Tetsushi; Izu, Yayoi; Oishi, Ayu; Mochida, Keiji; Ishikubo, Harumi; Usami, Takako; Aizawa, Hidenori; Yamamoto, Takashi; Tanaka, Kohichi

2016-11-28

Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining. Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. Our results provide a technical platform for high-throughput knock-in.
Dnmt3a deletion cooperates with the Flt3/ITD mutation to drive leukemogenesis in a murine model

PubMed Central

Poitras, Jennifer L.; Heiser, Diane; Li, Li; Nguyen, Bao; Nagai, Kozo; Duffield, Amy S.; Gamper, Christopher; Small, Donald

2016-01-01

Internal tandem duplications of the juxtamembrane domain of FLT3 (FLT3/ITD) are among the most common mutations in Acute Myeloid Leukemia (AML). Resulting in constitutive activation of the kinase, FLT3/ITD portends a particularly poor prognosis, with reduced overall survival and increased rates of relapse. We previously generated a knock-in mouse, harboring an internal tandem duplication at the endogenous Flt3 locus, which develops a fatal myeloproliferative neoplasm (MPN), but fails to develop acute leukemia, suggesting additional mutations are necessary for transformation. To investigate the potential cooperativity of FLT3/ITD and mutant DNMT3A, we bred a conditional Dnmt3a knockout to a substrain of our Flt3/ITD knock-in mice, and found deletion of Dnmt3a significantly reduced median survival of Flt3ITD/+ mice in a dose dependent manner. As expected, pIpC treated Flt3ITD/+ mice solely developed MPN, while Flt3ITD/+;Dnmt3af/f and Flt3ITD/+;Dnmt3af/+ developed a spectrum of neoplasms, including MPN, T-ALL, and AML. Functionally, FLT3/ITD and DNMT3A deletion cooperate to expand LT-HSCs, which exhibit enhanced self-renewal in serial re-plating assays. These results illustrate that DNMT3A loss cooperates with FLT3/ITD to generate hematopoietic neoplasms, including AML. In combination with FLT3/ITD, homozygous Dnmt3a knock-out results in reduced time to disease onset, LT-HSC expansion, and a higher incidence of T-ALL compared with loss of just one allele. The co-occurrence of FLT3 and DNMT3A mutations in AML, as well as subsets of T-ALL, suggests the Flt3ITD/+;Dnmt3af/f model may serve as a valuable resource for delineating effective therapeutic strategies in two clinically relevant contexts. PMID:27636998
Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.

PubMed

Miura, Hiromi; Quadros, Rolen M; Gurumurthy, Channabasavaiah B; Ohtsuka, Masato

2018-01-01

CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.
Characterisation of a C1qtnf5 Ser163Arg Knock-In Mouse Model of Late-Onset Retinal Macular Degeneration

PubMed Central

Shu, Xinhua; Luhmann, Ulrich F. O.; Aleman, Tomas S.; Barker, Susan E.; Lennon, Alan; Tulloch, Brian; Chen, Mei; Xu, Heping; Jacobson, Samuel G.; Ali, Robin; Wright, Alan F.

2011-01-01

A single founder mutation resulting in a Ser163Arg substitution in the C1QTNF5 gene product causes autosomal dominant late-onset retinal macular degeneration (L-ORMD) in humans, which has clinical and pathological features resembling age-related macular degeneration. We generated and characterised a mouse “knock-in” model carrying the Ser163Arg mutation in the orthologous murine C1qtnf5 gene by site-directed mutagenesis and homologous recombination into mouse embryonic stem cells. Biochemical, immunological, electron microscopic, fundus autofluorescence, electroretinography and laser photocoagulation analyses were used to characterise the mouse model. Heterozygous and homozygous knock-in mice showed no significant abnormality in any of the above measures at time points up to 2 years. This result contrasts with another C1qtnf5 Ser163Arg knock-in mouse which showed most of the features of L-ORMD but differed in genetic background and targeting construct. PMID:22110650
Role of VGF-derived carboxy-terminal peptides in energy balance and reproduction: analysis of "humanized" knockin mice expressing full-length or truncated VGF.

PubMed

Sadahiro, Masato; Erickson, Connor; Lin, Wei-Jye; Shin, Andrew C; Razzoli, Maria; Jiang, Cheng; Fargali, Samira; Gurney, Allison; Kelley, Kevin A; Buettner, Christoph; Bartolomucci, Alessandro; Salton, Stephen R

2015-05-01

Targeted deletion of VGF, a secreted neuronal and endocrine peptide precursor, produces lean, hypermetabolic, and infertile mice that are resistant to diet-, lesion-, and genetically-induced obesity and diabetes. Previous studies suggest that VGF controls energy expenditure (EE), fat storage, and lipolysis, whereas VGF C-terminal peptides also regulate reproductive behavior and glucose homeostasis. To assess the functional equivalence of human VGF(1-615) (hVGF) and mouse VGF(1-617) (mVGF), and to elucidate the function of the VGF C-terminal region in the regulation of energy balance and susceptibility to obesity, we generated humanized VGF knockin mouse models expressing full-length hVGF or a C-terminally deleted human VGF(1-524) (hSNP), encoded by a single nucleotide polymorphism (rs35400704). We show that homozygous male and female hVGF and hSNP mice are fertile. hVGF female mice had significantly increased body weight compared with wild-type mice, whereas hSNP mice have reduced adiposity, increased activity- and nonactivity-related EE, and improved glucose tolerance, indicating that VGF C-terminal peptides are not required for reproductive function, but 1 or more specific VGF C-terminal peptides are likely to be critical regulators of EE. Taken together, our results suggest that human and mouse VGF proteins are largely functionally conserved but that species-specific differences in VGF peptide function, perhaps a result of known differences in receptor binding affinity, likely alter the metabolic phenotype of hVGF compared with mVGF mice, and in hSNP mice in which several C-terminal VGF peptides are ablated, result in significantly increased activity- and nonactivity-related EE.
Improvement of neuropathology and transcriptional deficits in CAG 140 knock-in mice supports a beneficial effect of dietary curcumin in Huntington's disease

PubMed Central

2012-01-01

Backgound No disease modifying treatment currently exists for Huntington's disease (HD), a fatal neurodegenerative disorder characterized by the formation of amyloid-like aggregates of the mutated huntingtin protein. Curcumin is a naturally occurring polyphenolic compound with Congo red-like amyloid binding properties and the ability to cross the blood brain barrier. CAG140 mice, a knock-in (KI) mouse model of HD, display abnormal aggregates of mutant huntingtin and striatal transcriptional deficits, as well as early motor, cognitive and affective abnormalities, many months prior to exhibiting spontaneous gait deficits, decreased striatal volume, and neuronal loss. We have examined the ability of life-long dietary curcumin to improve the early pathological phenotype of CAG140 mice. Results KI mice fed a curcumin-containing diet since conception showed decreased huntingtin aggregates and increased striatal DARPP-32 and D1 receptor mRNAs, as well as an amelioration of rearing deficits. However, similar to other antioxidants, curcumin impaired rotarod behavior in both WT and KI mice and climbing in WT mice. These behavioral effects were also noted in WT C57Bl/6 J mice exposed to the same curcumin regime as adults. However, neither locomotor function, behavioral despair, muscle strength or food utilization were affected by curcumin in this latter study. The clinical significance of curcumin's impairment of motor performance in mice remains unclear because curcumin has an excellent blood chemistry and adverse event safety profile, even in the elderly and in patients with Alzheimer's disease. Conclusion Together with this clinical experience, the improvement in several transgene-dependent parameters by curcumin in our study supports a net beneficial effect of dietary curcumin in HD. PMID:22475209
Role of VGF-Derived Carboxy-Terminal Peptides in Energy Balance and Reproduction: Analysis of “Humanized” Knockin Mice Expressing Full-Length or Truncated VGF

PubMed Central

Sadahiro, Masato; Erickson, Connor; Lin, Wei-Jye; Shin, Andrew C.; Razzoli, Maria; Jiang, Cheng; Fargali, Samira; Gurney, Allison; Kelley, Kevin A.; Buettner, Christoph

2015-01-01

Targeted deletion of VGF, a secreted neuronal and endocrine peptide precursor, produces lean, hypermetabolic, and infertile mice that are resistant to diet-, lesion-, and genetically-induced obesity and diabetes. Previous studies suggest that VGF controls energy expenditure (EE), fat storage, and lipolysis, whereas VGF C-terminal peptides also regulate reproductive behavior and glucose homeostasis. To assess the functional equivalence of human VGF1–615 (hVGF) and mouse VGF1–617 (mVGF), and to elucidate the function of the VGF C-terminal region in the regulation of energy balance and susceptibility to obesity, we generated humanized VGF knockin mouse models expressing full-length hVGF or a C-terminally deleted human VGF1–524 (hSNP), encoded by a single nucleotide polymorphism (rs35400704). We show that homozygous male and female hVGF and hSNP mice are fertile. hVGF female mice had significantly increased body weight compared with wild-type mice, whereas hSNP mice have reduced adiposity, increased activity- and nonactivity-related EE, and improved glucose tolerance, indicating that VGF C-terminal peptides are not required for reproductive function, but 1 or more specific VGF C-terminal peptides are likely to be critical regulators of EE. Taken together, our results suggest that human and mouse VGF proteins are largely functionally conserved but that species-specific differences in VGF peptide function, perhaps a result of known differences in receptor binding affinity, likely alter the metabolic phenotype of hVGF compared with mVGF mice, and in hSNP mice in which several C-terminal VGF peptides are ablated, result in significantly increased activity- and nonactivity-related EE. PMID:25675362
Developmental regulation of GABAergic signalling in the hippocampus of neuroligin 3 R451C knock-in mice: an animal model of Autism.

PubMed

Pizzarelli, Rocco; Cherubini, Enrico

2013-01-01

Autism Spectrum Disorders (ASDs) comprise an heterogeneous group of neuro-developmental abnormalities, mainly of genetic origin, characterized by impaired social interactions, communications deficits, and stereotyped behaviors. In a small percentage of cases, ASDs have been found to be associated with single mutations in genes involved in synaptic function. One of these involves the postsynaptic cell adhesion molecule neuroligin (NL) 3. NLs interact with presynaptic neurexins (Nrxs) to ensure a correct cross talk between post and presynaptic specializations. Here, transgenic mice carrying the human R451C mutation of Nlgn3, were used to study GABAergic signaling in the hippocampus early in postnatal life. Whole cell recordings from CA3 pyramidal neurons in slices from NL3(R451C) knock-in mice revealed an enhanced frequency of Giant Depolarizing Potentials (GDPs), as compared to controls. This effect was probably dependent on an increased GABAergic drive to principal cells as demonstrated by the enhanced frequency of miniature GABAA-mediated (GPSCs), but not AMPA-mediated postsynaptic currents (EPSCs). Changes in frequency of mGPSCs were associated with an acceleration of their decay kinetics, in the absence of any change in unitary synaptic conductance or in the number of GABAA receptor channels, as assessed by peak scaled non-stationary fluctuation analysis. The enhanced GABAergic but not glutamatergic transmission early in postnatal life may change the excitatory/inhibitory balance known to play a key role in the construction and refinement of neuronal circuits during postnatal development. This may lead to behavioral deficits reminiscent of those observed in ASDs patients.
Developmental regulation of GABAergic signalling in the hippocampus of neuroligin 3 R451C knock-in mice: an animal model of Autism

PubMed Central

Pizzarelli, Rocco; Cherubini, Enrico

2013-01-01

Autism Spectrum Disorders (ASDs) comprise an heterogeneous group of neuro-developmental abnormalities, mainly of genetic origin, characterized by impaired social interactions, communications deficits, and stereotyped behaviors. In a small percentage of cases, ASDs have been found to be associated with single mutations in genes involved in synaptic function. One of these involves the postsynaptic cell adhesion molecule neuroligin (NL) 3. NLs interact with presynaptic neurexins (Nrxs) to ensure a correct cross talk between post and presynaptic specializations. Here, transgenic mice carrying the human R451C mutation of Nlgn3, were used to study GABAergic signaling in the hippocampus early in postnatal life. Whole cell recordings from CA3 pyramidal neurons in slices from NL3R451C knock-in mice revealed an enhanced frequency of Giant Depolarizing Potentials (GDPs), as compared to controls. This effect was probably dependent on an increased GABAergic drive to principal cells as demonstrated by the enhanced frequency of miniature GABAA-mediated (GPSCs), but not AMPA-mediated postsynaptic currents (EPSCs). Changes in frequency of mGPSCs were associated with an acceleration of their decay kinetics, in the absence of any change in unitary synaptic conductance or in the number of GABAA receptor channels, as assessed by peak scaled non-stationary fluctuation analysis. The enhanced GABAergic but not glutamatergic transmission early in postnatal life may change the excitatory/inhibitory balance known to play a key role in the construction and refinement of neuronal circuits during postnatal development. This may lead to behavioral deficits reminiscent of those observed in ASDs patients. PMID:23761734
Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

PubMed

Jeong, Young-Hee; Kim, Yeong Ji; Kim, Eun Young; Kim, Se Eun; Kim, Jiwoo; Park, Min Jee; Lee, Hong-Gu; Park, Se Pill; Kang, Man-Jong

2016-06-01

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene.
Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.

PubMed

Prykhozhij, Sergey V; Fuller, Charlotte; Steele, Shelby L; Veinotte, Chansey J; Razaghi, Babak; Robitaille, Johane M; McMaster, Christopher R; Shlien, Adam; Malkin, David; Berman, Jason N

2018-06-14

We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.
An Acvr1 R206H knock-in mouse has fibrodysplasia ossificans progressiva

PubMed Central

Chakkalakal, Salin A.; Zhang, Deyu; Culbert, Andria L.; Convente, Michael R.; Caron, Robert J.; Wright, Alexander C.; Maidment, Andrew D.A.; Kaplan, Frederick S.; Shore, Eileen M.

2013-01-01

Fibrodysplasia ossificans progressiva (FOP; MIM #135100) is a debilitating genetic disorder of dysregulated cellular differentiation characterized by malformation of the great toes during embryonic skeletal development and by progressive heterotopic endochondral ossification post-natally. Patients with these classic clinical features of FOP have the identical heterozygous single nucleotide substitution (c.617G>A; R206H) in the gene encoding ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor. Gene targeting was used to develop a knock-in mouse model for FOP (Acvr1R206H/+). Radiographic analysis of Acvr1R206H/+ chimeric mice revealed that this mutation induced malformed first digits in the hind limbs and post-natal extra-skeletal bone formation, recapitulating the human disease. Histological analysis of murine lesions showed inflammatory infiltration and apoptosis of skeletal muscle followed by robust formation of heterotopic bone through an endochondral pathway, identical to that seen in patients. Progenitor cells of a Tie2+ lineage participated in each stage of endochondral osteogenesis. We further determined that both wild-type and mutant cells are present within the ectopic bone tissue, an unexpected finding that indicates that although the mutation is necessary to induce the bone formation process, the mutation is not required for progenitor cell contribution to bone and cartilage. This unique knock-in mouse model provides novel insight into the genetic regulation of heterotopic ossification and establishes the first direct in vivo evidence that the R206H mutation in ACVR1 causes FOP. PMID:22508565
In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

PubMed Central

Panaroni, Cristina; Gioia, Roberta; Lupi, Anna; Besio, Roberta; Goldstein, Steven A.; Kreider, Jaclynn; Leikin, Sergey; Vera, Juan Carlos; Mertz, Edward L.; Perilli, Egon; Baruffaldi, Fabio; Villa, Isabella; Farina, Aurora; Casasco, Marco; Cetta, Giuseppe; Rossi, Antonio; Frattini, Annalisa; Marini, Joan C.; Vezzoni, Paolo

2009-01-01

Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [α1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases. PMID:19414862
Ketamine up-regulates a cluster of intronic miRNAs within the serotonin receptor 2C gene by inhibiting glycogen synthase kinase-3.

PubMed

Grieco, Steven F; Velmeshev, Dmitry; Magistri, Marco; Eldar-Finkelman, Hagit; Faghihi, Mohammad A; Jope, Richard S; Beurel, Eleonore

2017-09-01

We examined mechanisms that contribute to the rapid antidepressant effect of ketamine in mice that is dependent on glycogen synthase kinase-3 (GSK3) inhibition. We measured serotonergic (5HT)-2C-receptor (5HTR2C) cluster microRNA (miRNA) levels in mouse hippocampus after administering an antidepressant dose of ketamine (10 mg/kg) in wild-type and GSK3 knockin mice, after GSK3 inhibition with L803-mts, and in learned helpless mice. Ketamine up-regulated cluster miRNAs 448-3p, 764-5p, 1264-3p, 1298-5p and 1912-3p (2- to 11-fold). This up-regulation was abolished in GSK3 knockin mice that express mutant constitutively active GSK3. The GSK3 specific inhibitor L803-mts was antidepressant in the learned helplessness and novelty suppressed feeding depression-like behaviours and up-regulated the 5HTR2C miRNA cluster in mouse hippocampus. After administration of the learned helplessness paradigm mice were divided into cohorts that were resilient (non-depressed) or were susceptible (depressed) to learned helplessness. The resilient, but not depressed, mice displayed increased hippocampal levels of miRNAs 448-3p and 1264-3p. Administration of an antagonist to miRNA 448-3p diminished the antidepressant effect of ketamine in the learned helplessness paradigm, indicating that up-regulation of miRNA 448-3p provides an antidepressant action. These findings identify a new outcome of GSK3 inhibition by ketamine that may contribute to antidepressant effects.
CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice.

PubMed

Meca-Cortés, Oscar; Guerra-Rebollo, Marta; Garrido, Cristina; Borrós, Salvador; Rubio, Nuria; Blanco, Jeronimo

2017-09-15

The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs), using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker) and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene), under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Atm knock-in mice harboring an in-frame deletion corresponding to the human ATM 7636del9 common mutation exhibit a variant phenotype.

PubMed

Spring, K; Cross, S; Li, C; Watters, D; Ben-Senior, L; Waring, P; Ahangari, F; Lu, S L; Chen, P; Misko, I; Paterson, C; Kay, G; Smorodinsky, N I; Shiloh, Y; Lavin, M F

2001-06-01

ATM, the gene mutated in the human immunodeficiency disorder ataxia-telangiectasia (A-T), plays a central role in recognizing ionizing radiation damage in DNA and in controlling several cell cycle checkpoints. We describe here a murine model in which a nine-nucleotide in-frame deletion has been introduced into the Atm gene by homologous recombination followed by removal of the selectable marker cassette by Cre-loxP site-specific, recombination-mediated excision. This mouse, Atm-DeltaSRI, was designed as a model of one of the most common deletion mutations (7636del9) found in A-T patients. The murine Atm deletion results in the loss of three amino acid residues (SRI; 2556-2558) but produces near full-length detectable Atm protein that lacks protein kinase activity. Radiosensitivity was observed in Atm-DeltaSRI mice, whereas the immunological profile of these mice showed greater heterogeneity of T-cell subsets than observed in Atm(-/-) mice. The life span of Atm-DeltaSRI mice was significantly longer than that of Atm(-/-) mice when maintained under nonspecific pathogen-free conditions. This can be accounted for by a lower incidence of thymic lymphomas in Atm-DeltaSRI mice up to 40 weeks, after which time the animals died of other causes. The thymic lymphomas in Atm-DeltaSRI mice were characterized by extensive apoptosis, which appears to be attributable to an increased number of cells expressing Fas ligand. A variety of other tumors including B-cell lymphomas, sarcomas, and carcinomas not seen in Atm(-/-) mice were observed in older Atm-DeltaSRI animals. Thus, expression of mutant protein in Atm-DeltaSRI knock-in mice gives rise to a discernibly different phenotype to Atm(-/-) mice, which may account for the heterogeneity seen in A-T patients with different mutations.
Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay.

PubMed

Nguyen, Andrew D; Nguyen, Thi A; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M; Xu, Xialian; Miller, Bruce L; Rigo, Frank; Ferguson, Shawn M; Huang, Eric J; Walther, Tobias C; Farese, Robert V

2018-03-20

Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized Grn R493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous Grn R493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in Grn R493X mice and cell lines and in fibroblasts from patients containing the GRN R493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. Copyright © 2018 the Author(s). Published by PNAS.
CCR2 antagonist CCX140-B provides renal and glycemic benefits in diabetic transgenic human CCR2 knockin mice

PubMed Central

Sullivan, Timothy; Miao, Zhenhua; Dairaghi, Daniel J.; Krasinski, Antoni; Wang, Yu; Zhao, Bin N.; Baumgart, Trageen; Ertl, Linda S.; Pennell, Andrew; Seitz, Lisa; Powers, Jay; Zhao, Ruiping; Ungashe, Solomon; Wei, Zheng; Boring, Landin; Tsou, Chia-Lin; Charo, Israel; Schall, Thomas J.; Jaen, Juan C.

2013-01-01

Chemokine (C-C motif) receptor 2 (CCR2) is central for the migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate phase 2 clinical trials in diabetic nephropathy, has recently been shown to reduce hemoglobin A1c and fasting blood glucose levels in type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has a low affinity for mouse CCR2, transgenic human CCR2 knockin mice were generated and rendered diabetic with either a high-fat diet (diet-induced obesity) or by deletion of the leptin receptor gene (db/db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of diet-induced obese mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of homeostatic model assessment of insulin resistance values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function. PMID:23986513

Rescue of cardiomyopathy through U7snRNA-mediated exon skipping in Mybpc3-targeted knock-in mice.

PubMed

Gedicke-Hornung, Christina; Behrens-Gawlik, Verena; Reischmann, Silke; Geertz, Birgit; Stimpel, Doreen; Weinberger, Florian; Schlossarek, Saskia; Précigout, Guillaume; Braren, Ingke; Eschenhagen, Thomas; Mearini, Giulia; Lorain, Stéphanie; Voit, Thomas; Dreyfus, Patrick A; Garcia, Luis; Carrier, Lucie

2013-07-01

Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5-6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5+6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5+6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.
Missense Mutations in LRP5 Associated with High Bone Mass Protect the Mouse Skeleton from Disuse- and Ovariectomy-Induced Osteopenia.

PubMed

Niziolek, Paul J; Bullock, Whitney; Warman, Matthew L; Robling, Alexander G

2015-01-01

The low density lipoprotein receptor-related protein-5 (LRP5), a co-receptor in the Wnt signaling pathway, modulates bone mass in humans and in mice. Lrp5 knock-out mice have severely impaired responsiveness to mechanical stimulation whereas Lrp5 gain-of-function knock-in and transgenic mice have enhanced responsiveness to mechanical stimulation. Those observations highlight the importance of Lrp5 protein in bone cell mechanotransduction. It is unclear if and how high bone mass-causing (HBM) point mutations in Lrp5 alter the bone-wasting effects of mechanical disuse. To address this issue we explored the skeletal effects of mechanical disuse using two models, tail suspension and Botulinum toxin-induced muscle paralysis, in two different Lrp5 HBM knock-in mouse models. A separate experiment employing estrogen withdrawal-induced bone loss by ovariectomy was also conducted as a control. Both disuse stimuli induced significant bone loss in WT mice, but Lrp5 A214V and G171V were partially or fully protected from the bone loss that normally results from disuse. Trabecular bone parameters among HBM mice were significantly affected by disuse in both models, but these data are consistent with DEXA data showing a failure to continue growing in HBM mice, rather than a loss of pre-existing bone. Ovariectomy in Lrp5 HBM mice resulted in similar protection from catabolism as was observed for the disuse experiments. In conclusion, the Lrp5 HBM alleles offer significant protection from the resorptive effects of disuse and from estrogen withdrawal, and consequently, present a potential mechanism to mimic with pharmaceutical intervention to protect against various bone-wasting stimuli.
Examination of methylphenidate-mediated behavior regulation by glycogen synthase kinase-3 in mice.

PubMed

Mines, Marjelo A; Beurel, Eleonore; Jope, Richard S

2013-01-05

Abnormalities in dopaminergic activity have been implicated in psychiatric diseases, such as attention deficit hyperactivity disorder (ADHD), and are treated with therapeutic stimulants, commonly methylphenidate or amphetamine. Amphetamine administration increases glycogen synthase kinase-3 (GSK3) activation, which is necessary for certain acute behavioral responses to amphetamine, including increased locomotor activity and impaired sensorimotor gating. Here, we tested if modulating GSK3 by administration of the GSK3 inhibitor lithium or expression of constitutively active GSK3 altered behavioral responses to methylphenidate administered to mice acutely or daily for 8 days. Methylphenidate or amphetamine was administered to mice intraperitoneally for 1 or 8 days. Open-field activity and pre-pulse inhibition (PPI) were measured. In contrast to lithium's blockade of acute amphetamine-induced locomotor hyperactivity, lithium treatment did not significantly reduce methylphenidate-induced locomotor hyperactivity in wild-type mice after acute or 8 days of repeated methylphenidate administration. Lithium treatment significantly increased the impairment in PPI caused by methylphenidate, but significantly reduced the amphetamine-induced PPI deficit. In GSK3 knockin mice, expression of constitutively active GSK3β, but not GSK3α, significantly increased locomotor hyperactivity after acute methylphenidate treatment, and significantly impaired PPI, preventing further methylphenidate-induced impairment of PPI that was evident in wild-type mice and GSK3α knockin mice. Lithium does not counteract locomotor activity and PPI responses to methylphenidate as it does these responses to amphetamine, indicating that different mechanisms mediate these behavioral responses to methylphenidate and amphetamine. Only active GSK3β, not GSK3α, modulates behavioral responses to MPH, indicating selectivity in the actions of GSK3 isoforms. Copyright © 2012 Elsevier B.V. All rights reserved.
Distinct effects of loss of classical estrogen receptor signaling versus complete deletion of estrogen receptor alpha on bone

PubMed Central

Syed, Farhan A.; Fraser, Daniel G.; Monroe, David G.; Khosla, Sundeep

2011-01-01

Estrogen receptor (ER)α is a major regulator of bone metabolism which can modulate gene expression via a “classical” pathway involving direct DNA binding to estrogen-response elements (EREs) or via “non-classical” pathways involving protein-protein interactions. While the skeletal consequences of loss of ERE binding by ERα have been described, a significant unresolved question is how loss of ERE binding differs from complete loss of ERα. Thus, we compared the skeletal phenotype of wild-type (ERα+/+) and ERα knock out (ERα−/−) mice with that of mice in which the only ERα present had a knock-in mutation abolishing ERE binding (non-classical ERα knock-in [NERKI], ERα−/NERKI). All three groups were in the same genetic background (C57BL/6). As compared to both ERα+/+ and ERα−/− mice, ERα−/NERKI mice had significantly reduced cortical volumetric bone mineral density and thickness at the tibial diaphysis; this was accompanied by significant decreases in periosteal and endocortical mineral apposition rates. Colony forming unit (CFU)-fibroblast, CFU-alkaline phosphatase, and CFU-osteoblast numbers were all increased in ERα−/− compared to ERα+/+ mice, but reduced in ERα−/NERKI mice compared to the two other groups. Thus, using mice in identical genetic backgrounds, our data indicate that the presence of an ERα that cannot bind DNA but can function through protein-protein interactions may have more deleterious skeletal effects than complete loss of ERα. These findings suggest that shifting the balance of classical versus non-classical ERα signaling triggers pathways that impair bone formation. Further studies defining these pathways may lead to novel approaches to selectively modulate ER signaling for beneficial skeletal effects. PMID:21458604
Progression of behavioural despair in R6/2 and Hdh knock-in mouse models recapitulates depression in Huntington's disease.

PubMed

Ciamei, Alessandro; Detloff, Peter J; Morton, A Jennifer

2015-09-15

In Huntington's disease (HD) depression is observed before the disease is diagnosed, and is likely to be a component of the disease, rather than a consequence. Depression in HD patients does not progress in parallel with other symptoms; rather it peaks at early- to mid-stages of the disease and declines thereafter. In mice, depressive-like behaviours can be measured as an increase in behavioural despair (floating) observed in the forced swim test (FST). Floating in the FST is modulated differently by antidepressants with different mechanisms of action. Drugs that increase levels of serotonin inhibit floating by promoting horizontal swimming, whereas drugs that increase levels of noradrenaline inhibit floating by enhancing vertical swimming (climbing). We compared the FST behavioural profiles of two different allelic series of HD mice, a fragment model (R6/2 mice carrying 120, 250, or 350 CAG repeats), and a knock-in model (Hdh mice carrying 50, 150, or 250 CAG repeats). The FST behavioural profile was similar in both lines. It was characterized by an early-stage increase in floating, and then, as the mice aged, floating decreased, whereas active behaviours of swimming and climbing increased. Our results show that, as with depression in HD patients, floating in HD mice does not progress linearly, suggesting that, at the late stages of the disease, an increase in serotonergic and noradrenergic activity might contribute to lower floating levels in HD mice. If similar compensatory changes occur in humans, this should be taken into account when considering the treatment of depression in HD patients. Copyright © 2015 Elsevier B.V. All rights reserved.
Enhanced extinction of cocaine seeking in brain-derived neurotrophic factor Val66Met knock-in mice.

PubMed

Briand, Lisa A; Lee, Francis S; Blendy, Julie A; Pierce, R Christopher

2012-03-01

The Val66Met polymorphism in the brain-derived neurotropic factor (BDNF) gene results in alterations in fear extinction behavior in both human populations and mouse models. However, it is not clear whether this polymorphism plays a similar role in extinction of appetitive behaviors. Therefore, we examined operant learning and extinction of both food and cocaine self-administration behavior in an inbred genetic knock-in mouse strain expressing the variant Bdnf. These mice provide a unique opportunity to relate alterations in aversive and appetitive extinction learning as well as provide insight into how human genetic variation can lead to differences in behavior. BDNF(Met/Met) mice exhibited a severe deficit in operant learning as demonstrated by an inability to learn the food self-administration task. Therefore, extinction experiments were performed comparing wildtype (BDNF(Val/Val) ) animals to mice heterozygous for the Met allele (BDNF(Val/Met) ), which did not differ in food or cocaine self-administration behavior. In contrast to the deficit in fear extinction previously demonstrated in these mice, we found that BDNF(Val/Met) mice exhibited more rapid extinction of cocaine responding compared to wildtype mice. No differences were found between the genotypes in the extinction of food self-administration behavior or the reinstatement of cocaine seeking, indicating that the effect is specific to extinction of cocaine responding. These results suggest that the molecular mechanisms underlying aversive and appetitive extinction are distinct from one another and BDNF may play opposing roles in the two phenomena. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.
Functional Analysis of Dopaminergic Systems in a DYT1 Knock-in Mouse Model of Dystonia

PubMed Central

Song, Chang-Hyun; Fan, Xueliang; Exeter, Cicely J.; Hess, Ellen J.; Jinnah, H. A.

2012-01-01

The dystonias are a group of disorders characterized by involuntary twisting movements and abnormal posturing. The most common of the inherited dystonias is DYT1 dystonia, which is due to deletion of a single GAG codon (ΔE) in the TOR1A gene that encodes torsinA. Since some forms of dystonia have been linked with dysfunction of brain dopamine pathways, the integrity of these pathways was explored in a knock-in mouse model of DYT1 dystonia. In DYT1(ΔE) knock-in mice, neurochemical measures revealed only small changes in the content of dopamine or its metabolites in tissue homogenates from caudoputamen or midbrain, but microdialysis studies revealed robust decreases in baseline and amphetamine-stimulated extracellular dopamine in the caudoputamen. Quantitative stereological methods revealed no evidence for striatal or midbrain atrophy, but substantia nigra neurons immunopositive for tyrosine hydroxylase were slightly reduced in numbers and enlarged in size. Behavioral studies revealed subtle abnormalities in gross motor activity and motor coordination without overt dystonia. Neuropharmacological challenges of dopamine systems revealed normal behavioral responses to amphetamine and a minor increase in sensitivity to haloperidol. These results demonstrate that this DYT1(ΔE) knock-in mouse model of dystonia harbors neurochemical and structural changes of the dopamine pathways, as well as motor abnormalities. PMID:22659308
Accelerated leukemogenesis by truncated CBFβ-SMMHC defective in high-affinity binding with RUNX1

PubMed Central

Kamikubo, Yasuhiko; Zhao, Ling; Wunderlich, Mark; Corpora, Takeshi; Hyde, R. Katherine; Paul, Thomas A.; Kundu, Mondira; Garrett, Lisa; Compton, Sheila; Huang, Gang; Wolff, Linda; Ito, Yoshiaki; Bushweller, John; Mulloy, James C.; Liu, P. Paul

2010-01-01

SUMMARY Dominant RUNX1 inhibition has been proposed as a common pathway for CBF-leukemia. CBFβ-SMMHC, a fusion protein in human acute myeloid leukemia (AML), dominantly inhibits RUNX1 largely through its RUNX1 high-affinity binding domain (HABD). However, the type I CBFβ-SMMHC fusion in AML patients lacks HABD. Here we report that the type I CBFβ-SMMHC protein binds RUNX1 inefficiently. Knock-in mice expressing CBFβ-SMMHC with a HABD deletion developed leukemia quickly, even though hematopoietic defects associated with Runx1-inhibition were partially rescued. A larger pool of leukemia initiating cells, increased MN1 expression, and retention of RUNX1 phosphorylation are potential mechanisms for accelerated leukemia development in these mice. Our data suggest that RUNX1 dominant inhibition may not be a critical step for leukemogenesis by CBFβ-SMMHC. PMID:20478528
Pathomechanisms of Dopamine Dysregulation in DYT1 Dystonia: Targets for Therapeutics

DTIC Science & Technology

2016-10-01

DA release in DYT1(ΔE) knockin mice by assessing VMAT2 function, vesicle utilization, the ultrastructure of DA terminals, and D2 DA...in slice, the ultrastructure of DA terminals, D2 DA autoreceptor function nicotinic AChR (nAChR) heteroreceptors function. 2) To determine the
Mass Spectrometry Analysis of Wild-Type and Knock-in Q140/Q140 Huntington's Disease Mouse Brains Reveals Changes in Glycerophospholipids Including Alterations in Phosphatidic Acid and Lyso-Phosphatidic Acid.

PubMed

Vodicka, Petr; Mo, Shunyan; Tousley, Adelaide; Green, Karin M; Sapp, Ellen; Iuliano, Maria; Sadri-Vakili, Ghazaleh; Shaffer, Scott A; Aronin, Neil; DiFiglia, Marian; Kegel-Gleason, Kimberly B

2015-01-01

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG expansion in the HD gene, which encodes the protein Huntingtin. Huntingtin associates with membranes and can interact directly with glycerophospholipids in membranes. We analyzed glycerophospholipid profiles from brains of 11 month old wild-type (WT) and Q140/Q140 HD knock-in mice to assess potential changes in glycerophospholipid metabolism. Polar lipids from cerebellum, cortex, and striatum were extracted and analyzed by liquid chromatography and negative ion electrospray tandem mass spectrometry analysis (LC-MS/MS). Gene products involved in polar lipid metabolism were studied using western blotting, immuno-electron microscopy and qPCR. Significant changes in numerous species of glycerophosphate (phosphatidic acid, PA) were found in striatum, cerebellum and cortex from Q140/Q140 HD mice compared to WT mice at 11 months. Changes in specific species could also be detected for other glycerophospholipids. Increases in species of lyso-PA (LPA) were measured in striatum of Q140/Q140 HD mice compared to WT. Protein levels for c-terminal binding protein 1 (CtBP1), a regulator of PA biosynthesis, were reduced in striatal synaptosomes from HD mice compared to wild-type at 6 and 12 months. Immunoreactivity for CtBP1 was detected on membranes of synaptic vesicles in striatal axon terminals in the globus pallidus. These novel results identify a potential site of molecular pathology caused by mutant Huntingtin that may impart early changes in HD.
Glutamate receptor 1 phosphorylation at serine 831 and 845 modulates seizure susceptibility and hippocampal hyperexcitability after early life seizures.

PubMed

Rakhade, Sanjay N; Fitzgerald, Erin F; Klein, Peter M; Zhou, Chengwen; Sun, Hongyu; Huganir, Richard L; Hunganir, Richard L; Jensen, Frances E

2012-12-05

Neonatal seizures can lead to later life epilepsy and neurobehavioral deficits, and there are no treatments to prevent these sequelae. We showed previously that hypoxia-induced seizures in a neonatal rat model induce rapid phosphorylation of serine-831 (S831) and Serine 845 (S845) sites of the AMPA receptor GluR1 subunit and later neuronal hyperexcitability and epilepsy, suggesting that seizure-induced posttranslational modifications may represent a novel therapeutic target. To unambiguously assess the contribution of these sites, we examined seizure susceptibility in wild-type mice versus transgenic knock-in mice with deficits in GluR1 S831 and S845 phosphorylation [GluR1 double-phosphomutant (GluR1 DPM) mice]. Phosphorylation of the GluR1 S831 and S845 sites was significantly increased in the hippocampus and cortex after a single episode of pentyleneterazol-induced seizures in postnatal day 7 (P7) wild-type mouse pups and that transgenic knock-in mice have a higher threshold and longer latencies to seizures. Like the rat, hypoxic seizures in P9 C57BL/6N wild-type mice resulted in transient increases in GluR1 S831 and GluR1 S845 phosphorylation in cortex and were associated with enhanced seizure susceptibility to later-life kainic-acid-induced seizures. In contrast, later-life seizure susceptibility after hypoxia-induced seizures was attenuated in GluR1 DPM mice, supporting a role for posttranslational modifications in seizure-induced network excitability. Finally, human hippocampal samples from neonatal seizure autopsy cases also showed an increase in GluR1 S831 and S845, supporting the validation of this potential therapeutic target in human tissue.
Glutamate receptor 1 phosphorylation at Serine 831 and 845 modulates seizure susceptibility and hippocampal hyperexcitability following early life seizures

PubMed Central

Rakhade, S.N.; Fitzgerald, E.F.; Klein, P.M.; Zhou, C.; Sun, H; Huganir, R.L.; Jensen, F.E.

2012-01-01

Neonatal seizures can lead to later life epilepsy and neurobehavioral deficits, and there are no treatments to prevent these sequelae. We previously showed that hypoxia-induced seizures in a neonatal rat model induce rapid phosphorylation of S831 and S845 sites of the AMPA receptor GluR1 subunit and later neuronal hyperexcitability and epilepsy, suggesting that seizure-induced post-translational modifications may represent a novel therapeutic target. To unambiguously assess the contribution of these sites, we examined seizure susceptibility in wild type mice versus transgenic knock-in mice with deficits in GluR1 S831 and S845 phosphorylation (GluR1 double phosphomutant (GluR1DPM) mice). Phosphorylation of the GluR1 S831 and S845 sites was significantly increased in the hippocampus and cortex following a single episode of pentyleneterazol (PTZ) induced seizures in postnatal day 9 (P9) wild type mouse pups, and that transgenic knock-in mice have a higher threshold and longer latencies to seizures. Like the rat, hypoxic seizures in P9 C57BL/6N wild type mice resulted in transient increases in GluR1 S831 and GluR1 S845 phosphorylation in cortex, and were associated with enhanced seizure susceptibility to later-life kainic acid induced seizures. In contrast, later-life seizure susceptibility following hypoxia-induced seizures was attenuated in GluR1 DPM mice, supporting a role for post-translational modifications in seizure-induced network excitability. Finally, human hippocampal samples from neonatal seizure autopsy cases also showed an increase in GluR1 S831 and S845, supporting the validation of this potential therapeutic target in human tissue. PMID:23223299
Mechanisms of anaphylaxis in human low-affinity IgG receptor locus knock-in mice.

PubMed

Gillis, Caitlin M; Jönsson, Friederike; Mancardi, David A; Tu, Naxin; Beutier, Héloïse; Van Rooijen, Nico; Macdonald, Lynn E; Murphy, Andrew J; Bruhns, Pierre

2017-04-01

Anaphylaxis can proceed through distinct IgE- or IgG-dependent pathways, which have been investigated in various mouse models. We developed a novel mouse strain in which the human low-affinity IgG receptor locus, comprising both activating (hFcγRIIA, hFcγRIIIA, and hFcγRIIIB) and inhibitory (hFcγRIIB) hFcγR genes, has been inserted into the equivalent murine locus, corresponding to a locus swap. We sought to determine the capabilities of hFcγRs to induce systemic anaphylaxis and identify the cell types and mediators involved. hFcγR expression on mouse and human cells was compared to validate the model. Passive systemic anaphylaxis was induced by injection of heat-aggregated human intravenous immunoglobulin and active systemic anaphylaxis after immunization and challenge. Anaphylaxis severity was evaluated based on hypothermia and mortality. The contribution of receptors, mediators, or cell types was assessed based on receptor blockade or depletion. The human-to-mouse low-affinity FcγR locus swap engendered hFcγRIIA/IIB/IIIA/IIIB expression in mice comparable with that seen in human subjects. Knock-in mice were susceptible to passive and active anaphylaxis, accompanied by downregulation of both activating and inhibitory hFcγR expression on specific myeloid cells. The contribution of hFcγRIIA was predominant. Depletion of neutrophils protected against hypothermia and mortality. Basophils contributed to a lesser extent. Anaphylaxis was inhibited by platelet-activating factor receptor or histamine receptor 1 blockade. Low-affinity FcγR locus-switched mice represent an unprecedented model of cognate hFcγR expression. Importantly, IgG-related anaphylaxis proceeds within a native context of activating and inhibitory hFcγRs, indicating that, despite robust hFcγRIIB expression, activating signals can dominate to initiate a severe anaphylactic reaction. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Modulation of NCC activity by low and high K(+) intake: insights into the signaling pathways involved.

PubMed

Castañeda-Bueno, María; Cervantes-Perez, Luz Graciela; Rojas-Vega, Lorena; Arroyo-Garza, Isidora; Vázquez, Norma; Moreno, Erika; Gamba, Gerardo

2014-06-15

Modulation of Na(+)-Cl(-) cotransporter (NCC) activity is essential to adjust K(+) excretion in the face of changes in dietary K(+) intake. We used previously characterized genetic mouse models to assess the role of Ste20-related proline-alanine-rich kinase (SPAK) and with-no-lysine kinase (WNK)4 in the modulation of NCC by K(+) diets. SPAK knockin and WNK4 knockout mice were placed on normal-, low-, or high-K(+)-citrate diets for 4 days. The low-K(+) diet decreased and high-K(+) diet increased plasma aldosterone levels, but both diets were associated with increased phosphorylation of NCC (phospho-NCC, Thr(44)/Thr(48)/Thr(53)) and phosphorylation of SPAK/oxidative stress responsive kinase 1 (phospho-SPAK/OSR1, Ser(383)/Ser(325)). The effect of the low-K(+) diet on SPAK phosphorylation persisted in WNK4 knockout and SPAK knockin mice, whereas the effects of ANG II on NCC and SPAK were lost in both mouse colonies. This suggests that for NCC activation by ANG II, integrity of the WNK4/SPAK pathway is required, whereas for the low-K(+) diet, SPAK phosphorylation occurred despite the absence of WNK4, suggesting the involvement of another WNK (WNK1 or WNK3). Additionally, because NCC activation also occurred in SPAK knockin mice, it is possible that loss of SPAK was compensated by OSR1. The positive effect of the high-K(+) diet was observed when the accompanying anion was citrate, whereas the high-KCl diet reduced NCC phosphorylation. However, the effect of the high-K(+)-citrate diet was aldosterone dependent, and neither metabolic alkalosis induced by bicarbonate, nor citrate administration in the absence of K(+) increased NCC phosphorylation, suggesting that it was not due to citrate-induced metabolic alkalosis. Thus, the accompanying anion might modulate the NCC response to the high-K(+) diet. Copyright © 2014 the American Physiological Society.
Modulation of NCC activity by low and high K+ intake: insights into the signaling pathways involved

PubMed Central

Castañeda-Bueno, María; Cervantes-Perez, Luz Graciela; Rojas-Vega, Lorena; Arroyo-Garza, Isidora; Vázquez, Norma; Moreno, Erika

2014-01-01

Modulation of Na+-Cl− cotransporter (NCC) activity is essential to adjust K+ excretion in the face of changes in dietary K+ intake. We used previously characterized genetic mouse models to assess the role of Ste20-related proline-alanine-rich kinase (SPAK) and with-no-lysine kinase (WNK)4 in the modulation of NCC by K+ diets. SPAK knockin and WNK4 knockout mice were placed on normal-, low-, or high-K+-citrate diets for 4 days. The low-K+ diet decreased and high-K+ diet increased plasma aldosterone levels, but both diets were associated with increased phosphorylation of NCC (phospho-NCC, Thr44/Thr48/Thr53) and phosphorylation of SPAK/oxidative stress responsive kinase 1 (phospho-SPAK/OSR1, Ser383/Ser325). The effect of the low-K+ diet on SPAK phosphorylation persisted in WNK4 knockout and SPAK knockin mice, whereas the effects of ANG II on NCC and SPAK were lost in both mouse colonies. This suggests that for NCC activation by ANG II, integrity of the WNK4/SPAK pathway is required, whereas for the low-K+ diet, SPAK phosphorylation occurred despite the absence of WNK4, suggesting the involvement of another WNK (WNK1 or WNK3). Additionally, because NCC activation also occurred in SPAK knockin mice, it is possible that loss of SPAK was compensated by OSR1. The positive effect of the high-K+ diet was observed when the accompanying anion was citrate, whereas the high-KCl diet reduced NCC phosphorylation. However, the effect of the high-K+-citrate diet was aldosterone dependent, and neither metabolic alkalosis induced by bicarbonate, nor citrate administration in the absence of K+ increased NCC phosphorylation, suggesting that it was not due to citrate-induced metabolic alkalosis. Thus, the accompanying anion might modulate the NCC response to the high-K+ diet. PMID:24761002
Characterization of neurophysiological and behavioral changes, MRI brain volumetry and 1H MRS in zQ175 knock-in mouse model of Huntington's disease.

PubMed

Heikkinen, Taneli; Lehtimäki, Kimmo; Vartiainen, Nina; Puoliväli, Jukka; Hendricks, Susan J; Glaser, Jack R; Bradaia, Amyaouch; Wadel, Kristian; Touller, Chrystelle; Kontkanen, Outi; Yrjänheikki, Juha M; Buisson, Bruno; Howland, David; Beaumont, Vahri; Munoz-Sanjuan, Ignacio; Park, Larry C

2012-01-01

Huntington's disease (HD) is an autosomal neurodegenerative disorder, characterized by severe behavioral, cognitive, and motor deficits. Since the discovery of the huntingtin gene (HTT) mutation that causes the disease, several mouse lines have been developed using different gene constructs of Htt. Recently, a new model, the zQ175 knock-in (KI) mouse, was developed (see description by Menalled et al, [1]) in an attempt to have the Htt gene in a context and causing a phenotype that more closely mimics HD in humans. Here we confirm the behavioral phenotypes reported by Menalled et al [1], and extend the characterization to include brain volumetry, striatal metabolite concentration, and early neurophysiological changes. The overall reproducibility of the behavioral phenotype across the two independent laboratories demonstrates the utility of this new model. Further, important features reminiscent of human HD pathology are observed in zQ175 mice: compared to wild-type neurons, electrophysiological recordings from acute brain slices reveal that medium spiny neurons from zQ175 mice display a progressive hyperexcitability; glutamatergic transmission in the striatum is severely attenuated; decreased striatal and cortical volumes from 3 and 4 months of age in homo- and heterozygous mice, respectively, with whole brain volumes only decreased in homozygotes. MR spectroscopy reveals decreased concentrations of N-acetylaspartate and increased concentrations of glutamine, taurine and creatine + phosphocreatine in the striatum of 12-month old homozygotes, the latter also measured in 12-month-old heterozygotes. Motor, behavioral, and cognitive deficits in homozygotes occur concurrently with the structural and metabolic changes observed. In sum, the zQ175 KI model has robust behavioral, electrophysiological, and histopathological features that may be valuable in both furthering our understanding of HD-like pathophyisology and the evaluation of potential therapeutic strategies to slow the progression of disease.
Critical role of the SPAK protein kinase CCT domain in controlling blood pressure

PubMed Central

Zhang, Jinwei; Siew, Keith; Macartney, Thomas; O'Shaughnessy, Kevin M.; Alessi, Dario R.

2015-01-01

The STE20/SPS1-related proline/alanine-rich kinase (SPAK) controls blood pressure (BP) by phosphorylating and stimulating the Na-Cl (NCC) and Na-K-2Cl (NKCC2) co-transporters, which regulate salt reabsorption in the kidney. SPAK possesses a conserved carboxy-terminal (CCT) domain, which recognises RFXV/I motifs present in its upstream activator [isoforms of the With-No-lysine (K) kinases (WNKs)] as well as its substrates (NCC and NKCC2). To define the physiological importance of the CCT domain, we generated knock-in mice in which the critical CCT domain Leu502 residue required for high affinity recognition of the RFXI/V motif was mutated to Alanine. The SPAK CCT domain defective knock-in animals are viable, and the Leu502Ala mutation abolished co-immunoprecipitation of SPAK with WNK1, NCC and NKCC2. The CCT domain defective animals displayed markedly reduced SPAK activity and phosphorylation of NCC and NKCC2 co-transporters at the residues phosphorylated by SPAK. This was also accompanied by a reduction in the expression of NCC and NKCC2 protein without changes in mRNA levels. The SPAK CCT domain knock-in mice showed typical features of Gitelman Syndrome with mild hypokalaemia, hypomagnesaemia, hypocalciuria and displayed salt wasting on switching to a low-Na diet. These observations establish that the CCT domain plays a crucial role in controlling SPAK activity and BP. Our results indicate that CCT domain inhibitors would be effective at reducing BP by lowering phosphorylation as well as expression of NCC and NKCC2. PMID:25994507
Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models

PubMed Central

Cebrian-Serrano, Alberto; Zha, Shijun; Hanssen, Lars; Biggs, Daniel; Preece, Christopher

2017-01-01

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply. PMID:28081254
Prestin-based outer hair cell electromotility in knockin mice does not appear to adjust the operating point of a cilia-based amplifier

PubMed Central

Gao, Jiangang; Wang, Xiang; Wu, Xudong; Aguinaga, Sal; Huynh, Kristin; Jia, Shuping; Matsuda, Keiji; Patel, Manish; Zheng, Jing; Cheatham, MaryAnn; He, David Z.; Dallos, Peter; Zuo, Jian

2007-01-01

The remarkable sensitivity and frequency selectivity of the mammalian cochlea is attributed to a unique amplification process that resides in outer hair cells (OHCs). Although the mammalian-specific somatic motility is considered a substrate of cochlear amplification, it has also been proposed that somatic motility in mammals simply acts as an operating-point adjustment for the ubiquitous stereocilia-based amplifier. To address this issue, we created a mouse model in which a mutation (C1) was introduced into the OHC motor protein prestin, based on previous results in transfected cells. In C1/C1 knockin mice, localization of C1-prestin, as well as the length and number of OHCs, were all normal. In OHCs isolated from C1/C1 mice, nonlinear capacitance and somatic motility were both shifted toward hyperpolarization, so that, compared with WT controls, the amplitude of cycle-by-cycle (alternating, or AC) somatic motility remained the same, but the unidirectional (DC) component reversed polarity near the OHC's presumed in vivo resting membrane potential. No physiological defects in cochlear sensitivity or frequency selectivity were detected in C1/C1 or C1/+ mice. Hence, our results do not support the idea that OHC somatic motility adjusts the operating point of a stereocilia-based amplifier. However, they are consistent with the notion that the AC component of OHC somatic motility plays a dominant role in mammalian cochlear amplification. PMID:17640919
Reduced TCR signaling potential impairs negative selection but does not result in autoimmune disease

PubMed Central

Hwang, SuJin; Song, Ki-Duk; Lesourne, Renaud; Lee, Jan; Pinkhasov, Julia; Li, LiQi; El-Khoury, Dalal

2012-01-01

Negative selection and regulatory T (T reg) cell development are two thymus-dependent processes necessary for the enforcement of self-tolerance, and both require high-affinity interactions between the T cell receptor (TCR) and self-ligands. However, it remains unclear if they are similarly impacted by alterations in TCR signaling potential. We generated a knock-in allele (6F) of the TCR ζ chain gene encoding a mutant protein lacking signaling capability whose expression is controlled by endogenous ζ regulatory sequences. Although negative selection was defective in 6F/6F mice, leading to the survival of autoreactive T cells, 6F/6F mice did not develop autoimmune disease. We found that 6F/6F mice generated increased numbers of thymus-derived T reg cells. We show that attenuation of TCR signaling potential selectively impacts downstream signaling responses and that this differential effect favors Foxp3 expression and T reg cell lineage commitment. These results identify a potential compensatory pathway for the enforcement of immune tolerance in response to defective negative selection caused by reduced TCR signaling capability. PMID:22945921

Expression of Wild-Type Rp1 Protein in Rp1 Knock-in Mice Rescues the Retinal Degeneration Phenotype

PubMed Central

Liu, Qin; Collin, Rob W. J.; Cremers, Frans P. M.; den Hollander, Anneke I.; van den Born, L. Ingeborgh; Pierce, Eric A.

2012-01-01

Mutations in the retinitis pigmentosa 1 (RP1) gene are a common cause of autosomal dominant retinitis pigmentosa (adRP), and have also been found to cause autosomal recessive RP (arRP) in a few families. The 33 dominant mutations and 6 recessive RP1 mutations identified to date are all nonsense or frameshift mutations, and almost exclusively (38 out of 39) are located in the 4th and final exon of RP1. To better understand the underlying disease mechanisms of and help develop therapeutic strategies for RP1 disease, we performed a series of human genetic and animal studies using gene targeted and transgenic mice. Here we report that a frameshift mutation in the 3rd exon of RP1 (c.686delC; p.P229QfsX35) found in a patient with recessive RP1 disease causes RP in the homozygous state, whereas the heterozygous carriers are unaffected, confirming that haploinsufficiency is not the causative mechanism for RP1 disease. We then generated Rp1 knock-in mice with a nonsense Q662X mutation in exon 4, as well as Rp1 transgenic mice carrying a wild-type BAC Rp1 transgene. The Rp1-Q662X allele produces a truncated Rp1 protein, and homozygous Rp1-Q662X mice experience a progressive photoreceptor degeneration characterized disorganization of photoreceptor outer segments. This phenotype could be prevented by expression of a normal amount of Rp1 protein from the BAC transgene without removal of the mutant Rp1-Q662X protein. Over-expression of Rp1 protein in additional BAC Rp1 transgenic lines resulted in retinal degeneration. These findings suggest that the truncated Rp1-Q662X protein does not exert a toxic gain-of-function effect. These results also imply that in principle gene augmentation therapy could be beneficial for both recessive and dominant RP1 patients, but the levels of RP1 protein delivered for therapy will have to be carefully controlled. PMID:22927954
Establishment of Nephrin Reporter Mice and Use for Chemical Screening

PubMed Central

Tsuchida, Junichi; Matsusaka, Taiji; Ohtsuka, Masato; Miura, Hiromi; Okuno, Yukiko; Asanuma, Katsuhiko; Nakagawa, Takahiko; Yanagita, Motoko

2016-01-01

Nephrin is a critical component of glomerular filtration barrier, which is important to maintain glomerular structure and avoid proteinuria. Downregulation of nephrin expression is commonly observed at early stage of glomerular disorders, suggesting that methods to increase nephrin expression in podocytes may have therapeutic utility. Here, we generated a knockin mouse line carrying single copy of 5.5 kb nephrin promoter controlling expression of enhanced green fluorescent protein (EGFP) at Rosa26 genomic locus (Nephrin-EGFP mouse). In these mice, EGFP was specifically expressed in podocytes. Next, we isolated and cultivated glomeruli from these mice, and developed a protocol to automatically quantitate EGFP expression in cultured glomeruli. EGFP signal was markedly reduced after 5 days of culture but reduction was inhibited by vitamin D treatment. We confirmed that vitamin D increased mRNA and protein expression of endogenous nephrin in cultivated glomeruli. Thus, we generated a mouse line converting nephrin promoter activity into fluorescence, which can be used to screen compounds having activity to enhance nephrin gene expression. PMID:27362433
Autophagy Differentially Regulates Insulin Production and Insulin Sensitivity.

PubMed

Yamamoto, Soh; Kuramoto, Kenta; Wang, Nan; Situ, Xiaolei; Priyadarshini, Medha; Zhang, Weiran; Cordoba-Chacon, Jose; Layden, Brian T; He, Congcong

2018-06-12

Autophagy, a stress-induced lysosomal degradative pathway, has been assumed to exert similar metabolic effects in different organs. Here, we establish a model where autophagy plays different roles in insulin-producing β cells versus insulin-responsive cells, utilizing knockin (Becn1 F121A ) mice manifesting constitutively active autophagy. With a high-fat-diet challenge, the autophagy-hyperactive mice unexpectedly show impaired glucose tolerance, but improved insulin sensitivity, compared to mice with normal autophagy. Autophagy hyperactivation enhances insulin signaling, via suppressing ER stress in insulin-responsive cells, but decreases insulin secretion by selectively sequestrating and degrading insulin granule vesicles in β cells, a process we term "vesicophagy." The reduction in insulin storage, insulin secretion, and glucose tolerance is reversed by transient treatment of autophagy inhibitors. Thus, β cells and insulin-responsive tissues require different autophagy levels for optimal function. To improve insulin sensitivity without hampering secretion, acute or intermittent, rather than chronic, activation of autophagy should be considered in diabetic therapy development. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance.

PubMed

van Oers, Johanna M M; Roa, Sergio; Werling, Uwe; Liu, Yiyong; Genschel, Jochen; Hou, Harry; Sellers, Rani S; Modrich, Paul; Scharff, Matthew D; Edelmann, Winfried

2010-07-27

The DNA mismatch repair protein PMS2 was recently found to encode a novel endonuclease activity. To determine the biological functions of this activity in mammals, we generated endonuclease-deficient Pms2E702K knock-in mice. Pms2EK/EK mice displayed increased genomic mutation rates and a strong cancer predisposition. In addition, class switch recombination, but not somatic hypermutation, was impaired in Pms2EK/EK B cells, indicating a specific role in Ig diversity. In contrast to Pms2-/- mice, Pms2EK/EK male mice were fertile, indicating that this activity is dispensable in spermatogenesis. Therefore, the PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance and tumor suppression.
Development of a new knock-in mouse model and evaluation of pharmacological activities of lusutrombopag, a novel, nonpeptidyl small-molecule agonist of the human thrombopoietin receptor c-Mpl.

PubMed

Yoshida, Hiroshi; Yamada, Hajime; Nogami, Wataru; Dohi, Keiji; Kurino-Yamada, Tomomi; Sugiyama, Koji; Takahashi, Koji; Gahara, Yoshinari; Kitaura, Motoji; Hasegawa, Minoru; Oshima, Itsuki; Kuwabara, Kenji

2018-03-01

Lusutrombopag (S-888711), an oral small-molecule thrombopoietin receptor (TPOR) agonist, has gained first approval as a drug to treat thrombocytopenia of chronic liver disease in patients undergoing elective invasive procedures in Japan. Preclinical studies were performed to evaluate its efficacy against megakaryopoiesis and thrombopoiesis. To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production. Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.
Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa.

PubMed

Arno, Gavin; Agrawal, Smriti A; Eblimit, Aiden; Bellingham, James; Xu, Mingchu; Wang, Feng; Chakarova, Christina; Parfitt, David A; Lane, Amelia; Burgoyne, Thomas; Hull, Sarah; Carss, Keren J; Fiorentino, Alessia; Hayes, Matthew J; Munro, Peter M; Nicols, Ralph; Pontikos, Nikolas; Holder, Graham E; Asomugha, Chinwe; Raymond, F Lucy; Moore, Anthony T; Plagnol, Vincent; Michaelides, Michel; Hardcastle, Alison J; Li, Yumei; Cukras, Catherine; Webster, Andrew R; Cheetham, Michael E; Chen, Rui

2016-12-01

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy. Copyright Â© 2016 The Authors. Published by Elsevier Inc. All rights reserved.
Control of Mycobacterial Infections in Mice Expressing Human Tumor Necrosis Factor (TNF) but Not Mouse TNF.

PubMed

Olleros, Maria L; Chavez-Galan, Leslie; Segueni, Noria; Bourigault, Marie L; Vesin, Dominique; Kruglov, Andrey A; Drutskaya, Marina S; Bisig, Ruth; Ehlers, Stefan; Aly, Sahar; Walter, Kerstin; Kuprash, Dmitry V; Chouchkova, Miliana; Kozlov, Sergei V; Erard, François; Ryffel, Bernard; Quesniaux, Valérie F J; Nedospasov, Sergei A; Garcia, Irene

2015-09-01

Tumor necrosis factor (TNF) is an important cytokine for host defense against pathogens but is also associated with the development of human immunopathologies. TNF blockade effectively ameliorates many chronic inflammatory conditions but compromises host immunity to tuberculosis. The search for novel, more specific human TNF blockers requires the development of a reliable animal model. We used a novel mouse model with complete replacement of the mouse TNF gene by its human ortholog (human TNF [huTNF] knock-in [KI] mice) to determine resistance to Mycobacterium bovis BCG and M. tuberculosis infections and to investigate whether TNF inhibitors in clinical use reduce host immunity. Our results show that macrophages from huTNF KI mice responded to BCG and lipopolysaccharide similarly to wild-type macrophages by NF-κB activation and cytokine production. While TNF-deficient mice rapidly succumbed to mycobacterial infection, huTNF KI mice survived, controlling the bacterial burden and activating bactericidal mechanisms. Administration of TNF-neutralizing biologics disrupted the control of mycobacterial infection in huTNF KI mice, leading to an increased bacterial burden and hyperinflammation. Thus, our findings demonstrate that human TNF can functionally replace murine TNF in vivo, providing mycobacterial resistance that could be compromised by TNF neutralization. This new animal model will be helpful for the testing of specific biologics neutralizing human TNF. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Gene targeting and subsequent site-specific transgenesis at the β-actin (ACTB) locus in common marmoset embryonic stem cells.

PubMed

Shiozawa, Seiji; Kawai, Kenji; Okada, Yohei; Tomioka, Ikuo; Maeda, Takuji; Kanda, Akifumi; Shinohara, Haruka; Suemizu, Hiroshi; James Okano, Hirotaka; Sotomaru, Yusuke; Sasaki, Erika; Okano, Hideyuki

2011-09-01

Nonhuman primate embryonic stem (ES) cells have vast promise for preclinical studies. Genetic modification in nonhuman primate ES cells is an essential technique for maximizing the potential of these cells. The common marmoset (Callithrix jacchus), a nonhuman primate, is expected to be a useful transgenic model for preclinical studies. However, genetic modification in common marmoset ES (cmES) cells has not yet been adequately developed. To establish efficient and stable genetic modifications in cmES cells, we inserted the enhanced green fluorescent protein (EGFP) gene with heterotypic lox sites into the β-actin (ACTB) locus of the cmES cells using gene targeting. The resulting knock-in ES cells expressed EGFP ubiquitously under the control of the endogenous ACTB promoter. Using inserted heterotypic lox sites, we demonstrated Cre recombinase-mediated cassette exchange (RMCE) and successfully established a monomeric red fluorescent protein (mRFP) knock-in cmES cell line. Further, a herpes simplex virus-thymidine kinase (HSV-tk) knock-in cmES cell line was established using RMCE. The growth of tumor cells originating from the cell line was significantly suppressed by the administration of ganciclovir. Therefore, the HSV-tk/ganciclovir system is promising as a safeguard for stem cell therapy. The stable and ubiquitous expression of EGFP before RMCE enables cell fate to be tracked when the cells are transplanted into an animal. Moreover, the creation of a transgene acceptor locus for site-specific transgenesis will be a powerful tool, similar to the ROSA26 locus in mice.
Glutamate-system defects behind psychiatric manifestations in a familial hemiplegic migraine type 2 disease-mutation mouse model

PubMed Central

Bøttger, Pernille; Glerup, Simon; Gesslein, Bodil; Illarionova, Nina B.; Isaksen, Toke J.; Heuck, Anders; Clausen, Bettina H.; Füchtbauer, Ernst-Martin; Gramsbergen, Jan B.; Gunnarson, Eli; Aperia, Anita; Lauritzen, Martin; Lambertsen, Kate L.; Nissen, Poul; Lykke-Hartmann, Karin

2016-01-01

Migraine is a complex brain disorder, and understanding the complexity of this prevalent disease could improve quality of life for millions of people. Familial Hemiplegic Migraine type 2 (FHM2) is a subtype of migraine with aura and co-morbidities like epilepsy/seizures, cognitive impairments and psychiatric manifestations, such as obsessive-compulsive disorder (OCD). FHM2 disease-mutations locate to the ATP1A2 gene encoding the astrocyte-located α2-isoform of the sodium-potassium pump (α2Na+/K+-ATPase). We show that knock-in mice heterozygous for the FHM2-associated G301R-mutation (α2+/G301R) phenocopy several FHM2-relevant disease traits e.g., by mimicking mood depression and OCD. In vitro studies showed impaired glutamate uptake in hippocampal mixed astrocyte-neuron cultures from α2G301R/G301R E17 embryonic mice, and moreover, induction of cortical spreading depression (CSD) resulted in reduced recovery in α2+/G301R male mice. Moreover, NMDA-type glutamate receptor antagonists or progestin-only treatment reverted specific α2+/G301R behavioral phenotypes. Our findings demonstrate that studies of an in vivo relevant FHM2 disease knock-in mouse model provide a link between the female sex hormone cycle and the glutamate system and a link to co-morbid psychiatric manifestations of FHM2. PMID:26911348
Deletion of the γ-secretase subunits Aph1B/C impairs memory and worsens the deficits of knock-in mice modeling the Alzheimer-like familial Danish dementia

PubMed Central

Biundo, Fabrizio; Ishiwari, Keita; Del Prete, Dolores; D'Adamio, Luciano

2016-01-01

Mutations in BRI2/ITM2b genes cause Familial British and Danish Dementias (FBD and FDD), which are pathogenically similar to Familial Alzheimer Disease (FAD). BRI2 inhibits processing of Amyloid precursor protein (APP), a protein involved in FAD pathogenesis. Accumulation of a carboxyl-terminal APP metabolite –β-CTF- causes memory deficits in a knock-in mouse model of FDD, called FDDKI. We have investigated further the pathogenic function of β-CTF studying the effect of Aph1B/C deletion on FDDKI mice. This strategy is based on the evidence that deletion of Aph1B/C proteins, which are components of the γ-secretase that cleaves β-CTF, results in stabilization of β-CTF and a reduction of Aβ. We found that both the FDD mutation and the Aph1B/C deficiency mildly interfered with spatial long term memory, spatial working/short-term memory and long-term contextual fear memory. In addition, the Aph1BC deficiency induced deficits in long-term cued fear memory. Moreover, the two mutations have additive adverse effects as they compromise the accuracy of spatial long-term memory and induce spatial memory retention deficits in young mice. Overall, the data are consistent with a role for β-CTF in the genesis of memory deficits. PMID:26942869
Deletion of the γ-secretase subunits Aph1B/C impairs memory and worsens the deficits of knock-in mice modeling the Alzheimer-like familial Danish dementia.

PubMed

Biundo, Fabrizio; Ishiwari, Keita; Del Prete, Dolores; D'Adamio, Luciano

2016-03-15

Mutations in BRI2/ITM2b genes cause Familial British and Danish Dementias (FBD and FDD), which are pathogenically similar to Familial Alzheimer Disease (FAD). BRI2 inhibits processing of Amyloid precursor protein (APP), a protein involved in FAD pathogenesis. Accumulation of a carboxyl-terminal APP metabolite -ß-CTF- causes memory deficits in a knock-in mouse model of FDD, called FDDKI.We have investigated further the pathogenic function of ß-CTF studying the effect of Aph1B/C deletion on FDDKI mice. This strategy is based on the evidence that deletion of Aph1B/C proteins, which are components of the γ-secretase that cleaves ß-CTF, results in stabilization of ß-CTF and a reduction of Aβ. We found that both the FDD mutation and the Aph1B/C deficiency mildly interfered with spatial long term memory, spatial working/short-term memory and long-term contextual fear memory. In addition, the Aph1BC deficiency induced deficits in long-term cued fear memory. Moreover, the two mutations have additive adverse effects as they compromise the accuracy of spatial long-term memory and induce spatial memory retention deficits in young mice. Overall, the data are consistent with a role for β-CTF in the genesis of memory deficits.
RNA Binding Protein CUGBP1 Inhibits Liver Cancer in a Phosphorylation-Dependent Manner.

PubMed

Lewis, Kyle; Valanejad, Leila; Cast, Ashley; Wright, Mary; Wei, Christina; Iakova, Polina; Stock, Lauren; Karns, Rebekah; Timchenko, Lubov; Timchenko, Nikolai

2017-08-15

Despite intensive investigations, mechanisms of liver cancer are not known. Here, we identified an important step of liver cancer, which is the neutralization of tumor suppressor activities of an RNA binding protein, CUGBP1. The translational activity of CUGBP1 is activated by dephosphorylation at Ser302. We generated CUGBP1-S302A knock-in mice and found that the reduction of translational activity of CUGBP1 causes development of a fatty liver phenotype in young S302A mice. Examination of liver cancer in diethylnitrosamine (DEN)-treated CUGBP1-S302A mice showed these mice develop much more severe liver cancer that is associated with elimination of the mutant CUGBP1. Searching for mechanisms of this elimination, we found that the oncoprotein gankyrin (Gank) preferentially binds to and triggers degradation of dephosphorylated CUGBP1 (de-ph-S302-CUGBP1) or S302A mutant CUGBP1. To test the role of Gank in degradation of CUGBP1, we generated mice with liver-specific deletion of Gank. In these mice, the tumor suppressor isoform of CUGBP1 is protected from Gank-mediated degradation. Consistent with reduction of CUGBP1 in animal models, CUGBP1 is reduced in patients with pediatric liver cancer. Thus, this work presents evidence that de-ph-S302-CUGBP1 is a tumor suppressor protein and that the Gank-UPS-mediated reduction of CUGBP1 is a key event in the development of liver cancer. Copyright © 2017 American Society for Microbiology.
The common missense mutation D489N in TRIM32 causing limb girdle muscular dystrophy 2H leads to loss of the mutated protein in knock-in mice resulting in a Trim32-null phenotype.

PubMed

Kudryashova, Elena; Struyk, Arie; Mokhonova, Ekaterina; Cannon, Stephen C; Spencer, Melissa J

2011-10-15

Mutations in tripartite motif protein 32 (TRIM32) are responsible for several hereditary disorders that include limb girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathy (STM) and Bardet Biedl syndrome. Most LGMD2H mutations in TRIM32 are clustered in the NHL β-propeller domain at the C-terminus and are predicted to interfere with homodimerization. To get insight into TRIM32's role in the pathogenesis of LGMD2H and to create an accurate model of disease, we have generated a knock-in mouse (T32KI) carrying the c.1465G > A (p.D489N) mutation in murine Trim32 corresponding to the human LGMD2H/STM pathogenic mutation c.1459G > A (p.D487N). Our data indicate that T32KI mice have both a myopathic and a neurogenic phenotype, very similar to the one described in the Trim32-null mice that we created previously. Analysis of Trim32 gene expression in T32KI mice revealed normal mRNA levels, but a severe reduction in mutant TRIM32 (D489N) at the protein level. Our results suggest that the D489N pathogenic mutation destabilizes the protein, leading to its degradation, and results in the same mild myopathic and neurogenic phenotype as that found in Trim32-null mice. Thus, one potential mechanism of LGMD2H might be destabilization of mutated TRIM32 protein leading to a null phenotype.
Impaired activity-dependent FMRP translation and enhanced mGluR-dependent LTD in Fragile X premutation mice

PubMed Central

Iliff, Adam J.; Renoux, Abigail J.; Krans, Amy; Usdin, Karen; Sutton, Michael A.; Todd, Peter K.

2013-01-01

Fragile X premutation-associated disorders, including Fragile X-associated Tremor Ataxia Syndrome, result from unmethylated CGG repeat expansions in the 5′ untranslated region (UTR) of the FMR1 gene. Premutation-sized repeats increase FMR1 transcription but impair rapid translation of the Fragile X mental retardation protein (FMRP), which is absent in Fragile X Syndrome (FXS). Normally, FMRP binds to RNA and regulates metabotropic glutamate receptor (mGluR)-mediated synaptic translation, allowing for dendritic synthesis of several proteins. FMRP itself is also synthesized at synapses in response to mGluR activation. However, the role of activity-dependent translation of FMRP in synaptic plasticity and Fragile X-premutation-associated disorders is unknown. To investigate this question, we utilized a CGG knock-in mouse model of the Fragile X premutation with 120–150 CGG repeats in the mouse Fmr1 5′ UTR. These mice exhibit increased Fmr1 mRNA production but impaired FMRP translational efficiency, leading to a modest reduction in basal FMRP expression. Cultured hippocampal neurons and synaptoneurosomes derived from CGG KI mice demonstrate impaired FMRP translation in response to the group I mGluR agonist 3,5-dihydroxyphenylglycine. Electrophysiological analysis reveals enhanced mGluR-mediated long-term depression (mGluR-LTD) at CA3–CA1 synapses in acute hippocampal slices prepared from CGG KI mice relative to wild-type littermates, similar to Fmr1 knockout mice. However, unlike mGluR-LTD in mice completely lacking FMRP, mGluR-LTD in CGG knock-in mice remains dependent on new protein synthesis. These studies demonstrate partially overlapping synaptic plasticity phenotypes in mouse models of FXS and Fragile X premutation disorders and support a role for activity-dependent synthesis of FMRP in enduring forms of synaptic plasticity. PMID:23250915
Insights into the pathogenesis of dominant retinitis pigmentosa associated with a D477G mutation in RPE65.

PubMed

Choi, Elliot H; Suh, Susie; Sander, Christopher L; Hernandez, Christian J Ortiz; Bulman, Elizabeth R; Khadka, Nimesh; Dong, Zhiqian; Shi, Wuxian; Palczewski, Krzysztof; Kiser, Philip D

2018-04-12

RPE65 is the essential trans-cis isomerase of the classical retinoid (visual) cycle. Mutations in RPE65 give rise to severe retinal dystrophies, most of which are associated with loss of protein function and recessive inheritance. The only known exception is a c.1430G>A (D477G) mutation that gives rise to dominant retinitis pigmentosa with delayed onset and choroidal and macular involvement. Position 477 is distant from functionally critical regions of RPE65. Hence, the mechanism of D477G pathogenicity remains unclear, although protein misfolding and aggregation mechanisms have been suggested. We characterized a D477G knock-in mouse model which exhibited mild age-dependent changes in retinal structure and function. Immunoblot analysis of protein extracts from the eyes of the knock-in mice demonstrated the presence of ubiquitinated RPE65 and reduced RPE65 expression. We observed an accumulation of retinyl esters in the knock-in mice as well as a delay in rhodopsin regeneration kinetics and diminished electroretinography responses, indicative of RPE65 functional impairment induced by the D477G mutation in vivo. However, a cell line expressing D477G RPE65 revealed protein expression levels, cellular localization, and retinoid isomerase activity comparable to cells expressing wild-type protein. Structural analysis of an RPE65 chimera suggested that the D477G mutation does not perturb protein folding or tertiary structure. Instead, the mutation generates an aggregation-prone surface that could induce cellular toxicity through abnormal complex formation as suggested by crystal packing analysis. These results indicate that a toxic gain-of-function induced by the D477G RPE65 substitution may play a role in the pathogenesis of this form of dominant retinitis pigmentosa.
HttQ111/+ Huntington's Disease Knock-in Mice Exhibit Brain Region-Specific Morphological Changes and Synaptic Dysfunction.

PubMed

Kovalenko, Marina; Milnerwood, Austen; Giordano, James; St Claire, Jason; Guide, Jolene R; Stromberg, Mary; Gillis, Tammy; Sapp, Ellen; DiFiglia, Marian; MacDonald, Marcy E; Carroll, Jeffrey B; Lee, Jong-Min; Tappan, Susan; Raymond, Lynn; Wheeler, Vanessa C

2018-01-01

Successful disease-modifying therapy for Huntington's disease (HD) will require therapeutic intervention early in the pathogenic process. Achieving this goal requires identifying phenotypes that are proximal to the HTT CAG repeat expansion. To use Htt CAG knock-in mice, precise genetic replicas of the HTT mutation in patients, as models to study proximal disease events. Using cohorts of B6J.HttQ111/+ mice from 2 to 18 months of age, we analyzed pathological markers, including immunohistochemistry, brain regional volumes and cortical thickness, CAG instability, electron microscopy of striatal synapses, and acute slice electrophysiology to record glutamatergic transmission at striatal synapses. We also incorporated a diet perturbation paradigm for some of these analyses. B6J.HttQ111/+ mice did not exhibit significant neurodegeneration or gliosis but revealed decreased striatal DARPP-32 as well as subtle but regional-specific changes in brain volumes and cortical thickness that parallel those in HD patients. Ultrastructural analyses of the striatum showed reduced synapse density, increased postsynaptic density thickness and increased synaptic cleft width. Acute slice electrophysiology showed alterations in spontaneous AMPA receptor-mediated postsynaptic currents, evoked NMDA receptor-mediated excitatory postsynaptic currents, and elevated extrasynaptic NMDA currents. Diet influenced cortical thickness, but did not impact somatic CAG expansion, nor did it show any significant interaction with genotype on immunohistochemical, brain volume or cortical thickness measures. These data show that a single HttQ111 allele is sufficient to elicit brain region-specific morphological changes and early neuronal dysfunction, highlighting an insidious disease process already apparent in the first few months of life.
Establishment of expanded and streamlined pipeline of PITCh knock-in – a web-based design tool for MMEJ-mediated gene knock-in, PITCh designer, and the variations of PITCh, PITCh-TG and PITCh-KIKO

PubMed Central

Nakamae, Kazuki; Nishimura, Yuki; Takenaga, Mitsumasa; Sakamoto, Naoaki; Ide, Hiroshi; Sakuma, Tetsushi; Yamamoto, Takashi

2017-01-01

ABSTRACT The emerging genome editing technology has enabled the creation of gene knock-in cells easily, efficiently, and rapidly, which has dramatically accelerated research in the field of mammalian functional genomics, including in humans. We recently developed a microhomology-mediated end-joining-based gene knock-in method, termed the PITCh system, and presented various examples of its application. Since the PITCh system only requires very short microhomologies (up to 40 bp) and single-guide RNA target sites on the donor vector, the targeting construct can be rapidly prepared compared with the conventional targeting vector for homologous recombination-based knock-in. Here, we established a streamlined pipeline to design and perform PITCh knock-in to further expand the availability of this method by creating web-based design software, PITCh designer (http://www.mls.sci.hiroshima-u.ac.jp/smg/PITChdesigner/index.html), as well as presenting an experimental example of versatile gene cassette knock-in. PITCh designer can automatically design not only the appropriate microhomologies but also the primers to construct locus-specific donor vectors for PITCh knock-in. By using our newly established pipeline, a reporter cell line for monitoring endogenous gene expression, and transgenesis (TG) or knock-in/knockout (KIKO) cell line can be produced systematically. Using these new variations of PITCh, an exogenous promoter-driven gene cassette expressing fluorescent protein gene and drug resistance gene can be integrated into a safe harbor or a specific gene locus to create transgenic reporter cells (PITCh-TG) or knockout cells with reporter knock-in (PITCh-KIKO), respectively. PMID:28453368
Establishment of expanded and streamlined pipeline of PITCh knock-in - a web-based design tool for MMEJ-mediated gene knock-in, PITCh designer, and the variations of PITCh, PITCh-TG and PITCh-KIKO.

PubMed

Nakamae, Kazuki; Nishimura, Yuki; Takenaga, Mitsumasa; Nakade, Shota; Sakamoto, Naoaki; Ide, Hiroshi; Sakuma, Tetsushi; Yamamoto, Takashi

2017-05-04

The emerging genome editing technology has enabled the creation of gene knock-in cells easily, efficiently, and rapidly, which has dramatically accelerated research in the field of mammalian functional genomics, including in humans. We recently developed a microhomology-mediated end-joining-based gene knock-in method, termed the PITCh system, and presented various examples of its application. Since the PITCh system only requires very short microhomologies (up to 40 bp) and single-guide RNA target sites on the donor vector, the targeting construct can be rapidly prepared compared with the conventional targeting vector for homologous recombination-based knock-in. Here, we established a streamlined pipeline to design and perform PITCh knock-in to further expand the availability of this method by creating web-based design software, PITCh designer ( http://www.mls.sci.hiroshima-u.ac.jp/smg/PITChdesigner/index.html ), as well as presenting an experimental example of versatile gene cassette knock-in. PITCh designer can automatically design not only the appropriate microhomologies but also the primers to construct locus-specific donor vectors for PITCh knock-in. By using our newly established pipeline, a reporter cell line for monitoring endogenous gene expression, and transgenesis (TG) or knock-in/knockout (KIKO) cell line can be produced systematically. Using these new variations of PITCh, an exogenous promoter-driven gene cassette expressing fluorescent protein gene and drug resistance gene can be integrated into a safe harbor or a specific gene locus to create transgenic reporter cells (PITCh-TG) or knockout cells with reporter knock-in (PITCh-KIKO), respectively.
A post-transcriptional compensatory pathway in heterozygous ventricular myosin light chain 2-deficient mice results in lack of gene dosage effect during normal cardiac growth or hypertrophy.

PubMed

Minamisawa, S; Gu, Y; Ross, J; Chien, K R; Chen, J

1999-04-09

Our previous study of homozygous mutants of the ventricular specific isoform of myosin light chain 2 (mlc-2v) demonstrated that mlc-2v plays an essential role in murine heart development (Chen, J., Kubalak, S. W., Minamisawa, S., Price, R. L., Becker, K. D., Hickey, R., Ross, J., Jr., and Chien, K. R. (1998) J. Biol. Chem. 273, 1252-1256). As gene dosage of some myofibrillar proteins can affect muscle function, we have analyzed heterozygous mutants in depth. Ventricles of heterozygous mutants displayed a 50% reduction in mlc-2v mRNA, yet expressed normal levels of protein both under basal conditions and following induction of cardiac hypertrophy by aortic constriction. Heterozygous mutants exhibited cardiac function comparable to that of wild-type littermate controls both prior to and following aortic constriction. There were no significant differences in contractility and responses to calcium between wild-type and heterozygous unloaded cardiomyocytes. We conclude that heterozygous mutants show neither a molecular nor a physiological cardiac phenotype either at base line or following hypertrophic stimuli. These results suggest that post-transcriptional compensatory mechanisms play a major role in maintaining the level of MLC-2v protein in murine hearts. In addition, as our mlc-2v knockout mutants were created by a knock-in of Cre recombinase into the endogenous mlc-2v locus, this study demonstrates that heterozygous mlc-2v cre knock-in mice are appropriate for ventricular specific gene targeting.
NMDA receptor agonists reverse impaired psychomotor and cognitive functions associated with hippocampal Hbegf-deficiency in mice.

PubMed

Sasaki, Keita; Omotuyi, Olaposi Idowu; Ueda, Mutsumi; Shinohara, Kazuyuki; Ueda, Hiroshi

2015-12-04

Structural and functional changes of the hippocampus are correlated with psychiatric disorders and cognitive dysfunctions. Genetic deletion of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is predominantly expressed in cortex and hippocampus, also causes similar psychiatric and cognitive dysfunctions, accompanying down-regulated NMDA receptor signaling. However, little is known of such dysfunctions in hippocampus-specific Hbegf cKO mice. We successfully developed hippocampus-specific cKO mice by crossbreeding floxed Hbegf and Gng7-Cre knock-in mice, as Gng7 promoter-driven Cre is highly expressed in hippocampal neurons as well as striatal medium spiny neurons. In mice lacking hippocampus Hbegf gene, there was a decreased neurogenesis in the subgranular zone (SGZ) of the dentate gyrus as well as down-regulation of PSD-95/NMDA-receptor-NR1/NR2B subunits and related NMDA receptor signaling. Psychiatric, social-behavioral and cognitive abnormalities were also observed in hippocampal cKO mice. Interestingly, D-cycloserine and nefiracetam, positive allosteric modulators (PAMs) of NMDA receptor reversed the apparent reduction in NMDA receptor signaling and most behavioral abnormalities. Furthermore, decreased SGZ neurogenesis in hippocampal cKO mice was reversed by nefiracetam. The present study demonstrates that PAMs of NMDA receptor have pharmacotherapeutic potentials to reverse down-regulated NMDA receptor signaling, neuro-socio-cognitive abnormalities and decreased neurogenesis in hippocampal cKO mice.

PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance

PubMed Central

van Oers, Johanna M. M.; Roa, Sergio; Werling, Uwe; Liu, Yiyong; Genschel, Jochen; Sellers, Rani S.; Modrich, Paul; Scharff, Matthew D.; Edelmann, Winfried

2010-01-01

The DNA mismatch repair protein PMS2 was recently found to encode a novel endonuclease activity. To determine the biological functions of this activity in mammals, we generated endonuclease-deficient Pms2E702K knock-in mice. Pms2EK/EK mice displayed increased genomic mutation rates and a strong cancer predisposition. In addition, class switch recombination, but not somatic hypermutation, was impaired in Pms2EK/EK B cells, indicating a specific role in Ig diversity. In contrast to Pms2−/− mice, Pms2EK/EK male mice were fertile, indicating that this activity is dispensable in spermatogenesis. Therefore, the PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance and tumor suppression. PMID:20624957
Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models.

PubMed

Uno, Narumi; Abe, Satoshi; Oshimura, Mitsuo; Kazuki, Yasuhiro

2018-02-01

Chromosome transfer technology, including chromosome modification, enables the introduction of Mb-sized or multiple genes to desired cells or animals. This technology has allowed innovative developments to be made for models of human disease and humanized animals, including Down syndrome model mice and humanized transchromosomic (Tc) immunoglobulin mice. Genome editing techniques are developing rapidly, and permit modifications such as gene knockout and knockin to be performed in various cell lines and animals. This review summarizes chromosome transfer-related technologies and the combined technologies of chromosome transfer and genome editing mainly for the production of cell/animal models of human disease and humanized animal models. Specifically, these include: (1) chromosome modification with genome editing in Chinese hamster ovary cells and mouse A9 cells for efficient transfer to desired cell types; (2) single-nucleotide polymorphism modification in humanized Tc mice with genome editing; and (3) generation of a disease model of Down syndrome-associated hematopoiesis abnormalities by the transfer of human chromosome 21 to normal human embryonic stem cells and the induction of mutation(s) in the endogenous gene(s) with genome editing. These combinations of chromosome transfer and genome editing open up new avenues for drug development and therapy as well as for basic research.
Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes.

PubMed

Nystoriak, Matthew A; Nieves-Cintrón, Madeline; Patriarchi, Tommaso; Buonarati, Olivia R; Prada, Maria Paz; Morotti, Stefano; Grandi, Eleonora; Fernandes, Julia Dos Santos; Forbush, Katherine; Hofmann, Franz; Sasse, Kent C; Scott, John D; Ward, Sean M; Hell, Johannes W; Navedo, Manuel F

2017-01-24

Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type Ca V 1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in Ca V 1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the Ca V 1.2 channel pore-forming subunit (α1 C ) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in Ca V 1.2 channel activity were associated with PKA activity, leading to α1 C phosphorylation at Ser 1928 Compared to arteries from low-fat diet (LFD)-fed mice and nondiabetic patients, arteries from high-fat diet (HFD)-fed mice and from diabetic patients had increased Ser 1928 phosphorylation and Ca V 1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser 1928 phosphorylation and Ca V 1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser 1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a Ca V 1.2 with Ser 1928 mutated to alanine (S1928A) lacked glucose-mediated increases in Ca V 1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in Ca V 1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1 C phosphorylation at Ser 1928 in stimulating Ca V 1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes. Copyright © 2017, American Association for the Advancement of Science.
Impaired synaptic clustering of postsynaptic density proteins and altered signal transmission in hippocampal neurons, and disrupted learning behavior in PDZ1 and PDZ2 ligand binding-deficient PSD-95 knockin mice

PubMed Central

2012-01-01

Background Postsynaptic density (PSD)-95-like membrane-associated guanylate kinases (PSD-MAGUKs) are scaffold proteins in PSDs that cluster signaling molecules near NMDA receptors. PSD-MAGUKs share a common domain structure, including three PDZ (PDZ1/2/3) domains in their N-terminus. While multiple domains enable the PSD-MAGUKs to bind various ligands, the contribution of each PDZ domain to synaptic organization and function is not fully understood. Here, we focused on the PDZ1/2 domains of PSD-95 that bind NMDA-type receptors, and studied the specific roles of the ligand binding of these domains in the assembly of PSD proteins, synaptic properties of hippocampal neurons, and behavior, using ligand binding-deficient PSD-95 cDNA knockin (KI) mice. Results The KI mice showed decreased accumulation of mutant PSD-95, PSD-93 and AMPA receptor subunits in the PSD fraction of the hippocampus. In the hippocampal CA1 region of young KI mice, basal synaptic efficacy was reduced and long-term potentiation (LTP) was enhanced with intact long-term depression. In adult KI mice, there was no significant change in the magnitude of LTP in CA1, but robustly enhanced LTP was induced at the medial perforant path-dentate gyrus synapses, suggesting that PSD-95 has an age- and subregion-dependent role. In a battery of behavioral tests, KI mice showed markedly abnormal anxiety-like behavior, impaired spatial reference and working memory, and impaired remote memory and pattern separation in fear conditioning test. Conclusions These findings reveal that PSD-95 including its ligand binding of the PDZ1/2 domains controls the synaptic clustering of PSD-MAGUKs and AMPA receptors, which may have an essential role in regulating hippocampal synaptic transmission, plasticity, and hippocampus-dependent behavior. PMID:23268962
Impaired synaptic clustering of postsynaptic density proteins and altered signal transmission in hippocampal neurons, and disrupted learning behavior in PDZ1 and PDZ2 ligand binding-deficient PSD-95 knockin mice.

PubMed

Nagura, Hitoshi; Ishikawa, Yasuyuki; Kobayashi, Katsunori; Takao, Keizo; Tanaka, Tomo; Nishikawa, Kouki; Tamura, Hideki; Shiosaka, Sadao; Suzuki, Hidenori; Miyakawa, Tsuyoshi; Fujiyoshi, Yoshinori; Doi, Tomoko

2012-12-26

Postsynaptic density (PSD)-95-like membrane-associated guanylate kinases (PSD-MAGUKs) are scaffold proteins in PSDs that cluster signaling molecules near NMDA receptors. PSD-MAGUKs share a common domain structure, including three PDZ (PDZ1/2/3) domains in their N-terminus. While multiple domains enable the PSD-MAGUKs to bind various ligands, the contribution of each PDZ domain to synaptic organization and function is not fully understood. Here, we focused on the PDZ1/2 domains of PSD-95 that bind NMDA-type receptors, and studied the specific roles of the ligand binding of these domains in the assembly of PSD proteins, synaptic properties of hippocampal neurons, and behavior, using ligand binding-deficient PSD-95 cDNA knockin (KI) mice. The KI mice showed decreased accumulation of mutant PSD-95, PSD-93 and AMPA receptor subunits in the PSD fraction of the hippocampus. In the hippocampal CA1 region of young KI mice, basal synaptic efficacy was reduced and long-term potentiation (LTP) was enhanced with intact long-term depression. In adult KI mice, there was no significant change in the magnitude of LTP in CA1, but robustly enhanced LTP was induced at the medial perforant path-dentate gyrus synapses, suggesting that PSD-95 has an age- and subregion-dependent role. In a battery of behavioral tests, KI mice showed markedly abnormal anxiety-like behavior, impaired spatial reference and working memory, and impaired remote memory and pattern separation in fear conditioning test. These findings reveal that PSD-95 including its ligand binding of the PDZ1/2 domains controls the synaptic clustering of PSD-MAGUKs and AMPA receptors, which may have an essential role in regulating hippocampal synaptic transmission, plasticity, and hippocampus-dependent behavior.
Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes

PubMed Central

Nystoriak, Matthew A.; Nieves-Cintrón, Madeline; Patriarchi, Tommaso; Buonarati, Olivia R.; Prada, Maria Paz; Morotti, Stefano; Grandi, Eleonora; Fernandes, Julia Dos Santos; Forbush, Katherine; Hofmann, Franz; Sasse, Kent C.; Scott, John D.; Ward, Sean M.; Hell, Johannes W.; Navedo, Manuel F.

2017-01-01

Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type CaV1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in CaV1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the CaV1.2 channel pore-forming subunit (α1C) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in CaV1.2 channel activity were associated with PKA activity, leading to α1C phosphorylation at Ser1928. Compared to arteries from low-fat diet (LFD)–fed mice and nondiabetic patients, arteries from high-fat diet (HFD)–fed mice and from diabetic patients had increased Ser1928 phosphorylation and CaV1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser1928 phosphorylation and CaV1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a CaV1.2 with Ser1928 mutated to alanine (S1928A) lacked glucose-mediated increases in CaV1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in CaV1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1C phosphorylation at Ser1928 in stimulating CaV1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes. PMID:28119464
Skeletal muscle ACC2 S212 phosphorylation is not required for the control of fatty acid oxidation during exercise.

PubMed

O'Neill, Hayley M; Lally, James S; Galic, Sandra; Pulinilkunnil, Thomas; Ford, Rebecca J; Dyck, Jason R B; van Denderen, Bryce J; Kemp, Bruce E; Steinberg, Gregory R

2015-07-01

During submaximal exercise fatty acids are a predominant energy source for muscle contractions. An important regulator of fatty acid oxidation is acetyl-CoA carboxylase (ACC), which exists as two isoforms (ACC1 and ACC2) with ACC2 predominating in skeletal muscle. Both ACC isoforms regulate malonyl-CoA production, an allosteric inhibitor of carnitine palmitoyltransferase 1 (CPT-1); the primary enzyme controlling fatty acyl-CoA flux into mitochondria for oxidation. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is activated during exercise or by pharmacological agents such as metformin and AICAR. In resting muscle the activation of AMPK with AICAR leads to increased phosphorylation of ACC (S79 on ACC1 and S221 on ACC2), which reduces ACC activity and malonyl-CoA; effects associated with increased fatty acid oxidation. However, whether this pathway is vital for regulating skeletal muscle fatty acid oxidation during conditions of increased metabolic flux such as exercise/muscle contractions remains unknown. To examine this we characterized mice lacking AMPK phosphorylation sites on ACC2 (S212 in mice/S221 in humans-ACC2-knock-in [ACC2-KI]) or both ACC1 (S79) and ACC2 (S212) (ACC double knock-in [ACCD-KI]) during submaximal treadmill exercise and/or ex vivo muscle contractions. We find that surprisingly, ACC2-KI mice had normal exercise capacity and whole-body fatty acid oxidation during treadmill running despite elevated muscle ACC2 activity and malonyl-CoA. Similar results were observed in ACCD-KI mice. Fatty acid oxidation was also maintained in muscles from ACC2-KI mice contracted ex vivo. These findings indicate that pathways independent of ACC phosphorylation are important for regulating skeletal muscle fatty acid oxidation during exercise/muscle contractions. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.
Genetic Rescue of Glycosylation-deficient Fgf23 in the Galnt3 Knockout Mouse

PubMed Central

Gray, Amie K.; Padgett, Leah R.; Allen, Matthew R.; Clinkenbeard, Erica L.; Sarpa, Nicole M.; White, Kenneth E.; Econs, Michael J.

2014-01-01

Fibroblast growth factor 23 (FGF23) is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. The FGF23 subtilisin-like proprotein convertase recognition sequence (176RHTR179↓) is protected by O-glycosylation through ppGalNAc-T3 (GALNT3) activity. Thus, inactivating GALNT3 mutations render FGF23 susceptible to proteolysis, thereby reducing circulating intact hormone levels and leading to hyperphosphatemic familial tumoral calcinosis. To further delineate the role of glycosylation in the Fgf23 function, we generated an inducible FGF23 transgenic mouse expressing human mutant FGF23 (R176Q and R179Q) found in patients with autosomal dominant hypophosphatemic rickets (ADHR) and bred this animal to Galnt3 knockout mice, a model of familial tumoral calcinosis. Due to the low intact Fgf23 level, Galnt3 knockout mice with wild-type Fgf23 alleles were hyperphosphatemic. In contrast, carriers of the mutant FGF23 transgene, regardless of Galnt3 mutation status, had significantly higher serum intact FGF23, resulting in severe hypophosphatemia. Importantly, serum phosphorus and FGF23 were comparable between transgenic mice with or without normal Galnt3 alleles. To determine whether the presence of the ADHR mutation could improve biochemical and skeletal abnormalities in Galnt3-null mice, these mice were also mated to Fgf23 knock-in mice, carrying heterozygous or homozygous R176Q ADHR Fgf23 mutations. The knock-in mice with functional Galnt3 had normal Fgf23 but were slightly hypophosphatemic. The stabilized Fgf23 ADHR allele reversed the Galnt3-null phenotype and normalized total Fgf23, serum phosphorus, and bone Fgf23 mRNA. However, the skeletal phenotype was unaffected. In summary, these data demonstrate that O-glycosylation by ppGaINAc-T3 is only necessary for proper secretion of intact Fgf23 and, once secreted, does not affect Fgf23 function. Furthermore, the more stable Fgf23 ADHR mutant protein could normalize serum phosphorus in Galnt3 knockout mice. PMID:25051439
Genetic rescue of glycosylation-deficient Fgf23 in the Galnt3 knockout mouse.

PubMed

Ichikawa, Shoji; Gray, Amie K; Padgett, Leah R; Allen, Matthew R; Clinkenbeard, Erica L; Sarpa, Nicole M; White, Kenneth E; Econs, Michael J

2014-10-01

Fibroblast growth factor 23 (FGF23) is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. The FGF23 subtilisin-like proprotein convertase recognition sequence ((176)RHTR(179)↓) is protected by O-glycosylation through ppGalNAc-T3 (GALNT3) activity. Thus, inactivating GALNT3 mutations render FGF23 susceptible to proteolysis, thereby reducing circulating intact hormone levels and leading to hyperphosphatemic familial tumoral calcinosis. To further delineate the role of glycosylation in the Fgf23 function, we generated an inducible FGF23 transgenic mouse expressing human mutant FGF23 (R176Q and R179Q) found in patients with autosomal dominant hypophosphatemic rickets (ADHR) and bred this animal to Galnt3 knockout mice, a model of familial tumoral calcinosis. Due to the low intact Fgf23 level, Galnt3 knockout mice with wild-type Fgf23 alleles were hyperphosphatemic. In contrast, carriers of the mutant FGF23 transgene, regardless of Galnt3 mutation status, had significantly higher serum intact FGF23, resulting in severe hypophosphatemia. Importantly, serum phosphorus and FGF23 were comparable between transgenic mice with or without normal Galnt3 alleles. To determine whether the presence of the ADHR mutation could improve biochemical and skeletal abnormalities in Galnt3-null mice, these mice were also mated to Fgf23 knock-in mice, carrying heterozygous or homozygous R176Q ADHR Fgf23 mutations. The knock-in mice with functional Galnt3 had normal Fgf23 but were slightly hypophosphatemic. The stabilized Fgf23 ADHR allele reversed the Galnt3-null phenotype and normalized total Fgf23, serum phosphorus, and bone Fgf23 mRNA. However, the skeletal phenotype was unaffected. In summary, these data demonstrate that O-glycosylation by ppGaINAc-T3 is only necessary for proper secretion of intact Fgf23 and, once secreted, does not affect Fgf23 function. Furthermore, the more stable Fgf23 ADHR mutant protein could normalize serum phosphorus in Galnt3 knockout mice.
Interaction of PDK1 with Phosphoinositides Is Essential for Neuronal Differentiation but Dispensable for Neuronal Survival

PubMed Central

Zurashvili, Tinatin; Cordón-Barris, Lluís; Ruiz-Babot, Gerard; Zhou, Xiangyu; Lizcano, Jose M.; Gómez, Nestor; Giménez-Llort, Lydia

2013-01-01

3-Phosphoinositide-dependent protein kinase 1 (PDK1) operates in cells in response to phosphoinositide 3-kinase activation and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] production by activating a number of AGC kinases, including protein kinase B (PKB)/Akt. Both PDK1 and PKB contain pleckstrin homology (PH) domains that interact with the PtdIns(3,4,5)P3 second messenger. Disrupting the interaction of the PDK1 PH domain with phosphoinositides by expressing the PDK1 K465E knock-in mutation resulted in mice with reduced PKB activation. We explored the physiological consequences of this biochemical lesion in the central nervous system. The PDK1 knock-in mice displayed a reduced brain size due to a reduction in neuronal cell size rather than cell number. Reduced BDNF-induced phosphorylation of PKB at Thr308, the PDK1 site, was observed in the mutant neurons, which was not rate limiting for the phosphorylation of those PKB substrates governing neuronal survival and apoptosis, such as FOXO1 or glycogen synthase kinase 3 (GSK3). Accordingly, the integrity of the PDK1 PH domain was not essential to support the survival of different embryonic neuronal populations analyzed. In contrast, PKB-mediated phosphorylation of PRAS40 and TSC2, allowing optimal mTORC1 activation and brain-specific kinase (BRSK) protein synthesis, was markedly reduced in the mutant mice, leading to impaired neuronal growth and differentiation. PMID:23275438
Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

PubMed Central

Bartalucci, Niccolò; Tozzi, Lorenzo; Bogani, Costanza; Martinelli, Serena; Rotunno, Giada; Villeval, Jean-Luc; Vannucchi, Alessandro M

2013-01-01

Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation-specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single-agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild-type counterpart, and preferentially prevented colony formation from JAK2V617F knock-in mice and patients' progenitor cells compared with normal ones. Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models. Co-treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F-mutated Ba/F3-EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock-in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN. PMID:24237791
Consequences of SPAK inactivation on Hyperkalemic Hypertension caused by WNK1 mutations: evidence for differential roles of WNK1 and WNK4.

PubMed

Rafael, Chloé; Soukaseum, Christelle; Baudrie, Véronique; Frère, Perrine; Hadchouel, Juliette

2018-02-19

Mutations of the gene encoding WNK1 [With No lysine (K) kinase 1] or WNK4 cause Familial Hyperkalemic Hypertension (FHHt). Previous studies have shown that the activation of SPAK (Ste20-related Proline/Alanine-rich Kinase) plays a dominant role in the development of FHHt caused by WNK4 mutations. The implication of SPAK in FHHt caused by WNK1 mutation has never been investigated. To clarify this issue, we crossed WNK1 +/FHHt mice with SPAK knock-in mice in which the T-loop Thr243 residue was mutated to alanine to prevent activation by WNK kinases. We show that WNK1 +/FHHT :SPAK 243A/243A mice display an intermediate phenotype, between that of control and SPAK 243A/243A mice, with normal blood pressure but hypochloremic metabolic alkalosis. NCC abundance and phosphorylation levels also decrease below the wild-type level in the double-mutant mice but remain higher than in SPAK 243A/243A mice. This is different from what was observed in WNK4-FHHt mice in which SPAK inactivation completely restored the phenotype and NCC expression to wild-type levels. Although these results confirm that FHHt caused by WNK1 mutations is dependent on the activation of SPAK, they suggest that WNK1 and WNK4 play different roles in the distal nephron.
Mutant ataxin1 disrupts cerebellar development in spinocerebellar ataxia type 1.

PubMed

Edamakanti, Chandrakanth Reddy; Do, Jeehaeh; Didonna, Alessandro; Martina, Marco; Opal, Puneet

2018-06-01

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disease caused by a polyglutamine expansion in the protein ATXN1, which is involved in transcriptional regulation. Although symptoms appear relatively late in life, primarily from cerebellar dysfunction, pathogenesis begins early, with transcriptional changes detectable as early as a week after birth in SCA1-knockin mice. Given the importance of this postnatal period for cerebellar development, we asked whether this region might be developmentally altered by mutant ATXN1. We found that expanded ATXN1 stimulates the proliferation of postnatal cerebellar stem cells in SCA1 mice. These hyperproliferating stem cells tended to differentiate into GABAergic inhibitory interneurons rather than astrocytes; this significantly increased the GABAergic inhibitory interneuron synaptic connections, disrupting cerebellar Purkinje cell function in a non-cell autonomous manner. We confirmed the increased basket cell-Purkinje cell connectivity in human SCA1 patients. Mutant ATXN1 thus alters the neural circuitry of the developing cerebellum, setting the stage for the later vulnerability of Purkinje cells to SCA1. We propose that other late-onset degenerative diseases may also be rooted in subtle developmental derailments.
The TINS Lecture. Understanding the roles of Otx1 and Otx2 in the control of brain morphogenesis.

PubMed

Acampora, D; Simeone, A

1999-03-01

The murine homologs of the orthodenticle (otd) gene of Drosophila, Otx1 and Otx2, have an important role in brain morphogenesis. Analysis of Otx1 and Otx2 null mice reveals that Otx1 is required primarily for corticogenesis and sense-organ development,while Otx2 is necessary for specification and maintenance of anterior neural plate as well as for proper gastrulation. Cross-phylum recoveries of Otx1 abnormalities by Drosophila otd, and vice versa, indicate that genetic functions required in mammalian-brain development evolved in a primitive ancestor of flies and mice. Knock-in mouse models in which Otx2 was replaced with Otx1, and vice versa, provide evidence that the existence of Otx1-/- and Otx2-/- divergent phenotypes largely reflects differences in expression patterns rather than in the biochemical activity of OTX1 and OTX2. In evolutionary terms, some of these findings lead us to hypothesize a fascinating and crucial role for Otx genes that contributes to the genetic program required for the specification of the development of the vertebrate head.
NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome

PubMed Central

Asano, Masanao; Li, Yue-Sheng; Núñez, Gabriel

2017-01-01

Although B cell development requires expression of the B cell antigen receptor (BCR), it remains unclear whether engagement of self-antigen provides a positive impact for most B cells. Here, we show that BCR engagement by self-ligand during development in vivo results in up-regulation of the Nod-like receptor member Nod1, which recognizes the products of intestinal commensal bacteria. In anti-thymocyte/Thy-1 autoreactive BCR knock-in mice lacking self–Thy-1 ligand, immunoglobulin light chain editing occurred, generating B cells with up-regulated Nod1, including follicular and marginal zone B cells with natural autoreactivity. This BCR editing with increased Nod1 resulted in preferential survival. In normal adult mice, most mature B cells are enriched for Nod1 up-regulated cells, and signaling through Nod1 promotes competitive survival of mature B cells. These findings demonstrate a role for microbial products in promoting survival of mature B cells through up-regulated Nod1, providing a positive effect of BCR engagement on development of most B cells. PMID:28878001
Using Stimulus Frequency Emissions to Characterize Cochlear Function in Mice

NASA Astrophysics Data System (ADS)

Cheatham, M. A.; Katz, E. D.; Charaziak, K.; Dallos, P.; Siegel, J. H.

2011-11-01

Stimulus frequency otoacoustic emissions (SFOAE) were used to assay cochlear function in wildtype and prestin knockin (KI) mice. The latter contain a mutated form of the outer hair cell (OHC) motor protein (V499G/Y501H) with significantly reduced activity. Because several genetic mutations cause accelerated OHC death, it is beneficial to perform experiments in young mice without surgical intervention. Inasmuch as SFOAE thresholds are elevated by only 30 dB in KIs, it is possible to obtain SFOAE tuning functions in these animals. This approach allows sensitivity/frequency selectivity to be assayed within the basilar membane-OHC-tectorial membrane feedback loop, thereby providing information about signal processing prior to inner hair cell stimulation and auditory nerve activation.
Do Structural Missense Variants in the ATM Gene Found in Women With Breast Cancer Cause Breast Cancer in Knock-in Mouse Strains?

DTIC Science & Technology

2006-04-01

W81XWH-05-1-0282 TITLE: Do Structural Missense Variants in the ATM Gene Found in Women with Breast Cancer Cause Breast Cancer in "Knock-in...5a. CONTRACT NUMBER Do Structural Missense Variants in the ATM Gene Found in Women with Breast Cancer Cause Breast Cancer in "Knock-in" Mouse...human cohort-specific missense mutations will develop breast cancer with dominant inheritance in a subset of animals. It also is hypothesized that
Tild-CRISPR Allows for Efficient and Precise Gene Knockin in Mouse and Human Cells.

PubMed

Yao, Xuan; Zhang, Meiling; Wang, Xing; Ying, Wenqin; Hu, Xinde; Dai, Pengfei; Meng, Feilong; Shi, Linyu; Sun, Yun; Yao, Ning; Zhong, Wanxia; Li, Yun; Wu, Keliang; Li, Weiping; Chen, Zi-Jiang; Yang, Hui

2018-05-21

The targeting efficiency of knockin sequences via homologous recombination (HR) is generally low. Here we describe a method we call Tild-CRISPR (targeted integration with linearized dsDNA-CRISPR), a targeting strategy in which a PCR-amplified or precisely enzyme-cut transgene donor with 800-bp homology arms is injected with Cas9 mRNA and single guide RNA into mouse zygotes. Compared with existing targeting strategies, this method achieved much higher knockin efficiency in mouse embryos, as well as brain tissue. Importantly, the Tild-CRISPR method also yielded up to 12-fold higher knockin efficiency than HR-based methods in human embryos, making it suitable for studying gene functions in vivo and developing potential gene therapies. Copyright © 2018 Elsevier Inc. All rights reserved.
HttQ111/+ Huntington’s Disease Knock-in Mice Exhibit Brain Region-Specific Morphological Changes and Synaptic Dysfunction

PubMed Central

Kovalenko, Marina; Milnerwood, Austen; Giordano, James; St. Claire, Jason; Guide, Jolene R.; Stromberg, Mary; Gillis, Tammy; Sapp, Ellen; DiFiglia, Marian; MacDonald, Marcy E.; Carroll, Jeffrey B.; Lee, Jong-Min; Tappan, Susan; Raymond, Lynn; Wheeler, Vanessa C.

2018-01-01

Background: Successful disease-modifying therapy for Huntington’s disease (HD) will require therapeutic intervention early in the pathogenic process. Achieving this goal requires identifying phenotypes that are proximal to the HTT CAG repeat expansion. Objective: To use Htt CAG knock-in mice, precise genetic replicas of the HTT mutation in patients, as models to study proximal disease events. Methods: Using cohorts of B6J.HttQ111/+ mice from 2 to 18 months of age, we analyzed pathological markers, including immunohistochemistry, brain regional volumes and cortical thickness, CAG instability, electron microscopy of striatal synapses, and acute slice electrophysiology to record glutamatergic transmission at striatal synapses. We also incorporated a diet perturbation paradigm for some of these analyses. Results: B6J.HttQ111/+ mice did not exhibit significant neurodegeneration or gliosis but revealed decreased striatal DARPP-32 as well as subtle but regional-specific changes in brain volumes and cortical thickness that parallel those in HD patients. Ultrastructural analyses of the striatum showed reduced synapse density, increased postsynaptic density thickness and increased synaptic cleft width. Acute slice electrophysiology showed alterations in spontaneous AMPA receptor-mediated postsynaptic currents, evoked NMDA receptor-mediated excitatory postsynaptic currents, and elevated extrasynaptic NMDA currents. Diet influenced cortical thickness, but did not impact somatic CAG expansion, nor did it show any significant interaction with genotype on immunohistochemical, brain volume or cortical thickness measures. Conclusions: These data show that a single HttQ111 allele is sufficient to elicit brain region-specific morphological changes and early neuronal dysfunction, highlighting an insidious disease process already apparent in the first few months of life. PMID:29480209
[Role of the sweet taste receptor in glucose metabolism: no sweets for diabetes?].

PubMed

Nomura, Masatoshi; Kawahara, Yuta

2015-01-01

Type 2 diabetes is closely associated with our daily diets and has become a global health problem with increasing number of patients. Maintaining energy homeostasis is essentially required for the treatment of diabetes. Energy metabolism starts with taking in a meal. Nutrients including amino acids, fatty acids and glucose in the digest have been shown to act on the neuroendocrine cells in the gastrointestinal (GI) tract, and thereby play important roles in energy homeostasis. Therefore, the GI tract is now recognized as a sensor system for nutrient signals. Taste receptor type 1 member 2 (T1R2) is known to function as a co-receptor with T1R3 to detect sweet chemicals in the taste buds. It has been proposed that the T1R2/T1R3 receptor complex acts as sweet sensor in the intestine, and plays a pivotal role in sensing sugars and maintaining glucose homeostasis through incretin secretion. To clarify the physiological roles of T1R2 in glucose homeostasis, T1r2-lacZ knock-in/knock-out mice were generated. We found lacZ gene expression in the GI tract where T1r3 expression has been reported. Interestingly, the T1r2-lacZ knock-in mice showed impaired glucose tolerance on oral glucose challenge but not on intraperitoneal injection. However, the fasting glucose level in T1r2-lacZ knock-in mice was comparable to that in wild type mice. These results suggest an important role of the sweet taste receptor system in the intestine when stimulated by glucose. Therefore, the roles of T1R2 will be presented and the mechanism for metabolic homeostasis will be discussed.

Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain.

PubMed

Tsunekawa, Yuji; Terhune, Raymond Kunikane; Fujita, Ikumi; Shitamukai, Atsunori; Suetsugu, Taeko; Matsuzaki, Fumio

2016-09-01

Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues. © 2016. Published by The Company of Biologists Ltd.
Age-associated change of C/EBP family proteins causes severe liver injury and acceleration of liver proliferation after CCl4 treatments.

PubMed

Hong, Il-Hwa; Lewis, Kyle; Iakova, Polina; Jin, Jingling; Sullivan, Emily; Jawanmardi, Nicole; Timchenko, Lubov; Timchenko, Nikolai

2014-01-10

The aged liver is more sensitive to the drug treatments and has a high probability of developing liver disorders such as fibrosis, cirrhosis, and cancer. Here we present mechanisms underlying age-associated severe liver injury and acceleration of liver proliferation after CCl4 treatments. We have examined liver response to CCl4 treatments using old WT mice and young C/EBPα-S193D knockin mice, which express an aged-like isoform of C/EBPα. Both animal models have altered chromatin structure as well as increased liver injury and proliferation after acute CCl4 treatments. We found that these age-related changes are associated with the repression of key regulators of liver biology: C/EBPα, Farnesoid X Receptor (FXR) and telomere reverse transcriptase (TERT). In quiescent livers of old WT and young S193D mice, the inhibition of TERT is mediated by HDAC1-C/EBPα complexes. After CCl4 treatments, TERT, C/EBPα and FXR are repressed by different mechanisms. These mechanisms include the increase of a dominant negative isoform, C/EBPβ-LIP, and subsequent repression of C/EBPα, FXR, and TERT promoters. C/EBPβ-LIP also disrupts Rb-E2F1 complexes in C/EBPα-S193D mice after CCl4 treatments. To examine if these alterations are involved in drug-mediated liver diseases, we performed chronic treatments of mice with CCl4. We found that C/EBPα-S193D mice developed fibrosis much more rapidly than WT mice. Thus, our data show that the age-associated alterations of C/EBP proteins create favorable conditions for the increased liver proliferation after CCl4 treatments and for development of drug-mediated liver diseases.
Age-associated Change of C/EBP Family Proteins Causes Severe Liver Injury and Acceleration of Liver Proliferation after CCl4 Treatments*

PubMed Central

Hong, Il-Hwa; Lewis, Kyle; Iakova, Polina; Jin, Jingling; Sullivan, Emily; Jawanmardi, Nicole; Timchenko, Lubov; Timchenko, Nikolai

2014-01-01

The aged liver is more sensitive to the drug treatments and has a high probability of developing liver disorders such as fibrosis, cirrhosis, and cancer. Here we present mechanisms underlying age-associated severe liver injury and acceleration of liver proliferation after CCl4 treatments. We have examined liver response to CCl4 treatments using old WT mice and young C/EBPα-S193D knockin mice, which express an aged-like isoform of C/EBPα. Both animal models have altered chromatin structure as well as increased liver injury and proliferation after acute CCl4 treatments. We found that these age-related changes are associated with the repression of key regulators of liver biology: C/EBPα, Farnesoid X Receptor (FXR) and telomere reverse transcriptase (TERT). In quiescent livers of old WT and young S193D mice, the inhibition of TERT is mediated by HDAC1-C/EBPα complexes. After CCl4 treatments, TERT, C/EBPα and FXR are repressed by different mechanisms. These mechanisms include the increase of a dominant negative isoform, C/EBPβ-LIP, and subsequent repression of C/EBPα, FXR, and TERT promoters. C/EBPβ-LIP also disrupts Rb-E2F1 complexes in C/EBPα-S193D mice after CCl4 treatments. To examine if these alterations are involved in drug-mediated liver diseases, we performed chronic treatments of mice with CCl4. We found that C/EBPα-S193D mice developed fibrosis much more rapidly than WT mice. Thus, our data show that the age-associated alterations of C/EBP proteins create favorable conditions for the increased liver proliferation after CCl4 treatments and for development of drug-mediated liver diseases. PMID:24273171
Decreased bone formation and increased osteoclastogenesis cause bone loss in mucolipidosis II

PubMed Central

Kollmann, Katrin; Pestka, Jan Malte; Kühn, Sonja Christin; Schöne, Elisabeth; Schweizer, Michaela; Karkmann, Kathrin; Otomo, Takanobu; Catala-Lehnen, Philip; Failla, Antonio Virgilio; Marshall, Robert Percy; Krause, Matthias; Santer, Rene; Amling, Michael; Braulke, Thomas; Schinke, Thorsten

2013-01-01

Mucolipidosis type II (MLII) is a severe multi-systemic genetic disorder caused by missorting of lysosomal proteins and the subsequent lysosomal storage of undegraded macromolecules. Although affected children develop disabling skeletal abnormalities, their pathogenesis is not understood. Here we report that MLII knock-in mice, recapitulating the human storage disease, are runted with accompanying growth plate widening, low trabecular bone mass and cortical porosity. Intralysosomal deficiency of numerous acid hydrolases results in accumulation of storage material in chondrocytes and osteoblasts, and impaired bone formation. In osteoclasts, no morphological or functional abnormalities are detected whereas osteoclastogenesis is dramatically increased in MLII mice. The high number of osteoclasts in MLII is associated with enhanced osteoblastic expression of the pro-osteoclastogenic cytokine interleukin-6, and pharmacological inhibition of bone resorption prevented the osteoporotic phenotype of MLII mice. Our findings show that progressive bone loss in MLII is due to the presence of dysfunctional osteoblasts combined with excessive osteoclastogenesis. They further underscore the importance of a deep skeletal phenotyping approach for other lysosomal diseases in which bone loss is a prominent feature. PMID:24127423
Development of versatile non-homologous end joining-based knock-in module for genome editing.

PubMed

Sawatsubashi, Shun; Joko, Yudai; Fukumoto, Seiji; Matsumoto, Toshio; Sugano, Shigeo S

2018-01-12

CRISPR/Cas9-based genome editing has dramatically accelerated genome engineering. An important aspect of genome engineering is efficient knock-in technology. For improved knock-in efficiency, the non-homologous end joining (NHEJ) repair pathway has been used over the homology-dependent repair pathway, but there remains a need to reduce the complexity of the preparation of donor vectors. We developed the versatile NHEJ-based knock-in module for genome editing (VIKING). Using the consensus sequence of the time-honored pUC vector to cut donor vectors, any vector with a pUC backbone could be used as the donor vector without customization. Conditions required to minimize random integration rates of the donor vector were also investigated. We attempted to isolate null lines of the VDR gene in human HaCaT keratinocytes using knock-in/knock-out with a selection marker cassette, and found 75% of clones isolated were successfully knocked-in. Although HaCaT cells have hypotetraploid genome composition, the results suggest multiple clones have VDR null phenotypes. VIKING modules enabled highly efficient knock-in of any vectors harboring pUC vectors. Users now can insert various existing vectors into an arbitrary locus in the genome. VIKING will contribute to low-cost genome engineering.
Tagging methyl-CpG-binding domain proteins reveals different spatiotemporal expression and supports distinct functions.

PubMed

Wood, Kathleen H; Johnson, Brian S; Welsh, Sarah A; Lee, Jun Y; Cui, Yue; Krizman, Elizabeth; Brodkin, Edward S; Blendy, Julie A; Robinson, Michael B; Bartolomei, Marisa S; Zhou, Zhaolan

2016-04-01

DNA methylation is recognized by methyl-CpG-binding domain (MBD) proteins. Multiple MBDs are linked to neurodevelopmental disorders in humans and mice. However, the functions of MBD2 are poorly understood. We characterized Mbd2 knockout mice and determined spatiotemporal expression of MBDs and MBD2-NuRD (nucleosome remodeling deacetylase) interactions. We analyzed behavioral phenotypes, generated biotin-tagged MBD1 and MBD2 knockin mice, and performed biochemical studies of MBD2-NuRD. Most behavioral measures are minimally affected in Mbd2 knockout mice. In contrast to other MBDs, MBD2 shows distinct expression patterns. Unlike most MBDs, MBD2 is ubiquitously expressed in all tissues examined and appears dispensable for brain functions measured in this study. We provide novel genetic tools and reveal new directions to investigate MBD2 functions in vivo.
Constitutive Activation of Smoothened in the Renal Collecting Ducts Leads to Renal Hypoplasia, Hydronephrosis, and Hydroureter.

PubMed

Gupta, Deepak Prasad; Hwang, Jae-Won; Cho, Eui-Sic; Kim, Won; Song, Chang Ho; Chai, Ok Hee

2017-01-01

Sonic Hedgehog (Shh) signaling plays a major role in and is essential for regulation, patterning, and proliferation during renal development. Smoothened (Smo) plays a pivot role in transducing the Shh-glioma-associated oncogene Kruppel family member. However, the cellular and molecular mechanism underlying the role of sustained Smo activation in postnatal kidney development is still not clearly understood. Using a conditional knockin mouse model that expresses a constitutively activated form of Smo (SmoM2) upon Homeobox-B7-mediated recombination (Hoxb7-Cre), the effects of Shh signaling were determined in postnatal kidney development. SmoM2;Hoxb7-Cre mutant mice showed growth retardation with a reduction of body weight. Constitutive activation of Smo in the renal collecting ducts caused renal hypoplasia, hydronephrosis, and hydroureter. The parenchymal area and glomerular numbers were reduced, but the glomerular density was increased in SmoM2;Hoxb7-Cre mutant mice. The expression of Patched 1, the receptor of Shh and a downstream target gene of the Shh signaling pathway, was highly restricted and it was upregulated in the inner medullary collecting ducts of the kidney. The proliferative cells in the mesenchyme and collecting ducts were decreased in SmoM2;Hoxb7-Cre mutant mice. This study showed for the first time that sustained Smo inhibits postnatal kidney development by suppressing the proliferation of the mesenchyme and medullary collecting ducts in mice. © 2017 S. Karger AG, Basel.
Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein.

PubMed

Lee, Sang Mi; Kim, Ji Woo; Jeong, Young-Hee; Kim, Se Eun; Kim, Yeong Ji; Moon, Seung Ju; Lee, Ji-Hye; Kim, Keun-Jung; Kim, Min-Kyu; Kang, Man-Jong

2014-11-01

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine β-casein gene locus using a knock-in vector for the β-casein gene locus. We developed the knock-in vector on the porcine β-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine β-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using β-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous β-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.
Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein

PubMed Central

Lee, Sang Mi; Kim, Ji Woo; Jeong, Young-Hee; Kim, Se Eun; Kim, Yeong Ji; Moon, Seung Ju; Lee, Ji-Hye; Kim, Keun-Jung; Kim, Min-Kyu; Kang, Man-Jong

2014-01-01

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine β-casein gene locus using a knock-in vector for the β-casein gene locus. We developed the knock-in vector on the porcine β-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5′ arm and 3′ arm from the porcine β-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using β-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous β-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands. PMID:25358326
Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53-associated apoptosis.

PubMed

Breuss, Martin; Fritz, Tanja; Gstrein, Thomas; Chan, Kelvin; Ushakova, Lyubov; Yu, Nuo; Vonberg, Frederick W; Werner, Barbara; Elling, Ulrich; Keays, David A

2016-04-01

Microtubules play a crucial role in the generation, migration and differentiation of nascent neurons in the developing vertebrate brain. Mutations in the constituents of microtubules, the tubulins, are known to cause an array of neurological disorders, including lissencephaly, polymicrogyria and microcephaly. In this study we explore the genetic and cellular mechanisms that cause TUBB5-associated microcephaly by exploiting two new mouse models: a conditional E401K knock-in, and a conditional knockout animal. These mice present with profound microcephaly due to a loss of upper-layer neurons that correlates with massive apoptosis and upregulation of p53. This phenotype is associated with a delay in cell cycle progression and ectopic DNA elements in progenitors, which is dependent on the dosage of functional Tubb5. Strikingly, we report ectopic Sox2-positive progenitors and defects in spindle orientation in our knock-in mouse line, which are absent in knockout animals. This work sheds light on the functional repertoire of Tubb5, reveals that the E401K mutation acts by a complex mechanism, and demonstrates that the cellular pathology driving TUBB5-associated microcephaly is cell death. © 2016. Published by The Company of Biologists Ltd.
Single delivery of an adeno-associated viral construct to transfer the CASQ2 gene to knock-in mice affected by catecholaminergic polymorphic ventricular tachycardia is able to cure the disease from birth to advanced age.

PubMed

Denegri, Marco; Bongianino, Rossana; Lodola, Francesco; Boncompagni, Simona; De Giusti, Verónica C; Avelino-Cruz, José E; Liu, Nian; Persampieri, Simone; Curcio, Antonio; Esposito, Francesca; Pietrangelo, Laura; Marty, Isabelle; Villani, Laura; Moyaho, Alejandro; Baiardi, Paola; Auricchio, Alberto; Protasi, Feliciano; Napolitano, Carlo; Priori, Silvia G

2014-06-24

Catecholaminergic polymorphic ventricular tachycardia is an inherited arrhythmogenic disorder characterized by sudden cardiac death in children. Drug therapy is still insufficient to provide full protection against cardiac arrest, and the use of implantable defibrillators in the pediatric population is limited by side effects. There is therefore a need to explore the curative potential of gene therapy for this disease. We investigated the efficacy and durability of viral gene transfer of the calsequestrin 2 (CASQ2) wild-type gene in a catecholaminergic polymorphic ventricular tachycardia knock-in mouse model carrying the CASQ2(R33Q/R33Q) (R33Q) mutation. We engineered an adeno-associated viral vector serotype 9 (AAV9) containing cDNA of CASQ2 wild-type (AAV9-CASQ2) plus the green fluorescent protein (GFP) gene to infect newborn R33Q mice studied by in vivo and in vitro protocols at 6, 9, and 12 months to investigate the ability of the infection to prevent the disease and adult R33Q mice studied after 2 months to assess whether the AAV9-CASQ2 delivery could revert the catecholaminergic polymorphic ventricular tachycardia phenotype. In both protocols, we observed the restoration of physiological expression and interaction of CASQ2, junctin, and triadin; the rescue of electrophysiological and ultrastructural abnormalities in calcium release units present in R33Q mice; and the lack of life-threatening arrhythmias. Our data demonstrate that viral gene transfer of wild-type CASQ2 into the heart of R33Q mice prevents and reverts severe manifestations of catecholaminergic polymorphic ventricular tachycardia and that this curative effect lasts for 1 year after a single injection of the vector, thus posing the rationale for the design of a clinical trial. © 2014 American Heart Association, Inc.
Mice Deficient for Glucagon Gene-Derived Peptides Display Normoglycemia and Hyperplasia of Islet α-Cells But Not of Intestinal L-Cells

PubMed Central

Hayashi, Yoshitaka; Yamamoto, Michiyo; Mizoguchi, Hiroyuki; Watanabe, Chika; Ito, Ryoichi; Yamamoto, Shiori; Sun, Xiao-yang; Murata, Yoshiharu

2009-01-01

Multiple bioactive peptides, including glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2, are derived from the glucagon gene (Gcg). In the present study, we disrupted Gcg by introduction of GFP cDNA and established a knock-in mouse line. Gcggfp/gfp mice that lack most, if not all, of Gcg-derived peptides were born in an expected Mendelian ratio without gross abnormalities. Gcggfp/gfp mice showed lower blood glucose levels at 2 wk of age, but those in adult Gcggfp/gfp mice were not significantly different from those in Gcg+/+ and Gcggfp/+ mice, even after starvation for 16 h. Serum insulin levels in Gcggfp/gfp mice were lower than in Gcg+/+ and Gcggfp/+ on ad libitum feeding, but no significant differences were observed on starvation. Islet α-cells and intestinal L-cells were readily visualized in Gcggfp/gfp and Gcggfp/+ mice under fluorescence. The Gcggfp/gfp postnatally developed hyperplasia of islet α-cells, whereas the population of intestinal L-cells was not increased. In the Gcggfp/gfp, expression of Aristaless-related homeobox (Arx) was markedly increased in pancreas but not in intestine and suggested involvement of Arx in differential regulation of proliferation of Gcg-expressing cells. These results illustrated that Gcg-derived peptides are dispensable for survival and maintaining normoglycemia in adult mice and that Gcg-derived peptides differentially regulate proliferation/differentiation of α-cells and L-cells. The present model is useful for analyzing glucose/energy metabolism in the absence of Gcg-derived peptides. It is useful also for analysis of the development, differentiation, and function of Gcg-expressing cells, because such cells are readily visualized by fluorescence in this model. PMID:19819987
Usefulness of running wheel for detection of congestive heart failure in dilated cardiomyopathy mouse model.

PubMed

Sugihara, Masami; Odagiri, Fuminori; Suzuki, Takeshi; Murayama, Takashi; Nakazato, Yuji; Unuma, Kana; Yoshida, Ken-ichi; Daida, Hiroyuki; Sakurai, Takashi; Morimoto, Sachio; Kurebayashi, Nagomi

2013-01-01

Inherited dilated cardiomyopathy (DCM) is a progressive disease that often results in death from congestive heart failure (CHF) or sudden cardiac death (SCD). Mouse models with human DCM mutation are useful to investigate the developmental mechanisms of CHF and SCD, but knowledge of the severity of CHF in live mice is necessary. We aimed to diagnose CHF in live DCM model mice by measuring voluntary exercise using a running wheel and to determine causes of death in these mice. A knock-in mouse with a mutation in cardiac troponin T (ΔK210) (DCM mouse), which results in frequent death with a t(1/2) of 70 to 90 days, was used as a DCM model. Until 2 months of age, average wheel-running activity was similar between wild-type and DCM mice (approximately 7 km/day). At approximately 3 months, some DCM mice demonstrated low running activity (LO: <1 km/day) while others maintained high running activity (HI: >5 km/day). In the LO group, the lung weight/body weight ratio was much higher than that in the other groups, and the lungs were infiltrated with hemosiderin-loaded alveolar macrophages. Furthermore, echocardiography showed more severe ventricular dilation and a lower ejection fraction, whereas Electrocardiography (ECG) revealed QRS widening. There were two patterns in the time courses of running activity before death in DCM mice: deaths with maintained activity and deaths with decreased activity. Our results indicate that DCM mice with low running activity developed severe CHF and that running wheels are useful for detection of CHF in mouse models. We found that approximately half of ΔK210 DCM mice die suddenly before onset of CHF, whereas others develop CHF, deteriorate within 10 to 20 days, and die.
Deregulated Ca2+ cycling underlies the development of arrhythmia and heart disease due to mutant obscurin

PubMed Central

Hu, Li-Yen R.; Ackermann, Maegen A.; Hecker, Peter A.; Prosser, Benjamin L.; King, Brendan; O’Connell, Kelly A.; Grogan, Alyssa; Meyer, Logan C.; Berndsen, Christopher E.; Wright, Nathan T.; Jonathan Lederer, W.; Kontrogianni-Konstantopoulos, Aikaterini

2017-01-01

Obscurins are cytoskeletal proteins with structural and regulatory roles encoded by OBSCN. Mutations in OBSCN are associated with the development of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Specifically, the R4344Q mutation present in immunoglobulin domain 58 (Ig58) was the first to be linked with the development of HCM. To assess the effects of R4344Q in vivo, we generated the respective knock-in mouse model. Mutant obscurins are expressed and incorporated normally into sarcomeres. The expression patterns of sarcomeric and Ca2+-cycling proteins are unaltered in sedentary 1-year-old knock-in myocardia, with the exception of sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase 2 (SERCA2) and pentameric phospholamban whose levels are significantly increased and decreased, respectively. Isolated cardiomyocytes from 1-year-old knock-in hearts exhibit increased Ca2+-transients and Ca2+-load in the sarcoplasmic reticulum and faster contractility kinetics. Moreover, sedentary 1-year-old knock-in animals develop tachycardia accompanied by premature ventricular contractions, whereas 2-month-old knock-in animals subjected to pressure overload develop a DCM-like phenotype. Structural analysis revealed that the R4344Q mutation alters the distribution of electrostatic charges over the Ig58 surface, thus interfering with its binding capabilities. Consistent with this, wild-type Ig58 interacts with phospholamban modestly, and this interaction is markedly enhanced in the presence of R4344Q. Together, our studies demonstrate that under sedentary conditions, the R4344Q mutation results in Ca2+ deregulation and spontaneous arrhythmia, whereas in the presence of chronic, pathological stress, it leads to cardiac remodeling and dilation. We postulate that enhanced binding between mutant obscurins and phospholamban leads to SERCA2 disinhibition, which may underlie the observed pathological alterations. PMID:28630914
Expression of GAD67 and Dlx5 in the taste buds of mice genetically lacking Mash1.

PubMed

Kito-Shingaki, Ayae; Seta, Yuji; Toyono, Takashi; Kataoka, Shinji; Kakinoki, Yasuaki; Yanagawa, Yuchio; Toyoshima, Kuniaki

2014-06-01

It has been reported that a subset of type III taste cells express glutamate decarboxylase (GAD)67, which is a molecule that synthesizes gamma-aminobutyric acid (GABA), and that Mash1 could be a potential regulator of the development of GABAnergic neurons via Dlx transcription factors in the central nervous system. In this study, we investigated the expression of GAD67 and Dlx in the embryonic taste buds of the soft palate and circumvallate papilla using Mash1 knockout (KO)/GAD67-GFP knock-in mice. In the wild-type animal, a subset of type III taste cells contained GAD67 in the taste buds of the soft palate and the developing circumvallate papilla, whereas GAD67-expressing taste bud cells were missing from Mash1 KO mice. A subset of type III cells expressed mRNA for Dlx5 in the wild-type animals, whereas Dlx5-expressing cells were not evident in the apical part of the circumvallate papilla and taste buds in the soft palate of Mash1 KO mice. Our results suggest that Mash1 is required for the expression of GAD67 and Dlx5 in taste bud cells. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics.

PubMed

Sasaki, Masato; Knobbe, Christiane B; Munger, Joshua C; Lind, Evan F; Brenner, Dirk; Brüstle, Anne; Harris, Isaac S; Holmes, Roxanne; Wakeham, Andrew; Haight, Jillian; You-Ten, Annick; Li, Wanda Y; Schalm, Stefanie; Su, Shinsan M; Virtanen, Carl; Reifenberger, Guido; Ohashi, Pamela S; Barber, Dwayne L; Figueroa, Maria E; Melnick, Ari; Zúñiga-Pflücker, Juan-Carlos; Mak, Tak W

2012-08-30

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the ‘oncometabolite’ R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.
Mice that lack the C-terminal region of Reelin exhibit behavioral abnormalities related to neuropsychiatric disorders

PubMed Central

Sakai, Kaori; Shoji, Hirotaka; Kohno, Takao; Miyakawa, Tsuyoshi; Hattori, Mitsuharu

2016-01-01

The secreted glycoprotein Reelin is believed to play critical roles in the pathogenesis of several neuropsychiatric disorders. The highly basic C-terminal region (CTR) of Reelin is necessary for efficient activation of its downstream signaling, and the brain structure of knock-in mice that lack the CTR (ΔC-KI mice) is impaired. Here, we performed a comprehensive behavioral test battery on ΔC-KI mice, in order to evaluate the effects of partial loss-of-function of Reelin on brain functions. The ΔC-KI mice were hyperactive and exhibited reduced anxiety-like and social behaviors. The working memory in ΔC-KI mice was impaired in a T-maze test. There was little difference in spatial reference memory, depression-like behavior, prepulse inhibition, or fear memory between ΔC-KI and wild-type mice. These results suggest that CTR-dependent Reelin functions are required for some specific normal brain functions and that ΔC-KI mice recapitulate some aspects of neuropsychiatric disorders, such as schizophrenia, bipolar disorder, and autism spectrum disorder. PMID:27346785
Biological constraints limit the use of rapamycin-inducible FKBP12-Inp54p for depleting PIP2 in dorsal root ganglia neurons.

PubMed

Coutinho-Budd, Jaeda C; Snider, Samuel B; Fitzpatrick, Brendan J; Rittiner, Joseph E; Zylka, Mark J

2013-09-08

Rapamycin-induced translocation systems can be used to manipulate biological processes with precise temporal control. These systems are based on rapamycin-induced dimerization of FK506 Binding Protein 12 (FKBP12) with the FKBP Rapamycin Binding (FRB) domain of mammalian target of rapamycin (mTOR). Here, we sought to adapt a rapamycin-inducible phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphatase (Inp54p) system to deplete PIP2 in nociceptive dorsal root ganglia (DRG) neurons. We genetically targeted membrane-tethered CFP-FRBPLF (a destabilized FRB mutant) to the ubiquitously expressed Rosa26 locus, generating a Rosa26-FRBPLF knockin mouse. In a second knockin mouse line, we targeted Venus-FKBP12-Inp54p to the Calcitonin gene-related peptide-alpha (CGRPα) locus. We hypothesized that after intercrossing these mice, rapamycin treatment would induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in CGRP+ DRG neurons. In control experiments with cell lines, rapamycin induced translocation of Venus-FKBP12-Inp54p to the plasma membrane, and subsequent depletion of PIP2, as measured with a PIP2 biosensor. However, rapamycin did not induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in FRBPLF-expressing DRG neurons (in vitro or in vivo). Moreover, rapamycin treatment did not alter PIP2-dependent thermosensation in vivo. Instead, rapamycin treatment stabilized FRBPLF in cultured DRG neurons, suggesting that rapamycin promoted dimerization of FRBPLF with endogenous FKBP12. Taken together, our data indicate that these knockin mice cannot be used to inducibly deplete PIP2 in DRG neurons. Moreover, our data suggest that high levels of endogenous FKBP12 could compete for binding to FRBPLF, hence limiting the use of rapamycin-inducible systems to cells with low levels of endogenous FKBP12.
Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

PubMed

Lu, Yifei; Yan, Hongxiang; Deng, Jiezhong; Huang, Zhigang; Jin, Xurui; Yu, Yanlan; Hu, Qiwen; Hu, Fuquan; Wang, Jing

2017-09-18

Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.
Dominant-negative diabetes insipidus and other endocrinopathies

PubMed Central

Phillips, John A.

2003-01-01

Familial neurohypophyseal diabetes insipidus (FNDI) in humans is an autosomal dominant disorder caused by a variety of mutations in the arginine vasopressin (AVP) precursor. A new report demonstrates how heterozygosity for an AVP mutation causes FNDI (see the related article beginning on page 1697). Using an AVP knock-in mutation in mice, the study shows that FNDI is caused by retention of AVP precursors and progressive loss of AVP-producing neurons. PMID:14660740

A sodium channel knockin mutant (NaV1.4-R669H) mouse model of hypokalemic periodic paralysis

PubMed Central

Wu, Fenfen; Mi, Wentao; Burns, Dennis K.; Fu, Yu; Gray, Hillery F.; Struyk, Arie F.; Cannon, Stephen C.

2011-01-01

Hypokalemic periodic paralysis (HypoPP) is an ion channelopathy of skeletal muscle characterized by attacks of muscle weakness associated with low serum K+. HypoPP results from a transient failure of muscle fiber excitability. Mutations in the genes encoding a calcium channel (CaV1.1) and a sodium channel (NaV1.4) have been identified in HypoPP families. Mutations of NaV1.4 give rise to a heterogeneous group of muscle disorders, with gain-of-function defects causing myotonia or hyperkalemic periodic paralysis. To address the question of specificity for the allele encoding the NaV1.4-R669H variant as a cause of HypoPP and to produce a model system in which to characterize functional defects of the mutant channel and susceptibility to paralysis, we generated knockin mice carrying the ortholog of the gene encoding the NaV1.4-R669H variant (referred to herein as R669H mice). Homozygous R669H mice had a robust HypoPP phenotype, with transient loss of muscle excitability and weakness in low-K+ challenge, insensitivity to high-K+ challenge, dominant inheritance, and absence of myotonia. Recovery was sensitive to the Na+/K+-ATPase pump inhibitor ouabain. Affected fibers had an anomalous inward current at hyperpolarized potentials, consistent with the proposal that a leaky gating pore in R669H channels triggers attacks, whereas a reduction in the amplitude of action potentials implies additional loss-of-function changes for the mutant NaV1.4 channels. PMID:21881211
Rett Syndrome Mutation MeCP2 T158A Disrupts DNA Binding, Protein Stability and ERP Responses

PubMed Central

Goffin, Darren; Allen, Megan; Zhang, Le; Amorim, Maria; Wang, I-Ting Judy; Reyes, Arith-Ruth S.; Mercado-Berton, Amy; Ong, Caroline; Cohen, Sonia; Hu, Linda; Blendy, Julie A.; Carlson, Gregory C.; Siegel, Steve J.; Greenberg, Michael E.; Zhou, Zhaolan (Joe)

2011-01-01

Mutations in the MECP2 gene cause the autism spectrum disorder Rett Syndrome (RTT). One of the most common mutations associated with RTT occurs at MeCP2 Threonine 158 converting it to Methionine (T158M) or Alanine (T158A). To understand the role of T158 mutation in the pathogenesis of RTT, we generated knockin mice recapitulating MeCP2 T158A mutation. Here we show a causal role for T158A mutation in the development of RTT-like phenotypes including developmental regression, motor dysfunction, and learning and memory deficits. These phenotypes resemble those in Mecp2-null mice and manifest through a reduction in MeCP2 binding to methylated DNA and a decrease in MeCP2 protein stability. Importantly, the age-dependent development of event-related neuronal responses are disrupted by MeCP2 mutation, suggesting that impaired neuronal circuitry underlies the pathogenesis of RTT and that assessment of event-related potentials may serve as a biomarker for RTT and treatment evaluation. PMID:22119903
The Origin, Development and Molecular Diversity of Rodent Olfactory Bulb Glutamatergic Neurons Distinguished by Expression of Transcription Factor NeuroD1.

PubMed

Roybon, Laurent; Mastracci, Teresa L; Li, Joyce; Stott, Simon R W; Leiter, Andrew B; Sussel, Lori; Brundin, Patrik; Li, Jia-Yi

2015-01-01

Production of olfactory bulb neurons occurs continuously in the rodent brain. Little is known, however, about cellular diversity in the glutamatergic neuron subpopulation. In the central nervous system, the basic helix-loop-helix transcription factor NeuroD1 (ND1) is commonly associated with glutamatergic neuron development. In this study, we utilized ND1 to identify the different subpopulations of olfactory bulb glutamategic neurons and their progenitors, both in the embryo and postnatally. Using knock-in mice, transgenic mice and retroviral transgene delivery, we demonstrate the existence of several different populations of glutamatergic olfactory bulb neurons, the progenitors of which are ND1+ and ND1- lineage-restricted, and are temporally and regionally separated. We show that the first olfactory bulb glutamatergic neurons produced - the mitral cells - can be divided into molecularly diverse subpopulations. Our findings illustrate the complexity of neuronal diversity in the olfactory bulb and that seemingly homogenous neuronal populations can consist of multiple subpopulations with unique molecular signatures of transcription factors and expressing neuronal subtype-specific markers.
The p27 Pathway Modulates the Regulation of Skeletal Growth and Osteoblastic Bone Formation by Parathyroid Hormone-Related Peptide.

PubMed

Zhu, Min; Zhang, Jing; Dong, Zhan; Zhang, Ying; Wang, Rong; Karaplis, Andrew; Goltzman, David; Miao, Dengshun

2015-11-01

Parathyroid hormone-related peptide (PTHrP) 1-84 knock-in mice (Pthrp KI) develop skeletal growth retardation and defective osteoblastic bone formation. To further examine the mechanisms underlying this phenotype, microarray analyses of differential gene expression profiles were performed in long bone extracts from Pthrp KI mice and their wild-type (WT) littermates. We found that the expression levels of p27, p16, and p53 were significantly upregulated in Pthrp KI mice relative to WT littermates. To determine whether p27 was involved in the regulation by PTHrP of skeletal growth and development in vivo, we generated compound mutant mice, which were homozygous for both p27 deletion and the Pthrp KI mutation (p27(-/-) Pthrp KI). We then compared p27(-/-) Pthrp KI mice with p27(-/-), Pthrp KI, and WT littermates. Deletion of p27 in Pthrp KI mice resulted in a longer lifespan, increased body weight, and improvement in skeletal growth. At 2 weeks of age, skeletal parameters, including length of long bones, size of epiphyses, numbers of proliferating cell nuclear antigen (PCNA)-positive chondrocytes, bone mineral density, trabecular bone volume, osteoblast numbers, and alkaline phosphatase (ALP)-, type I collagen-, and osteocalcin-positive bone areas were increased in p27(-/-) mice and reduced in both Pthrp KI and p27(-/-) Pthrp KI mice compared with WT mice; however, these parameters were increased in p27(-/-) Pthrp KI mice compared with Pthrp KI mice. As well, protein expression levels of PTHR, IGF-1, and Bmi-1, and the numbers of total colony-forming unit fibroblastic (CFU-f) and ALP-positive CFU-f were similarly increased in p27(-/-) Pthrp KI mice compared with Pthrp KI mice. Our results demonstrate that deletion of p27 in Pthrp KI mice can partially rescue defects in skeletal growth and osteoblastic bone formation by enhancing endochondral bone formation and osteogenesis. These studies, therefore, indicate that the p27 pathway may function downstream in the action of PTHrP to regulate skeletal growth and development. © 2015 American Society for Bone and Mineral Research.
Imbalanced gp130 signalling in ApoE-deficient mice protects against atherosclerosis.

PubMed

Jones, Gareth W; McLeod, Louise; Kennedy, Catherine L; Bozinovski, Steven; Najdovska, Meri; Jenkins, Brendan J

2015-02-01

Interleukin (IL)-6 is a key modulator of the acute phase response (APR), and while both are implicated in atherosclerosis, the pathological role of specific IL-6 signalling cascades is ill-defined. Since IL-6 employs the cytokine receptor gp130 to primarily activate the STAT3 pathway, here we evaluate whether gp130-dependent STAT3 activation modulates atherosclerosis. High-fat diet-induced atherosclerosis was established in ApoE(-/-) mice crossed with gp130(F/F) knock-in mice displaying elevated gp130-dependent STAT3 activation and production of the APR protein, serum amyloid A (SAA). Also generated were gp130(F/F):Stat3(-/+):ApoE(-/-) mice displaying genetically-normalised STAT3 activation and SAA levels, and bone marrow chimeras involving ApoE(-/-) and gp130(F/F):ApoE(-/-) mice. At 10 weeks post high-fat diet, aortic atherosclerotic lesions, including the presence of CD68(+) macrophages, and plasma lipid and SAA profiles, were assessed. Aortic plaque development and plasma triglyceride levels in gp130(F/F):ApoE(-/-) mice were significantly reduced (3-fold, P < 0.001) compared to ApoE(-/-) littermates. By contrast, in gp130(F/F):ApoE(-/-) mice, atherosclerotic plaques contained augmented CD68(+) macrophage infiltrates, and plasma SAA levels were elevated, compared to ApoE(-/-) mice. Atherosclerotic lesion development and plasma triglyceride levels in gp130(F/F):ApoE(-/-) and gp130(F/F):Stat3(-/+):ApoE(-/-) mice were comparable, despite a significant (P < 0.05) reduction in macrophage numbers in lesions, and also plasma SAA levels, in gp130(F/F):Stat3(-/+):ApoE(-/-) mice. Aortic plaque development and plasma triglyceride levels were comparable in ApoE(-/-) mice reconstituted with gp130(F/F):ApoE(-/-) (ApoE(F/F:ApoE)) or ApoE(-/-) (ApoE(ApoE)) bone marrow cells. Deregulation of gp130/STAT3 signalling augments the APR and macrophage infiltration during atherosclerosis without impacting on the development of aortic plaques. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
Loss of Activity-Induced Phosphorylation of MeCP2 Enhances Synaptogenesis, LTP, and Spatial Memory

PubMed Central

Li, Hongda; Zhong, Xiaofen; Chau, Kevin Fongching; Williams, Emily Cunningham; Chang, Qiang

2012-01-01

DNA methylation-dependent epigenetic mechanisms underlie the development and function of the mammalian brain. MeCP2 expresses highly in neurons, and functions as a molecular linker between DNA methylation, chromatin remodeling and transcription regulation. Previous in vitro studies showed neuronal activity-induced phosphorylation (NAIP) of MeCP2 precedes its release from the Bdnf promoter and the ensuing Bdnf transcription. However, the in vivo function of this phosphorylation event remains elusive. We generated knockin mice that lack NAIP of MeCP2, and show here the Mecp2 phospho-mutant mice perform better in hippocampus-dependent memory tests, present enhanced LTP at two synapses in the hippocampus, and show increased excitatory synaptogenesis. At the molecular level, the phospho-mutant MeCP2 protein binds more tightly to several MeCP2 target gene promoters and alters the expression of these genes. Our results supply the first genetic evidence that NAIP of MeCP2 is required in modulating dynamic functions of the adult mouse brain. PMID:21765426
Animal Models of Inflammasomopathies Reveal Roles for Innate but not Adaptive Immunity

PubMed Central

Brydges, Susannah D; Mueller, James L; McGeough, Matthew D; Pena, Carla A; Misaghi, Amirhossein; Gandhi, Chhavi; Putnam, Chris D; Boyle, David L; Firestein, Gary S; Horner, Anthony A; Soroosh, Pejman; Watford, Wendy T; O’Shea, John J; Kastner, Daniel L; Hoffman, Hal M

2009-01-01

SUMMARY Cryopyrin (NALP3) mediates formation of the inflammasome, a protein complex responsible for cleavage of pro-IL-1β to its active form. Mutations in the cryopyrin gene, NLRP3, cause the autoinflammatory disease spectrum: cryopyrin-associated periodic syndromes (CAPS). The central role of IL-1β in CAPS is supported by the remarkable response to IL-1 targeted therapy. We developed two novel Nlrp3 mutant knock-in mouse strains to model CAPS to examine the role of other inflammatory mediators and adaptive immune responses in an innate immune driven disease. These mice had systemic inflammation and poor growth, similar to some human CAPS patients, and demonstrated early mortality, primarily mediated by myeloid cells. Mating these mutant mice to various knock-out backgrounds confirmed the mouse disease phenotype required an intact inflammasome, was only partially dependent on IL-1β, and was independent of T cells. This data suggests CAPS are true inflammasomopathies and provide insight for more common inflammatory disorders. PMID:19501000
In Vivo Regulation of NGF-Mediated Functions by Nedd4-2 Ubiquitination of TrkA

PubMed Central

Yu, Tao; Calvo, Laura; Anta, Begoña; López-Benito, Saray; López-Bellido, Roger; Vicente-García, Cristina; Tessarollo, Lino; Rodriguez, Raquel E.

2014-01-01

Trk neurotrophin receptor ubiquitination in response to ligand activation regulates signaling, trafficking, and degradation of the receptors. However, the in vivo consequences of Trk ubiquitination remain to be addressed. We have developed a mouse model with a mutation in the TrkA neurotrophin receptor (P782S) that results in reduced ubiquitination due to a lack of binding to the E3 ubiquitin ligase, Nedd4-2. In vivo analyses of TrkAP782S indicate that defective ubiquitination of the TrkA mutant results in an altered trafficking and degradation of the receptor that affects the survival of sensory neurons. The dorsal root ganglia from the TrkAP782S knock-in mice display an increased number of neurons expressing CGRP and substance P. Moreover, the mutant mice show enhanced sensitivity to thermal and inflammatory pain. Our results indicate that the ubiquitination of the TrkA neurotrophin receptor plays a critical role in NGF-mediated functions, such as neuronal survival and sensitivity to pain. PMID:24760869
The Akt1-eNOS axis illustrates the specificity of kinase-substrate relationships in vivo.

PubMed

Schleicher, Michael; Yu, Jun; Murata, Takahisa; Derakhshan, Berhad; Atochin, Dimitriy; Qian, Li; Kashiwagi, Satoshi; Di Lorenzo, Annarita; Harrison, Kenneth D; Huang, Paul L; Sessa, William C

2009-08-04

Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1(-/-) mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme "constitutively active" or "less active." The eNOS mutations did not influence several phenotypes in Akt1(-/-) mice; however, the defective postnatal angiogenesis characteristic of Akt1(-/-) mice was rescued by crossing the Akt1(-/-) mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased production of HIF-1alpha-responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling.
Bone marrow-specific knock-in of a non-activatable Ikkα kinase mutant influences haematopoiesis but not atherosclerosis in Apoe-deficient mice.

PubMed

Tilstam, Pathricia V; Gijbels, Marion J; Habbeddine, Mohamed; Cudejko, Céline; Asare, Yaw; Theelen, Wendy; Zhou, Baixue; Döring, Yvonne; Drechsler, Maik; Pawig, Lukas; Simsekyilmaz, Sakine; Koenen, Rory R; de Winther, Menno P J; Lawrence, Toby; Bernhagen, Jürgen; Zernecke, Alma; Weber, Christian; Noels, Heidi

2014-01-01

The Ikkα kinase, a subunit of the NF-κB-activating IKK complex, has emerged as an important regulator of inflammatory gene expression. However, the role of Ikkα-mediated phosphorylation in haematopoiesis and atherogenesis remains unexplored. In this study, we investigated the effect of a bone marrow (BM)-specific activation-resistant Ikkα mutant knock-in on haematopoiesis and atherosclerosis in mice. Apolipoprotein E (Apoe)-deficient mice were transplanted with BM carrying an activation-resistant Ikkα gene (Ikkα(AA/AA)Apoe(-/-) ) or with Ikkα(+/+)Apoe(-/-) BM as control and were fed a high-cholesterol diet for 8 or 13 weeks. Interestingly, haematopoietic profiling by flow cytometry revealed a significant decrease in B-cells, regulatory T-cells and effector memory T-cells in Ikkα(AA/AA)Apoe(-/-) BM-chimeras, whereas the naive T-cell population was increased. Surprisingly, no differences were observed in the size, stage or cellular composition of atherosclerotic lesions in the aorta and aortic root of Ikkα(AA/AA)Apoe(-/-) vs Ikkα(+/+)Apoe(-/-) BM-transplanted mice, as shown by histological and immunofluorescent stainings. Necrotic core sizes, apoptosis, and intracellular lipid deposits in aortic root lesions were unaltered. In vitro, BM-derived macrophages from Ikkα(AA/AA)Apoe(-/-) vs Ikkα(+/+)Apoe(-/-) mice did not show significant differences in the uptake of oxidized low-density lipoproteins (oxLDL), and, with the exception of Il-12, the secretion of inflammatory proteins in conditions of Tnf-α or oxLDL stimulation was not significantly altered. Furthermore, serum levels of inflammatory proteins as measured with a cytokine bead array were comparable. Our data reveal an important and previously unrecognized role of haematopoietic Ikkα kinase activation in the homeostasis of B-cells and regulatory T-cells. However, transplantation of Ikkα(AA) mutant BM did not affect atherosclerosis in Apoe(-/-) mice. This suggests that the diverse functions of Ikkα in haematopoietic cells may counterbalance each other or may not be strong enough to influence atherogenesis, and reveals that targeting haematopoietic Ikkα kinase activity alone does not represent a therapeutic approach.
Proteome and behavioral alterations in phosphorylation-deficient mutant Collapsin Response Mediator Protein2 knock-in mice.

PubMed

Nakamura, Haruko; Takahashi-Jitsuki, Aoi; Makihara, Hiroko; Asano, Tetsuya; Kimura, Yayoi; Nakabayashi, Jun; Yamashita, Naoya; Kawamoto, Yuko; Nakamura, Fumio; Ohshima, Toshio; Hirano, Hisashi; Tanaka, Fumiaki; Goshima, Yoshio

2018-05-11

CRMP2, alternatively designated as DPYSL2, was the first CRMP family member to be identified as an intracellular molecule mediating the signaling of the axon guidance molecule Semaphorin 3A (Sema3A). In Sema3A signaling, cyclin-dependent kinase 5 (Cdk5) primarily phosphorylates CRMP2 at Ser522. Glycogen synthase kinase-3β (GSK-3β) subsequently phosphorylates the residues of Thr509 and Thr514 of CRMP2. Previous studies showed that CRMP2 is involved in pathogenesis of neurological disorders such as Alzheimer's disease. In Alzheimer's disease, hyper-phosphorylated forms of CRMP2 are accumulated in the paired helical filaments. To get insight into the possible involvement of the phosphorylation of CRMP2 in pathogenesis of neurological disorders, we previously created CRMP2 S522A knock-in (crmp2 ki/ki ) mice and demonstrated that the phosphorylation of CRMP2 at Ser522 is involved in normal dendrite patterning in cortical neurons. However, the behavioral impact and in vivo signaling network of the CRMP2 phosphorylation are not fully understood. In this study, we performed behavioral and proteomics analysis of crmp2 ki/ki mice. The crmp2 ki/ki mice appeared healthy and showed no obvious differences in physical characteristics compared to wild-type mice, but they showed impaired emotional behavior, reduced sociality, and low sensitivity to pain stimulation. Through mass-spectrometry-based proteomic analysis, we found that 59 proteins were increased and 77 proteins were decreased in the prefrontal cortex of crmp2 ki/ki mice. Notably, CRMP3, CRMP4, and CRMP5, the other CRMP family proteins, were increased in crmp2 ki/ki mice. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses identified 14 pathways in increased total proteins and 13 pathways in decreased total proteins which are associated with the pathogenesis of Parkinson's, Alzheimer's, and Huntington's diseases. We also detected 20 pathways in increased phosphopeptides and 16 pathways in decreased phosphopeptides including "inflammatory mediator regulation of TRP channels" in crmp2 ki/ki mice. Our study suggests that the phosphorylation of CRMP2 at Ser522 is involved in the signaling pathways that may be related to neuropsychiatric and neurodegenerative diseases and pain. Copyright © 2018. Published by Elsevier Ltd.
Severe combined immunodeficiency in Sting V154M/WT mice.

PubMed

Bouis, Delphine; Kirstetter, Peggy; Arbogast, Florent; Lamon, Delphine; Delgado, Virginia; Jung, Sophie; Ebel, Claudine; Jacobs, Hugues; Knapp, Anne-Marie; Jeremiah, Nadia; Belot, Alexandre; Martin, Thierry; Crow, Yanick J; André-Schmutz, Isabelle; Korganow, Anne-Sophie; Rieux-Laucat, Frédéric; Soulas-Sprauel, Pauline

2018-05-23

Autosomal dominant gain-of-function (GOF) mutations in human STING (Stimulator of Interferon Genes) lead to a severe autoinflammatory disease called SAVI (STING Associated Vasculopathy with onset in Infancy), associated with enhanced expression of interferon (IFN) stimulated gene (ISG) transcripts. The goal of this study was to analyze the phenotype of a new mouse model of Sting hyperactivation, and the role of type I IFN in this system. We generated a knock-in model carrying an amino acid substitution (V154M) in mouse Sting, corresponding to a recurrent mutation seen in human patients with SAVI. Hematopoietic development and tissue histology were analyzed. Lymphocyte activation and proliferation were assessed in vitro. Sting V154M/WT mice were crossed to IFNAR (IFNα/β Receptor) knock-out mice in order to evaluate the type I IFN-dependence of the mutant Sting phenotype recorded. In Sting V154M/WT mice we detected variable expression of inflammatory infiltrates in the lungs and kidneys. These mice showed a marked decrease in survival and developed a severe combined immunodeficiency disease (SCID) affecting B, T and NK cells, with an almost complete lack of antibodies and a significant expansion of monocytes and granulocytes. The blockade in B and T cell development was present from early immature stages in bone marrow and thymus. In addition, in vitro experiments revealed an intrinsic proliferative defect of mature T cells. Whilst the V154M/WT mutant demonstrated increased expression of ISGs, the SCID phenotype was not reversed in Sting V154M/WT IFNAR knock-out mice. However, the anti-proliferative defect in T cells was partially rescued by IFNAR deficiency. Sting GOF mice developed an IFN-independent SCID phenotype with a T, B and NK cell developmental defect and hypogammaglobulinemia, associated with signs of inflammation in lungs and kidneys. Only the intrinsic proliferative defect of T cells was, partially, IFN-dependent. Copyright © 2018. Published by Elsevier Inc.
Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice.

PubMed

Kon, Ayana; Yamazaki, Satoshi; Nannya, Yasuhito; Kataoka, Keisuke; Ota, Yasunori; Nakagawa, Masahiro Marshall; Yoshida, Kenichi; Shiozawa, Yusuke; Morita, Maiko; Yoshizato, Tetsuichi; Sanada, Masashi; Nakayama, Manabu; Koseki, Haruhiko; Nakauchi, Hiromitsu; Ogawa, Seishi

2018-02-08

Splicing factor mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis, but their roles in the development of MDS have not been fully elucidated. In the present study, we investigated the consequence of mutant Srsf2 expression using newly generated Vav1-Cre -mediated conditional knockin mice. Mice carrying a heterozygous Srsf2 P95H mutation showed significantly reduced numbers of hematopoietic stem and progenitor cells (HSPCs) and differentiation defects both in the steady-state condition and transplantation settings. Srsf2 -mutated hematopoietic stem cells (HSCs) showed impaired long-term reconstitution compared with control mice in competitive repopulation assays. Although the Srsf2 mutant mice did not develop MDS under the steady-state condition, when their stem cells were transplanted into lethally irradiated mice, the recipients developed anemia, leukopenia, and erythroid dysplasia, which suggests the role of replicative stress in the development of an MDS-like phenotype in Srsf2 -mutated mice. RNA sequencing of the Srsf2 -mutated HSPCs revealed a number of abnormal splicing events and differentially expressed genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies, such as Csf3r , Fyn , Gnas , Nsd1 , Hnrnpa2b1 , and Trp53bp1 Among the mutant Srsf2 -associated splicing events, most commonly observed were the enhanced inclusion and/or exclusion of cassette exons, which were caused by the altered consensus motifs for the recognition of exonic splicing enhancers. Our findings suggest that the mutant Srsf2 leads to a compromised HSC function by causing abnormal RNA splicing and expression, contributing to the deregulated hematopoiesis that recapitulates the MDS phenotypes, possibly as a result of additional genetic and/or environmental insults. © 2018 by The American Society of Hematology.
Distinct Brca1 Mutations Differentially Reduce Hematopoietic Stem Cell Function.

PubMed

Mgbemena, Victoria E; Signer, Robert A J; Wijayatunge, Ranjula; Laxson, Travis; Morrison, Sean J; Ross, Theodora S

2017-01-24

BRCA1 is a well-known DNA repair pathway component and a tissue-specific tumor suppressor. However, its role in hematopoiesis is uncertain. Here, we report that a cohort of patients heterozygous for BRCA1 mutations experienced more hematopoietic toxicity from chemotherapy than those with BRCA2 mutations. To test whether this reflects a requirement for BRCA1 in hematopoiesis, we generated mice with Brca1 mutations in hematopoietic cells. Mice homozygous for a null Brca1 mutation in the embryonic hematopoietic system (Vav1-iCre;Brca1 F22-24/F22-24 ) developed hematopoietic defects in early adulthood that included reduced hematopoietic stem cells (HSCs). Although mice homozygous for a huBRCA1 knockin allele (Brca1 BRCA1/BRCA1 ) were normal, mice with a mutant huBRCA1/5382insC allele and a null allele (Mx1-Cre;Brca1 F22-24/5382insC ) had severe hematopoietic defects marked by a complete loss of hematopoietic stem and progenitor cells. Our data show that Brca1 is necessary for HSC maintenance and normal hematopoiesis and that distinct mutations lead to different degrees of hematopoietic dysfunction. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Translational Mouse Models of Autism: Advancing Toward Pharmacological Therapeutics

PubMed Central

Kazdoba, Tatiana M.; Leach, Prescott T.; Yang, Mu; Silverman, Jill L.; Solomon, Marjorie

2016-01-01

Animal models provide preclinical tools to investigate the causal role of genetic mutations and environmental factors in the etiology of autism spectrum disorder (ASD). Knockout and humanized knock-in mice, and more recently knockout rats, have been generated for many of the de novo single gene mutations and copy number variants (CNVs) detected in ASD and comorbid neurodevelopmental disorders. Mouse models incorporating genetic and environmental manipulations have been employed for preclinical testing of hypothesis-driven pharmacological targets, to begin to develop treatments for the diagnostic and associated symptoms of autism. In this review, we summarize rodent behavioral assays relevant to the core features of autism, preclinical and clinical evaluations of pharmacological interventions, and strategies to improve the translational value of rodent models of autism. PMID:27305922
Deletion of Snai2 and Snai3 Results in Impaired Physical Development Compounded by Lymphocyte Deficiency

PubMed Central

Pioli, Peter D.; Dahlem, Timothy J.; Weis, Janis J.; Weis, John H.

2013-01-01

The Snail family of transcriptional regulators consists of three highly conserved members. These proteins regulate (repress) transcription via the recruitment of histone deacetylases to target gene promoters that possess the appropriate E-box binding sequences. Murine Snai1 is required for mouse development while Snai2 deficient animals survive with some anomalies. Less is known about the third member of the family, Snai3. To investigate the function of Snai3, we generated a conditional knockin mouse. Utilizing Cre-mediated deletion to facilitate the ablation of Snai3 in T cells or the entire animal, we found little to no effect of the loss of Snai3 in the entire animal or in T cell lineages. This finding provided the hypothesis that absence of Snai3 was mitigated, in part, by the presence of Snai2. To test this hypothesis we created Snai2/Snai3 double deficient mice. The developmental consequences of lacking both of these proteins was manifested in stunted growth, a paucity of offspring including a dramatic deficiency of female mice, and impaired immune cell development within the lymphoid lineages. PMID:23874916
Dmp1 Null Mice Develop a Unique Osteoarthritis-like Phenotype

PubMed Central

Zhang, Qi; Lin, Shuxian; Liu, Ying; Yuan, Baozhi; Harris, Steph E; Feng, Jian Q.

2016-01-01

Patients with hypophosphatemia rickets (including DMP1 mutations) develop severe osteoarthritis (OA), although the mechanism is largely unknown. In this study, we first identified the expression of DMP1 in hypertrophic chondrocytes using immunohistochemistry (IHC) and X-gal analysis of Dmp1-knockout-lacZ-knockin heterozygous mice. Next, we characterized the OA-like phenotype in Dmp1 null mice from 7-week-old to one-year-old using multiple techniques, including X-ray, micro-CT, H&E staining, Goldner staining, scanning electronic microscopy, IHC assays, etc. We found a classical OA-like phenotype in Dmp1 null mice such as articular cartilage degradation, osteophyte formation, and subchondral osteosclerosis. These Dmp1 null mice also developed unique pathological changes, including a biphasic change in their articular cartilage from the initial expansion of hypertrophic chondrocytes at the age of 1-month to a quick diminished articular cartilage layer at the age of 3-months. Further, these null mice displayed severe enlarged knees and poorly formed bone with an expanded osteoid area. To address whether DMP1 plays a direct role in the articular cartilage, we deleted Dmp1 specifically in hypertrophic chondrocytes by crossing the Dmp1-loxP mice with Col X Cre mice. Interestingly, these conditional knockout mice didn't display notable defects in either the articular cartilage or the growth plate. Because of the hypophosphatemia remained in the entire life span of the Dmp1 null mice, we also investigated whether a high phosphate diet would improve the OA-like phenotype. A 8-week treatment of a high phosphate diet significantly rescued the OA-like defect in Dmp1 null mice, supporting the critical role of phosphate homeostasis in maintaining the healthy joint morphology and function. Taken together, this study demonstrates a unique OA-like phenotype in Dmp1 null mice, but a lack of the direct impact of DMP1 on chondrogenesis. Instead, the regulation of phosphate homeostasis by DMP1 via the axis of “FGF23-renal phosphorus reabsorption” is vital for maintaining a healthy joint. PMID:27766035
The granin VGF promotes genesis of secretory vesicles, and regulates circulating catecholamine levels and blood pressure.

PubMed

Fargali, Samira; Garcia, Angelo L; Sadahiro, Masato; Jiang, Cheng; Janssen, William G; Lin, Wei-Jye; Cogliani, Valeria; Elste, Alice; Mortillo, Steven; Cero, Cheryl; Veitenheimer, Britta; Graiani, Gallia; Pasinetti, Giulio M; Mahata, Sushil K; Osborn, John W; Huntley, George W; Phillips, Greg R; Benson, Deanna L; Bartolomucci, Alessandro; Salton, Stephen R

2014-05-01

Secretion of proteins and neurotransmitters from large dense core vesicles (LDCVs) is a highly regulated process. Adrenal LDCV formation involves the granin proteins chromogranin A (CgA) and chromogranin B (CgB); CgA- and CgB-derived peptides regulate catecholamine levels and blood pressure. We investigated function of the granin VGF (nonacronymic) in LDCV formation and the regulation of catecholamine levels and blood pressure. Expression of exogenous VGF in nonendocrine NIH 3T3 fibroblasts resulted in the formation of LDCV-like structures and depolarization-induced VGF secretion. Analysis of germline VGF-knockout mouse adrenal medulla revealed decreased LDCV size in noradrenergic chromaffin cells, increased adrenal norepinephrine and epinephrine content and circulating plasma epinephrine, and decreased adrenal CgB. These neurochemical changes in VGF-knockout mice were associated with hypertension. Germline knock-in of human VGF1-615 into the mouse Vgf locus rescued the hypertensive knockout phenotype, while knock-in of a truncated human VGF1-524 that lacks several C-terminal peptides, including TLQP-21, resulted in a small but significant increase in systolic blood pressure compared to hVGF1-615 mice. Finally, acute and chronic administration of the VGF-derived peptide TLQP-21 to rodents decreased blood pressure. Our studies establish a role for VGF in adrenal LDCV formation and the regulation of catecholamine levels and blood pressure.
Real-time visualization of macromolecule uptake by epidermal Langerhans cells in living animals.

PubMed

Frugé, Rachel E; Krout, Colleen; Lu, Ran; Matsushima, Hironori; Takashima, Akira

2012-03-01

As a skin-resident member of the dendritic cell family, Langerhans cells (LCs) are generally regarded to function as professional antigen-presenting cells. Here we report a simple method to visualize the endocytotic activity of LCs in living animals. BALB/c mice received subcutaneous injection of FITC-conjugated dextran (DX) probes into the ear skin and were then examined under confocal microscopy. Large numbers of FITC(+) epidermal cells became detectable 12-24 hours after injection as background fluorescence signals began to disappear. Most (>90%) of the FITC(+) epidermal cells expressed Langerin, and >95% of Langerin(+) epidermal cells exhibited significant FITC signals. To assess intracellular localization, Alexa Fluor 546-conjugated DX probes were locally injected into IAβ-enhanced green fluorescent protein (EGFP) knock-in mice and Langerin-EGFP-diphtheria toxin receptor mice--three dimensional rotation images showed close association of most of the internalized DX probes with major histocompatibility complex (MHC) class II molecules, but not with Langerin molecules. These observations support the current view that LCs constantly sample surrounding materials, including harmful and innocuous antigens, at the environmental interface. Our data also validate the potential utility of the newly developed imaging approach to monitor LC function in wild-type animals.
Myeloproliferative Neoplasm Animal Models

PubMed Central

Mullally, Ann; Lane, Steven W.; Brumme, Kristina; Ebert, Benjamin L.

2012-01-01

Synopsis Myeloproliferative neoplasm (MPN) animal models accurately re-capitulate human disease in mice and have been an important tool for the study of MPN biology and therapy. Transplantation of BCR-ABL transduced bone marrow cells into irradiated syngeneic mice established the field of MPN animal modeling and the retroviral bone marrow transplantation (BMT) assay has been used extensively since. Genetically engineered MPN animal models have enabled detailed characterization of the effects of specific MPN associated genetic abnormalities on the hematopoietic stem and progenitor cell (HSPC) compartment and xenograft models have allowed the study of primary human MPN-propagating cells in vivo. All models have facilitated the pre-clinical development of MPN therapies. JAK2V617F, the most common molecular abnormality in BCR-ABL negative MPN, has been extensively studied using retroviral, transgenic, knock-in and xenograft models. MPN animal models have also been used to investigate additional genetic lesions found in human MPN and to evaluate the bone marrow microenvironment in these diseases. Finally, several genetic lesions, although not common, somatically mutated drivers of MPN in humans induce a MPN phenotype in mice. Future uses for MPN animal models will include modeling compound genetic lesions in MPN and studying myelofibrotic transformation. PMID:23009938

Human CD22 cannot fully substitute murine CD22 functions in vivo, as shown in a new knockin mouse model.

PubMed

Wöhner, Miriam; Born, Stefanie; Nitschke, Lars

2012-11-01

CD22, an inhibitory co-receptor of the B-cell receptor, shows a B-cell-specific expression pattern and is expressed on most B-cell lymphomas. The anti-CD22 antibody Epratuzumab is in clinical trials for B-cell non-Hodgkin lymphoma and systemic lupus erythematosus, but shows a mostly unknown mode of action. We generated a new mouse model that expresses human CD22 instead of murine CD22 (Huki CD22 mice), in which human CD22 can be targeted. Expression of human CD22 on the B cells of Huki CD22 mice does not generally interfere with B-cell development. However, Huki CD22 mice show a reduction of the population of mature recirculating B cells in the bone marrow and reduced transitional and marginal zone B cells in the spleen, phenotypes resembling that of CD22-deficient mice. Similarly, enhanced BCR-induced Ca(2+) signalling is observed in Huki CD22 mice, which also mount normal immune responses toward different classes of antigens. Huki CD22 B cells show a normal anti-hCD22 antibody-mediated endocytosis. In conclusion, human CD22 cannot fully substitute for murine CD22 functions, possibly due to the changed intracellular tail of the protein or due to lower expression levels. Huki CD22 mice are a valuable new model for both antibody- and immunotoxin-mediated targeting of human CD22. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Neurodevelopmental Role for VGLUT2 in Pyramidal Neuron Plasticity, Dendritic Refinement, and in Spatial Learning

PubMed Central

He, Hongbo; Mahnke, Amanda H.; Doyle, Sukhjeevan; Fan, Ni; Wang, Chih-Chieh; Hall, Benjamin J.; Tang, Ya-Ping; Inglis, Fiona M.; Chen, Chu; Erickson, Jeffrey D.

2012-01-01

The level and integrity of glutamate transmission during critical periods of postnatal development plays an important role in the refinement of pyramidal neuron dendritic arbor, synaptic plasticity, and cognition. Presently, it is not clear how excitatory transmission via the two predominant isoforms of the vesicular glutamate transporter (VGLUT1 and VGLUT2) participate in this process. To assess a neurodevelopmental role for VGLUT2 in pyramidal neuron maturation we have generated recombinant VGLUT2 knockout mice and inactivated VGLUT2 throughout development using Emx1-Cre+/+ knockin mice. We show that VGLUT2-deficiency in cortico-limbic circuits results in reduced evoked glutamate transmission, release probability, and LTD at hippocampal CA3-CA1 synapses during a formative developmental period (postnatal days 11–14). In adults, we find a marked reduction in the amount of dendritic arbor across the span of the dendritic tree of CA1 pyramidal neurons, reduced LTP and levels of synaptic markers spinophilin and VGLUT1. Loss of dendritic arbor is accompanied by corresponding reductions in the number of dendritic spines, suggesting widespread alterations in synaptic connectivity. Conditional VGLUT2 knockout mice exhibit increased open-field exploratory activity, yet impaired spatial learning and memory; endophenotypes similar to NMDA receptor knockdown mice. Remarkably, the impairment in learning can be partially restored selectively increasing NMDA-receptor mediated glutamate transmission in adult mice by prolonged treatment with D-serine and a D-amino acid oxidase inhibitor. Our data indicate that VGLUT2 expression is pivotal to the proper development of mature pyramidal neuronal architecture and plasticity, and that such glutamatergic deficiency leads to cognitive malfunction as observed in several neurodevelopmental psychiatric disorders. PMID:23136427
Neurodevelopmental role for VGLUT2 in pyramidal neuron plasticity, dendritic refinement, and in spatial learning.

PubMed

He, Hongbo; Mahnke, Amanda H; Doyle, Sukhjeevan; Fan, Ni; Wang, Chih-Chieh; Hall, Benjamin J; Tang, Ya-Ping; Inglis, Fiona M; Chen, Chu; Erickson, Jeffrey D

2012-11-07

The level and integrity of glutamate transmission during critical periods of postnatal development plays an important role in the refinement of pyramidal neuron dendritic arbor, synaptic plasticity, and cognition. Presently, it is not clear how excitatory transmission via the two predominant isoforms of the vesicular glutamate transporter (VGLUT1 and VGLUT2) participate in this process. To assess a neurodevelopmental role for VGLUT2 in pyramidal neuron maturation, we generated recombinant VGLUT2 knock-out mice and inactivated VGLUT2 throughout development using Emx1-Cre(+/+) knock-in mice. We show that VGLUT2 deficiency in corticolimbic circuits results in reduced evoked glutamate transmission, release probability, and LTD at hippocampal CA3-CA1 synapses during a formative developmental period (postnatal days 11-14). In adults, we find a marked reduction in the amount of dendritic arbor across the span of the dendritic tree of CA1 pyramidal neurons and reduced long-term potentiation and levels of synaptic markers spinophilin and VGLUT1. Loss of dendritic arbor is accompanied by corresponding reductions in the number of dendritic spines, suggesting widespread alterations in synaptic connectivity. Conditional VGLUT2 knock-out mice exhibit increased open-field exploratory activity yet impaired spatial learning and memory, endophenotypes similar to those of NMDA receptor knock-down mice. Remarkably, the impairment in learning can be partially restored by selectively increasing NMDA receptor-mediated glutamate transmission in adult mice by prolonged treatment with d-serine and a d-amino acid oxidase inhibitor. Our data indicate that VGLUT2 expression is pivotal to the proper development of mature pyramidal neuronal architecture and plasticity, and that such glutamatergic deficiency leads to cognitive malfunction as observed in several neurodevelopmental psychiatric disorders.
Unbound (bioavailable) IGF1 enhances somatic growth

PubMed Central

Elis, Sebastien; Wu, Yingjie; Courtland, Hayden-William; Cannata, Dara; Sun, Hui; Beth-On, Mordechay; Liu, Chengyu; Jasper, Hector; Domené, Horacio; Karabatas, Liliana; Guida, Clara; Basta-Pljakic, Jelena; Cardoso, Luis; Rosen, Clifford J.; Frystyk, Jan; Yakar, Shoshana

2011-01-01

SUMMARY Understanding insulin-like growth factor-1 (IGF1) biology is of particular importance because, apart from its role in mediating growth, it plays key roles in cellular transformation, organ regeneration, immune function, development of the musculoskeletal system and aging. IGF1 bioactivity is modulated by its binding to IGF-binding proteins (IGFBPs) and the acid labile subunit (ALS), which are present in serum and tissues. To determine whether IGF1 binding to IGFBPs is necessary to facilitate normal growth and development, we used a gene-targeting approach and generated two novel knock-in mouse models of mutated IGF1, in which the native Igf1 gene was replaced by Des-Igf1 (KID mice) or R3-Igf1 (KIR mice). The KID and KIR mutant proteins have reduced affinity for the IGFBPs, and therefore present as unbound IGF1, or ‘free IGF1’. We found that both KID and KIR mice have reduced serum IGF1 levels and a concomitant increase in serum growth hormone levels. Ternary complex formation of IGF1 with the IGFBPs and the ALS was markedly reduced in sera from KID and KIR mice compared with wild type. Both mutant mice showed increased body weight, body and bone lengths, and relative lean mass. We found selective organomegaly of the spleen, kidneys and uterus, enhanced mammary gland complexity, and increased skeletal acquisition. The KID and KIR models show unequivocally that IGF1-complex formation with the IGFBPs is fundamental for establishing normal body and organ size, and that uncontrolled IGF bioactivity could lead to pathological conditions. PMID:21628395
Unbound (bioavailable) IGF1 enhances somatic growth.

PubMed

Elis, Sebastien; Wu, Yingjie; Courtland, Hayden-William; Cannata, Dara; Sun, Hui; Beth-On, Mordechay; Liu, Chengyu; Jasper, Hector; Domené, Horacio; Karabatas, Liliana; Guida, Clara; Basta-Pljakic, Jelena; Cardoso, Luis; Rosen, Clifford J; Frystyk, Jan; Yakar, Shoshana

2011-09-01

Understanding insulin-like growth factor-1 (IGF1) biology is of particular importance because, apart from its role in mediating growth, it plays key roles in cellular transformation, organ regeneration, immune function, development of the musculoskeletal system and aging. IGF1 bioactivity is modulated by its binding to IGF-binding proteins (IGFBPs) and the acid labile subunit (ALS), which are present in serum and tissues. To determine whether IGF1 binding to IGFBPs is necessary to facilitate normal growth and development, we used a gene-targeting approach and generated two novel knock-in mouse models of mutated IGF1, in which the native Igf1 gene was replaced by Des-Igf1 (KID mice) or R3-Igf1 (KIR mice). The KID and KIR mutant proteins have reduced affinity for the IGFBPs, and therefore present as unbound IGF1, or 'free IGF1'. We found that both KID and KIR mice have reduced serum IGF1 levels and a concomitant increase in serum growth hormone levels. Ternary complex formation of IGF1 with the IGFBPs and the ALS was markedly reduced in sera from KID and KIR mice compared with wild type. Both mutant mice showed increased body weight, body and bone lengths, and relative lean mass. We found selective organomegaly of the spleen, kidneys and uterus, enhanced mammary gland complexity, and increased skeletal acquisition. The KID and KIR models show unequivocally that IGF1-complex formation with the IGFBPs is fundamental for establishing normal body and organ size, and that uncontrolled IGF bioactivity could lead to pathological conditions.
Early Generated B-1-Derived B Cells Have the Capacity To Progress To Become Mantle Cell Lymphoma-like Neoplasia in Aged Mice.

PubMed

Hayakawa, Kyoko; Formica, Anthony M; Nakao, Yuka; Ichikawa, Daiju; Shinton, Susan A; Brill-Dashoff, Joni; Smith, Mitchell R; Morse, Herbert C; Hardy, Richard R

2018-06-13

In mice, fetal/neonatal B-1 cell development generates murine CD5 + B cells (B1a) with autoreactivity. We analyzed B1a cells at the neonatal stage in a V H 11/D/J H knock-in mouse line (V H 11t) that generates an autoreactive antiphosphatidylcholine BCR. Our study revealed that antiphosphatidylcholine B1a cells develop in liver, mature in spleen, and distribute in intestine/colon, mesenteric lymph node (mLN), and body cavity as the outcome of B-1 cell development before B-2 cell development. Throughout life, self-renewing B-1 B1a cells circulate through intestine, mesenteric vessel, and blood. The body cavity-deposited B1a cells also remigrate. In old age, some B1a cells proceed to monoclonal B cell lymphocytosis. When neonatal B-1 B1a cells express an antithymocyte/Thy-1 autoreactivity (ATA) BCR transgene in the C.B17 mouse background, ATA B cells increase in PBL and strongly develop lymphomas in aging mice that feature splenomegaly and mLN hyperplasia with heightened expression of CD11b, IL-10, and activated Stat3. At the adult stage, ATA B cells were normally present in the mantle zone area, including in intestine. Furthermore, frequent association with mLN hyperplasia suggests the influence by intestinal microenvironment on lymphoma development. When cyclin D1 was overexpressed by the Eμ-cyclin D1 transgene, ATA B cells progressed to further diffused lymphoma in aged mice, including in various lymph nodes with accumulation of IgM hi IgD lo CD5 + CD23 - CD43 + cells, resembling aggressive human mantle cell lymphoma. Thus, our findings reveal that early generated B cells, as an outcome of B-1 cell development, can progress to become lymphocytosis, lymphoma, and mantle cell lymphoma-like neoplasia in aged mice. Copyright © 2018 by The American Association of Immunologists, Inc.
Neuronal human BACE1 knockin induces systemic diabetes in mice.

PubMed

Plucińska, Kaja; Dekeryte, Ruta; Koss, David; Shearer, Kirsty; Mody, Nimesh; Whitfield, Phillip D; Doherty, Mary K; Mingarelli, Marco; Welch, Andy; Riedel, Gernot; Delibegovic, Mirela; Platt, Bettina

2016-07-01

β-Secretase 1 (BACE1) is a key enzyme in Alzheimer's disease pathogenesis that catalyses the amyloidogenic cleavage of amyloid precursor protein (APP). Recently, global Bace1 deletion was shown to protect against diet-induced obesity and diabetes, suggesting that BACE1 is a potential regulator of glucose homeostasis. Here, we investigated whether increased neuronal BACE1 is sufficient to alter systemic glucose metabolism, using a neuron-specific human BACE1 knockin mouse model (PLB4). Glucose homeostasis and adiposity were determined by glucose tolerance tests and EchoMRI, lipid species were measured by quantitative lipidomics, and biochemical and molecular alterations were assessed by western blotting, quantitative PCR and ELISAs. Glucose uptake in the brain and upper body was measured via (18)FDG-PET imaging. Physiological and molecular analyses demonstrated that centrally expressed human BACE1 induced systemic glucose intolerance in mice from 4 months of age onward, alongside a fatty liver phenotype and impaired hepatic glycogen storage. This diabetic phenotype was associated with hypothalamic pathology, i.e. deregulation of the melanocortin system, and advanced endoplasmic reticulum (ER) stress indicated by elevated central C/EBP homologous protein (CHOP) signalling and hyperphosphorylation of its regulator eukaryotic translation initiation factor 2α (eIF2α). In vivo (18)FDG-PET imaging further confirmed brain glucose hypometabolism in these mice; this corresponded with altered neuronal insulin-related signalling, enhanced protein tyrosine phosphatase 1B (PTP1B) and retinol-binding protein 4 (RBP4) levels, along with upregulation of the ribosomal protein and lipid translation machinery. Increased forebrain and plasma lipid accumulation (i.e. ceramides, triacylglycerols, phospholipids) was identified via lipidomics analysis. Our data reveal that neuronal BACE1 is a key regulator of metabolic homeostasis and provide a potential mechanism for the high prevalence of metabolic disturbance in Alzheimer's disease.
Reduced inhibitory gate in the barrel cortex of Neuroligin3R451C knock-in mice, an animal model of autism spectrum disorders.

PubMed

Cellot, Giada; Cherubini, Enrico

2014-07-16

Neuroligins are postsynaptic adhesion molecules that interacting with presynaptic neurexins ensure the cross-talk between pre- and postsynaptic specializations. Rare mutations in neurexin-neuroligin genes have been linked to autism spectrum disorders (ASDs). One of these, the R451C mutation of the gene encoding for Neuroligin3 (Nlgn3), has been found in patients with familial forms of ASDs. Animals carrying this mutation (NL3(R451C) knock-in mice) exhibit impaired social behaviors, reminiscent of those observed in ASD patients, associated with major alterations in both GABAergic and glutamatergic transmission, which vary among different brain regions and at different developmental stages. Here, pair recordings from parvalbumin- (PV) expressing basket cells and spiny neurons were used to study GABAergic synaptic signaling in layer IV barrel cortex of NL3(R451C) mutant mice. We found that the R451C mutation severely affects the probability of GABA release from PV-expressing basket cells, responsible for controlling via thalamo-cortical inputs the feed-forward inhibition. No changes in excitatory inputs to parvalbumin-positive basket cells or spiny neurons were detected. These data clearly show that primary targets of the NL3 mutation are PV-expressing basket cells, independently of the brain region where they are localized. Changes in the inhibitory gate of layer IV somatosensory cortex may alter sensory processing in ASD patients leading to misleading sensory representations with difficulties to combine pieces of information into a unified perceptual whole. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.
The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

PubMed Central

Kerr, Niall; Holmes, Fiona E.; Hobson, Sally-Ann; Vanderplank, Penny; Leard, Alan; Balthasar, Nina; Wynick, David

2015-01-01

The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal1, Gal2 and the less studied Gal3 (GalR1–3 gene products). There is a wealth of data on expression of Gal1–3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal1 or Gal2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs). PMID:26292267
Disruption of GluA2 phosphorylation potentiates stress responsivity.

PubMed

Ellis, Alexandra S; Fosnocht, Anne Q; Lucerne, Kelsey E; Briand, Lisa A

2017-08-30

Cocaine addiction is characterized by persistent craving and addicts frequently relapse even after long periods of abstinence. Exposure to stress can precipitate relapse in humans and rodents. Stress and drug use can lead to common alterations in synaptic plasticity and these commonalities may contribute to the ability of stress to elicit relapse. These common changes in synaptic plasticity are mediated, in part, by alterations in the trafficking and stabilization of AMPA receptors. Exposure to both cocaine and stress can lead to alterations in protein kinase C-mediated phosphorylation of GluA2 AMPA subunits and thus alter the trafficking of GluA2-containing AMPARs. However, it is not clear what role AMPAR trafficking plays in the interactions between stress and cocaine. The current study utilized a mouse with a point mutation within the GluA2 subunit c-terminus resulting in a disruption of PKC-mediated GluA2 phosphorylation to examine stress responsivity. Although no differences were seen in the response to a forced swim stress in naïve mice, GluA2 K882A knock-in mice exhibited an increased stress response following cocaine self-administration. Furthermore, we demonstrated that disrupting GluA2 phosphorylation increases vulnerability to stress-induced reinstatement of both cocaine seeking and cocaine-conditioned reward. Finally, GluA2 K882A knock-in mice exhibit an increased vulnerability to social defeat as indicated by increased social avoidance. Taken together these results indicate that disrupting GluA2 phosphorylation leads to increased responsivity to acute stress following cocaine exposure and increased vulnerability to chronic stress. These results highlight the GluA2 phosphorylation site as a novel target for the stress-related disorders. Copyright © 2017 Elsevier B.V. All rights reserved.
A Novel Intergenic ETnII-β Insertion Mutation Causes Multiple Malformations in Polypodia Mice

PubMed Central

Lehoczky, Jessica A.; Thomas, Peedikayil E.; Patrie, Kevin M.; Owens, Kailey M.; Villarreal, Lisa M.; Galbraith, Kenneth; Washburn, Joe; Johnson, Craig N.; Gavino, Bryant; Borowsky, Alexander D.; Millen, Kathleen J.; Wakenight, Paul; Law, William; Van Keuren, Margaret L.; Gavrilina, Galina; Hughes, Elizabeth D.; Saunders, Thomas L.; Brihn, Lesil; Nadeau, Joseph H.; Innis, Jeffrey W.

2013-01-01

Mouse early transposon insertions are responsible for ∼10% of spontaneous mutant phenotypes. We previously reported the phenotypes and genetic mapping of Polypodia, (Ppd), a spontaneous, X-linked dominant mutation with profound effects on body plan morphogenesis. Our new data shows that mutant mice are not born in expected Mendelian ratios secondary to loss after E9.5. In addition, we refined the Ppd genetic interval and discovered a novel ETnII-β early transposon insertion between the genes for Dusp9 and Pnck. The ETn inserted 1.6 kb downstream and antisense to Dusp9 and does not disrupt polyadenylation or splicing of either gene. Knock-in mice engineered to carry the ETn display Ppd characteristic ectopic caudal limb phenotypes, showing that the ETn insertion is the Ppd molecular lesion. Early transposons are actively expressed in the early blastocyst. To explore the consequences of the ETn on the genomic landscape at an early stage of development, we compared interval gene expression between wild-type and mutant ES cells. Mutant ES cell expression analysis revealed marked upregulation of Dusp9 mRNA and protein expression. Evaluation of the 5′ LTR CpG methylation state in adult mice revealed no correlation with the occurrence or severity of Ppd phenotypes at birth. Thus, the broad range of phenotypes observed in this mutant is secondary to a novel intergenic ETn insertion whose effects include dysregulation of nearby interval gene expression at early stages of development. PMID:24339789
A gain-of-function mutation in Tnni2 impeded bone development through increasing Hif3a expression in DA2B mice.

PubMed

Zhu, Xiaoquan; Wang, Fengchao; Zhao, Yanyang; Yang, Peng; Chen, Jun; Sun, Hanzi; Liu, Lei; Li, Wenjun; Pan, Lin; Guo, Yanru; Kou, Zhaohui; Zhang, Yu; Zhou, Cheng; He, Jiang; Zhang, Xue; Li, Jianxin; Han, Weitian; Li, Jian; Liu, Guanghui; Gao, Shaorong; Yang, Ze

2014-10-01

Distal arthrogryposis type 2B (DA2B) is an important genetic disorder in humans. However, the mechanisms governing this disease are not clearly understood. In this study, we generated knock-in mice carrying a DA2B mutation (K175del) in troponin I type 2 (skeletal, fast) (TNNI2), which encodes a fast-twitch skeletal muscle protein. Tnni2K175del mice (referred to as DA2B mice) showed typical DA2B phenotypes, including limb abnormality and small body size. However, the current knowledge concerning TNNI2 could not explain the small body phenotype of DA2B mice. We found that Tnni2 was expressed in the osteoblasts and chondrocytes of long bone growth plates. Expression profile analysis using radii and ulnae demonstrated that Hif3a expression was significantly increased in the Tnni2K175del mice. Chromatin immunoprecipitation assays indicated that both wild-type and mutant tnni2 protein can bind to the Hif3a promoter using mouse primary osteoblasts. Moreover, we showed that the mutant tnni2 protein had a higher capacity to transactivate Hif3a than the wild-type protein. The increased amount of hif3a resulted in impairment of angiogenesis, delay in endochondral ossification, and decrease in chondrocyte differentiation and osteoblast proliferation, suggesting that hif3a counteracted hif1a-induced Vegf expression in DA2B mice. Together, our data indicated that Tnni2K175del mutation led to abnormally increased hif3a and decreased vegf in bone, which explain, at least in part, the small body size of Tnni2K175del mice. Furthermore, our findings revealed a new function of tnni2 in the regulation of bone development, and the study of gain-of-function mutation in Tnni2 in transgenic mice opens a new avenue to understand the pathological mechanism of human DA2B disorder.
Osteoclastic miR-214 targets TRAF3 to contribute to osteolytic bone metastasis of breast cancer

PubMed Central

Liu, Jin; Li, Defang; Dang, Lei; Liang, Chao; Guo, Baosheng; Lu, Cheng; He, Xiaojuan; Cheung, Hilda Y. S.; He, Bing; Liu, Biao; Li, Fangfei; Lu, Jun; Wang, Luyao; Shaikh, Atik Badshah; Jiang, Feng; Lu, Changwei; Peng, Songlin; Zhang, Zongkang; Zhang, Bao-Ting; Pan, Xiaohua; Xiao, Lianbo; Lu, Aiping; Zhang, Ge

2017-01-01

The role of osteoclastic miRNAs in regulating osteolytic bone metastasis (OBM) of breast cancer is still underexplored. Here, we examined the expression profiles of osteoclastogenic miRNAs in human bone specimens and identified that miR-214-3p was significantly upregulated in breast cancer patients with OBM. Consistently, we found increased miR-214-3p within osteoclasts, which was associated with the elevated bone resorption, during the development of OBM in human breast cancer xenografted nude mice (BCX). Furthermore, genetic ablation of osteoclastic miR-214-3p in nude mice prevent the development of OBM. Conditioned medium from MDA-MB-231 cells dramatically stimulated miR-214-3p expression to promote osteoclast differentiation. Mechanistically, a series of in vitro study showed that miR-214-3p directly targeted Traf3 to promote osteoclast activity and bone-resorbing activity. In addition, osteoclast-specific miR-214-3p knock-in mice showed remarkably increased bone resorption when compared to the littermate controls, which was attenuated after osteoclast-targeted treatment with Traf3 3′UTR-containing plasmid. In BCX nude mice, osteoclast-targeted antagomir-214-3p delivery could recover the TRAF3 protein expression and attenuate the development of OBM, respectively. Collectively, inhibition of osteoclastic miR-214-3p may be a potential therapeutic strategy for breast cancer patients with OBM. Meanwhile, the intraosseous TRAF3 could be a promising biomarker for evaluation of the treatment response of antagomir-214-3p. PMID:28071724
Genome Editing in Mice Using TALE Nucleases.

PubMed

Wefers, Benedikt; Brandl, Christina; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

2016-01-01

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.
Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

PubMed Central

Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

2015-01-01

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins. PMID:26473830
Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

PubMed

Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

2015-10-09

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.
Altered activity-rest patterns in mice with a human autosomal-dominant nocturnal frontal lobe epilepsy mutation in the β2 nicotinic receptor

PubMed Central

Xu, Jian; Cohen, Bruce N.; Zhu, Yongling; Dziewczapolski, Gustavo; Panda, Satchidananda; Lester, Henry A.; Heinemann, Stephen F.; Contractor, Anis

2010-01-01

High-affinity nicotinic receptors containing beta2 subunits (β2*) are widely expressed in the brain, modulating many neuronal processes and contributing to neuropathologies such as Alzheimer’s disease, Parkinson’s disease and epilepsy. Mutations in both the α4 and β2 subunits are associated with a rare partial epilepsy, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Here we introduced one such human missense mutation into the mouse genome to generate a knock-in strain carrying a valine-to-leucine mutation β2V287L.β2V287L mice were viable and born at an expected Mendelian ratio. Surprisingly, mice did not display an overt seizure phenotype; however homozygous mice did display significant alterations in their activity-rest patterns. This was manifest as an increase in activity during the light cycle suggestive of disturbances in the normal sleep patterns of mice; a parallel phenotype to that found in human ADNFLE patients. Consistent with the role of nicotinic receptors in reward pathways, we found that β2V287L mice did not develop a normal proclivity to voluntary wheel running, a model for natural reward. Anxiety-related behaviors were also affected by the V287L mutation. Mutant mice spent more time in the open arms on the elevated plus maze (EPM) suggesting that they had reduced levels of anxiety. Together, these findings emphasize several important roles of β2* nicotinic receptors in complex biological processes including the activity-rest cycle, natural reward, and anxiety. PMID:20603624
Autoreactive T Cells and Chronic Fungal Infection Drive Esophageal Carcinogenesis

PubMed Central

Zhu, Feng; Willette-Brown, Jami; Song, Na-Young; Lomada, Dakshayani; Song, Yongmei; Xue, Liyan; Gray, Zane; Zhao, Zitong; Davis, Sean R.; Sun, Zhonghe; Zhang, Peilin; Wu, Xiaolin; Zhan, Qimin; Richie, Ellen R.; Hu, Yinling

2018-01-01

SUMMARY Humans with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a T cell–driven autoimmune disease caused by impaired central tolerance, are susceptible to developing chronic fungal infection and esophageal squamous cell carcinoma (ESCC). However, the relationship between autoreactive T cells and chronic fungal infection in ESCC development remains unclear. We find that kinase-dead Ikkα knockin mice develop phenotypes reminiscent of APECED, including impaired central tolerance, autoreactive T cells, chronic fungal infection, and ESCCs expressing specific human ESCC markers. Using this model, we investigated the potential link between ESCC and fungal infection. Autoreactive CD4 T cells permit fungal infection and incite tissue injury and inflammation. Antifungal treatment or depletion of autoreactive CD4 T cells rescues, whereas oral fungal administration promotes, ESCC development. Inhibition of inflammation or EGFR activity decreases fungal burden. Importantly, fungal infection is highly associated with ESCCs in non-autoimmune human patients. Therefore, autoreactive T cells and chronic fungal infection, fostered by inflammation and epithelial injury, promote ESCC development. PMID:28407484
Proteomic characterization of a mouse model of familial Danish dementia.

PubMed

Vitale, Monica; Renzone, Giovanni; Matsuda, Shuji; Scaloni, Andrea; D'Adamio, Luciano; Zambrano, Nicola

2012-01-01

A dominant mutation in the ITM2B/BRI2 gene causes familial Danish dementia (FDD) in humans. To model FDD in animal systems, a knock-in approach was recently implemented in mice expressing a wild-type and mutant allele, which bears the FDD-associated mutation. Since these FDD(KI) mice show behavioural alterations and impaired synaptic function, we characterized their synaptosomal proteome via two-dimensional differential in-gel electrophoresis. After identification by nanoliquid chromatography coupled to electrospray-linear ion trap tandem mass spectrometry, the differentially expressed proteins were classified according to their gene ontology descriptions and their predicted functional interactions. The Dlg4/Psd95 scaffold protein and additional signalling proteins, including protein phosphatases, were revealed by STRING analysis as potential players in the altered synaptic function of FDD(KI) mice. Immunoblotting analysis finally demonstrated the actual downregulation of the synaptosomal scaffold protein Dlg4/Psd95 and of the dual-specificity phosphatase Dusp3 in the synaptosomes of FDD(KI) mice.
Proteomic Characterization of a Mouse Model of Familial Danish Dementia

PubMed Central

Vitale, Monica; Renzone, Giovanni; Matsuda, Shuji; Scaloni, Andrea; D'Adamio, Luciano; Zambrano, Nicola

2012-01-01

A dominant mutation in the ITM2B/BRI2 gene causes familial Danish dementia (FDD) in humans. To model FDD in animal systems, a knock-in approach was recently implemented in mice expressing a wild-type and mutant allele, which bears the FDD-associated mutation. Since these FDDKI mice show behavioural alterations and impaired synaptic function, we characterized their synaptosomal proteome via two-dimensional differential in-gel electrophoresis. After identification by nanoliquid chromatography coupled to electrospray-linear ion trap tandem mass spectrometry, the differentially expressed proteins were classified according to their gene ontology descriptions and their predicted functional interactions. The Dlg4/Psd95 scaffold protein and additional signalling proteins, including protein phosphatases, were revealed by STRING analysis as potential players in the altered synaptic function of FDDKI mice. Immunoblotting analysis finally demonstrated the actual downregulation of the synaptosomal scaffold protein Dlg4/Psd95 and of the dual-specificity phosphatase Dusp3 in the synaptosomes of FDDKI mice. PMID:22619496

The Akt1-eNOS Axis Illustrates the Specificity of Kinase-Substrate Relationships in Vivo

PubMed Central

Schleicher, Michael; Yu, Jun; Murata, Takahisa; Derakhshan, Berhad; Atochin, Dimitriy; Qian, Li; Kashiwagi, Satoshi; Lorenzo, Annarita Di; Harrison, Kenneth D.; Huang, Paul L.; Sessa, William C.

2016-01-01

Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1−/− mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme “constitutively active” or “less active.” The eNOS mutations did not influence several phenotypes in Akt1−/− mice; however, the defective postnatal angiogenesis characteristic of Akt1−/− mice was rescued by crossing the Akt1−/− mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1α (HIF-1α) and increased production of HIF-1α–responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling. PMID:19654415
Cntnap2 expression in the cerebellum of Foxp2(R552H) mice, with a mutation related to speech-language disorder.

PubMed

Fujita, Eriko; Tanabe, Yuko; Momoi, Mariko Y; Momoi, Takashi

2012-01-11

Foxp2(R552H) knock-in (KI) mice carrying a mutation related to human speech-language disorder exhibit impaired ultrasonic vocalization and poor Purkinje cell development. Foxp2 is a forkhead domain-containing transcriptional repressor that associates with its co-repressor CtBP; Foxp2(R552H) displays reduced DNA binding activity. A genetic connection between FOXP2 and CNTNAP2 has been demonstrated in vitro, but not in vivo. Here we show that Cntnap2 mRNA levels significantly increased in the cerebellum of Foxp2(R552H) KI pups, although the cerebellar population of Foxp2-positive Purkinje cells was very small. Furthermore, Cntnap2 immunofluorescence did not decrease in the poorly developed Purkinje cells of Foxp2(R552H) KI pups, although synaptophysin immunofluorescence decreased. Cntnap2 and CtBP were ubiquitously expressed, while Foxp2 co-localized with CtBP only in Purkinje cells. Taken together, these observations suggest that Foxp2 may regulate ultrasonic vocalization by associating with CtBP in Purkinje cells; Cntnap2 may be a target of this co-repressor. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
Probing Mechanisms of Photoreceptor Degeneration in a New Mouse Model of the Common Form of Autosomal Dominant Retinitis Pigmentosa due to P23H Opsin Mutations*♦

PubMed Central

Sakami, Sanae; Maeda, Tadao; Bereta, Grzegorz; Okano, Kiichiro; Golczak, Marcin; Sumaroka, Alexander; Roman, Alejandro J.; Cideciyan, Artur V.; Jacobson, Samuel G.; Palczewski, Krzysztof

2011-01-01

Rhodopsin, the visual pigment mediating vision under dim light, is composed of the apoprotein opsin and the chromophore ligand 11-cis-retinal. A P23H mutation in the opsin gene is one of the most prevalent causes of the human blinding disease, autosomal dominant retinitis pigmentosa. Although P23H cultured cell and transgenic animal models have been developed, there remains controversy over whether they fully mimic the human phenotype; and the exact mechanism by which this mutation leads to photoreceptor cell degeneration remains unknown. By generating P23H opsin knock-in mice, we found that the P23H protein was inadequately glycosylated with levels 1–10% that of wild type opsin. Moreover, the P23H protein failed to accumulate in rod photoreceptor cell endoplasmic reticulum but instead disrupted rod photoreceptor disks. Genetically engineered P23H mice lacking the chromophore showed accelerated photoreceptor cell degeneration. These results indicate that most synthesized P23H protein is degraded, and its retinal cytotoxicity is enhanced by lack of the 11-cis-retinal chromophore during rod outer segment development. PMID:21224384
Infectious prions and proteinopathies.

PubMed

Barron, Rona M

2017-01-02

Transmissible spongiform encephalopathies (TSEs) are caused by an infectious agent that is thought to consist of only misfolded and aggregated prion protein (PrP). Unlike conventional micro-organisms, the agent spreads and propagates by binding to and converting normal host PrP into the abnormal conformer, increasing the infectious titre. Synthetic prions, composed of refolded fibrillar forms of recombinant PrP (rec-PrP) have been generated to address whether PrP aggregates alone are indeed infectious prions. In several reports, the development of TSE disease has been described following inoculation and passage of rec-PrP fibrils in transgenic mice and hamsters. However in studies described here we show that inoculation of rec-PrP fibrils does not always cause clinical TSE disease or increased infectious titre, but can seed the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). These data are reminiscent of the "prion-like" spread of misfolded protein in other models of neurodegenerative disease following inoculation of transgenic mice with pre-formed amyloid seeds. Protein misfolding, even when the protein is PrP, does not inevitably lead to the development of an infectious TSE disease. It is possible that most in vivo and in vitro produced misfolded PrP is not infectious and that only a specific subpopulation is associated with infectivity and neurotoxicity.
Mast cells play no role in the pathogenesis of postoperative ileus induced by intestinal manipulation.

PubMed

Gomez-Pinilla, Pedro J; Farro, Giovanna; Di Giovangiulio, Martina; Stakenborg, Nathalie; Némethova, Andrea; de Vries, Annick; Liston, Adrian; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Rodewald, Hans-Reimwer; Boeckxstaens, Guy E; Matteoli, Gianluca

2014-01-01

Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3(Cre/+) , devoid of mast cells but with intact Kit signaling. The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. Kit(W-sh/W-sh) and Cpa3(Cre/+) mice, and by use of the mast cell stabilizer cromolyn. Kit(W-sh/W-sh) mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3(Cre/+) mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3(Cre/+) mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3(+/+) ). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI. Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI.
Mast Cells Play No Role in the Pathogenesis of Postoperative Ileus Induced by Intestinal Manipulation

PubMed Central

Gomez-Pinilla, Pedro J.; Farro, Giovanna; Di Giovangiulio, Martina; Stakenborg, Nathalie; Némethova, Andrea; de Vries, Annick; Liston, Adrian; Feyerabend, Thorsten B.; Rodewald, Hans-Reimwer; Boeckxstaens, Guy E.; Matteoli, Gianluca

2014-01-01

Introduction Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3Cre/+, devoid of mast cells but with intact Kit signaling. Design The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. KitW-sh/W-sh and Cpa3Cre/+ mice, and by use of the mast cell stabilizer cromolyn. Results KitW-sh/W-sh mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3Cre/+ mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3Cre/+ mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3+/+). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI. Conclusions Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI. PMID:24416383
Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor.

PubMed

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Veach, Darren; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R; DeMatteo, Ronald P; Besmer, Peter

2017-10-03

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic Kit V558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant Kit V558Δ/+ mice, double-mutant Kit V558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in Kit V558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing Kit V558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant Kit V558Δ;T669I/+ tumors.
Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor

PubMed Central

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q.; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R.; DeMatteo, Ronald P.; Besmer, Peter

2017-01-01

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors. PMID:28923937
Mutated CaV2.1 channels dysregulate CASK/P2X3 signaling in mouse trigeminal sensory neurons of R192Q Cacna1a knock-in mice.

PubMed

Gnanasekaran, Aswini; Bele, Tanja; Hullugundi, Swathi; Simonetti, Manuela; Ferrari, Michael D; van den Maagdenberg, Arn M J M; Nistri, Andrea; Fabbretti, Elsa

2013-12-02

ATP-gated P2X3 receptors of sensory ganglion neurons are important transducers of pain as they adapt their expression and function in response to acute and chronic nociceptive signals. The present study investigated the role of calcium/calmodulin-dependent serine protein kinase (CASK) in controlling P2X3 receptor expression and function in trigeminal ganglia from Cacna1a R192Q-mutated knock-in (KI) mice, a genetic model for familial hemiplegic migraine type-1. KI ganglion neurons showed more abundant CASK/P2X3 receptor complex at membrane level, a result that likely originated from gain-of-function effects of R192Q-mutated CaV2.1 channels and downstream enhanced CaMKII activity. The selective CaV2.1 channel blocker ω-Agatoxin IVA and the CaMKII inhibitor KN-93 were sufficient to return CASK/P2X3 co-expression to WT levels. After CASK silencing, P2X3 receptor expression was decreased in both WT and KI ganglia, supporting the role of CASK in P2X3 receptor stabilization. This process was functionally observed as reduced P2X3 receptor currents. We propose that, in trigeminal sensory neurons, the CASK/P2X3 complex has a dynamic nature depending on intracellular calcium and related signaling, that are enhanced in a transgenic mouse model of genetic hemiplegic migraine.
Characterization of an MPS I-H Knock-In Mouse that Carries a Nonsense Mutation Analogous to the Human IDUA-W402X Mutation

PubMed Central

Wang, Dan; Shukla, Charu; Liu, Xiaoli; Schoeb, Trenton R.; Clarke, Lorne A.; Bedwell, David M.; Keeling, Kim M.

2009-01-01

Here we report the characterization of a knock-in mouse model for the autosomal recessive disorder mucopolysaccharidosis type I-Hurler (MPS I-H), also known as Hurler syndrome. MPS I-H is the most severe form of α-L-iduronidase deficiency. α-L-iduronidase (encoded by the IDUA gene) is a lysosomal enzyme that participates in the degradation of dermatan sulfate and heparan sulfate. Using gene replacement methodology, a nucleotide change was introduced into the mouse Idua locus that resulted in a nonsense mutation at codon W392. The Idua-W392X mutation is analogous to the human IDUA-W402X mutation commonly found in MPS I-H patients. We found that the phenotype in homozygous Idua-W392X mice closely correlated with the human MPS I-H disease. Homozygous W392X mice showed no detectable α-L-iduronidase activity. We observed a defect in GAG degradation as evidenced by an increase in sulfated GAGs excreted in the urine and stored in multiple tissues. Histology and electron microscopy also revealed evidence of GAG storage in all tissues examined. Additional assessment revealed bone abnormalities and altered metabolism within the Idua-W392X mouse. This new mouse will provide an important tool to investigate therapeutic approaches for MPS I-H that cannot be addressed using current MPS I-H animal models. PMID:19751987
Functional importance of evolutionally conserved Tbx6 binding sites in the presomitic mesoderm-specific enhancer of Mesp2.

PubMed

Yasuhiko, Yukuto; Kitajima, Satoshi; Takahashi, Yu; Oginuma, Masayuki; Kagiwada, Harumi; Kanno, Jun; Saga, Yumiko

2008-11-01

The T-box transcription factor Tbx6 controls the expression of Mesp2, which encodes a basic helix-loop-helix transcription factor that has crucial roles in somitogenesis. In cultured cells, Tbx6 binding to the Mesp2 enhancer region is essential for the activation of Mesp2 by Notch signaling. However, it is not known whether this binding is required in vivo. Here we report that an Mesp2 enhancer knockout mouse bearing mutations in two crucial Tbx6 binding sites does not express Mesp2 in the presomitic mesoderm. This absence leads to impaired skeletal segmentation identical to that reported for Mesp2-null mice, indicating that these Tbx6 binding sites are indispensable for Mesp2 expression. T-box binding to the consensus sequences in the Mesp2 upstream region was confirmed by chromatin immunoprecipitation assays. Further enhancer analyses indicated that the number and spatial organization of the T-box binding sites are critical for initiating Mesp2 transcription via Notch signaling. We also generated a knock-in mouse in which the endogenous Mesp2 enhancer was replaced by the core enhancer of medaka mespb, an ortholog of mouse Mesp2. The homozygous enhancer knock-in mouse was viable and showed normal skeletal segmentation, indicating that the medaka mespb enhancer functionally replaced the mouse Mesp2 enhancer. These results demonstrate that there is significant evolutionary conservation of Mesp regulatory mechanisms between fish and mice.
A Longitudinal Operant Assessment of Cognitive and Behavioural Changes in the HdhQ111 Mouse Model of Huntington’s Disease

PubMed Central

Dunnett, Stephen B.; Brooks, Simon P.

2016-01-01

Huntington’s disease (HD) is characterised by motor symptoms which are often preceded by cognitive and behavioural changes, that can significantly contribute to disease burden for people living with HD. Numerous knock-in mouse models of HD are currently available for scientific research. However, before their use, they must be behaviourally characterised to determine their suitability in recapitulating the symptoms of the human condition. Thus, we sought to longitudinally characterise the nature, severity and time course of cognitive and behavioural changes observed in HdhQ111 heterozygous knock-in mice.To determine changes in cognition and behaviour an extensive battery of operant tests including: fixed ratio, progressive ratio, the five choice serial reaction time task and the serial implicit learning task, were applied longitudinally to HdhQ111 and wild type mice. The operant test battery was conducted at 6, 12 and 18 months of age. Significant deficits were observed in HdhQ111 animals in comparison to wild type animals in all operant tests indicating altered cognition (attentional and executive function) and motivation. However, the cognitive and behavioural deficits observed were not shown to be progressive over time in the longitudinal testing paradigm that was utilised. The results therefore demonstrate that the HdhQ111 mouse model of HD reflects some features of the cognitive and behavioural changes shown in the human condition of HD. Although, the cognitive and behavioural deficits demonstrated were not shown to be progressive over time. PMID:27701442
Matrix Disruptions, Growth, and Degradation of Cartilage with Impaired Sulfation*

PubMed Central

Mertz, Edward L.; Facchini, Marcella; Pham, Anna T.; Gualeni, Benedetta; De Leonardis, Fabio; Rossi, Antonio; Forlino, Antonella

2012-01-01

Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in the SLC26A2 transporter responsible for sulfate uptake by chondrocytes. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally. To understand underlying mechanisms, we studied newborn dtd mice. We developed, verified and used high-definition infrared hyperspectral imaging of cartilage sections at physiological conditions, to quantify collagen and its orientation, noncollagenous proteins, and chondroitin chains, and their sulfation with 6-μm spatial resolution and without labeling. We found that chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with the chondroitin synthesis rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth. The results provide clues to in vivo sulfation, DTD treatment, and cartilage growth. PMID:22556422
Actions of the Japanese Pancreas and Islet Transplantation Association regarding transplanted human islets isolated using Liberase HI.

PubMed

Saito, T; Anazawa, T; Gotoh, M; Uemoto, S; Kenmochi, T; Kuroda, Y; Satomi, S; Itoh, T; Yasunami, Y; Kitamoto, T; Mohri, S; Teraoka, S

2010-12-01

The potential for introducing transmissible spongiform encephalopathy (TSE) into islet cells was indicated by recognizing that Liberase HI is isolated from Clostridium histolyticum grown in media containing brain-heart infusion broth. A national team within the Japanese Pancreas and Islet Transplantation Association implemented an islet transplantation program in Japan using Liberase HI. The program comprised 65 islet isolations from non-heart-beating donors and 34 transplants into 18 patients. Herein, we have summarized how the Association followed these recipients over the long term. We established an ad hoc committee to follow recipients transplanted with islets isolated using Liberase HI after becoming informed of the associated dangers of using this enzyme. We also stopped islet transplantations using Liberase. The committee addressed the major concerns of the risk of the collagenase being contaminated with TSE and of the recipient follow-up. All recipients were examined by diffusion MRI and EEG and then scheduled for evaluation and follow-up by specialists in Creutzfeldt-Jakob disease (CJD). Bioassays of bovine spongiform encephalopathy prions in the enzyme proceeded using knock-in mice expressing bovine prion protein. These assays could detect contaminating prions at a dilution of 1 × 10(4). After inactivating its collagenase activity, Liberase HI was injected into the abdominal cavities of knock-in mice. Four months later, prion infectivity in Liberase HI was evaluated by immunohistochemical staining and Western blotting of spleen homogenates using anti-prion protein antibodies. Western blotting and immunohistochemical staining did not detect prions in Liberase HI. Diffusion MRI and EEG evaluations performed by CJD specialists confirmed that none of the transplanted recipients had CJD. Three years of follow-up revealed that none of the Japanese recipients of islet transplants developed CJD. Prion bioassays showed that the Liberase HI used to isolate islets for transplantation was free of infectious TSE prions. Copyright © 2010 Elsevier Inc. All rights reserved.
GSDMD is critical for autoinflammatory pathology in a mouse model of Familial Mediterranean Fever.

PubMed

Kanneganti, Apurva; Malireddi, R K Subbarao; Saavedra, Pedro H V; Vande Walle, Lieselotte; Van Gorp, Hanne; Kambara, Hiroto; Tillman, Heather; Vogel, Peter; Luo, Hongbo R; Xavier, Ramnik J; Chi, Hongbo; Lamkanfi, Mohamed

2018-06-04

Pyroptosis is an inflammasome-induced lytic cell death mode, the physiological role of which in chronic inflammatory diseases is unknown. Familial Mediterranean Fever (FMF) is the most common monogenic autoinflammatory disease worldwide, affecting an estimated 150,000 patients. The disease is caused by missense mutations in Mefv that activate the Pyrin inflammasome, but the pathophysiologic mechanisms driving autoinflammation in FMF are incompletely understood. Here, we show that Clostridium difficile infection of FMF knock-in macrophages that express a chimeric FMF-associated Mefv V726A Pyrin elicited pyroptosis and gasdermin D (GSDMD)-mediated interleukin (IL)-1β secretion. Importantly, in vivo GSDMD deletion abolished spontaneous autoinflammatory disease. GSDMD-deficient FMF knock-in mice were fully protected from the runted growth, anemia, systemic inflammatory cytokine production, neutrophilia, and tissue damage that characterize this autoinflammatory disease model. Overall, this work identifies pyroptosis as a critical mechanism of IL-1β-dependent autoinflammation in FMF and highlights GSDMD inhibition as a potential antiinflammatory strategy in inflammasome-driven diseases. © 2018 Kanneganti et al.
A role for NF-κB activity in skin hyperplasia and the development of keratoacanthomata in mice.

PubMed

Poligone, Brian; Hayden, Matthew S; Chen, Luojing; Pentland, Alice P; Jimi, Eijiro; Ghosh, Sankar

2013-01-01

Previous studies have implicated NF-κB signaling in both cutaneous development and oncogenesis. However, these studies have been limited in part by the lethality that results from extreme over- or under-expression of NF-κB in available mouse models. Even cre-driven tissue specific expression of transgenes, or targeted deletion of NF-κB can cause cell death. Therefore, the present study was undertaken to evaluate a novel mouse model of enhanced NF-κB activity in the skin. A knock-in homologous recombination technique was utilized to develop a mouse model (referred to as PD mice) with increased NF-κB activity. The data show that increased NF-κB activity leads to hyperproliferation and dysplasia of the mouse epidermis. Chemical carcinogenesis in the context of enhanced NF-κB activity promotes the development of keratoacanthomata. Our findings support an important role for NF-κB in keratinocyte dysplasia. We have found that enhanced NF-κB activity renders keratinocytes susceptible to hyperproliferation and keratoacanthoma (KA) development but is not sufficient for transformation and SCC development. We therefore propose that NF-κB activation in the absence of additional oncogenic events can promote TNF-dependent, actinic keratosis-like dysplasia and TNF-independent, KAs upon chemical carcinogensis. These studies suggest that resolution of KA cannot occur when NF-κB activation is constitutively enforced.
Girdin Is an Intrinsic Regulator of Neuroblast Chain Migration in the Rostral Migratory Stream of the Postnatal Brain

PubMed Central

Wang, Yun; Kaneko, Naoko; Asai, Naoya; Enomoto, Atsushi; Isotani-Sakakibara, Mayu; Kato, Takuya; Asai, Masato; Murakumo, Yoshiki; Ota, Haruko; Hikita, Takao; Namba, Takashi; Kuroda, Keisuke; Kaibuchi, Kozo; Ming, Guo-li; Song, Hongjun; Sawamoto, Kazunobu; Takahashi, Masahide

2017-01-01

In postnatally developing and adult brains, interneurons of the olfactory bulb (OB) are continuously generated at the subventricular zone of the forebrain. The newborn neuroblasts migrate tangentially to the OB through a well defined pathway, the rostral migratory stream (RMS), where the neuroblasts undergo collective migration termed “chain migration.” The cell-intrinsic regulatory mechanism of neuroblast chain migration, however, has not been uncovered. Here we show that mice lacking the actin-binding Akt substrate Girdin (a protein that interacts with Disrupted-In-Schizophrenia 1 to regulate neurogenesis in the dentate gyrus) have profound defects in neuroblast chain migration along the RMS. Analysis of two gene knock-in mice harboring Girdin mutants identified unique amino acid residues in Girdin’s C-terminal domain that are responsible for the regulation of neuroblast chain migration but revealed no apparent requirement of Girdin phosphorylation by Akt. Electron microscopic analyses demonstrated the involvement of Girdin in neuroblast cell–cell interactions. These findings suggest that Girdin is an important intrinsic factor that specifically governs neuroblast chain migration along the RMS. PMID:21632933
Constitutive glycogen synthase kinase-3α/β activity protects against chronic β-adrenergic remodelling of the heart

PubMed Central

Webb, Ian G.; Nishino, Yasuhiro; Clark, James E.; Murdoch, Colin; Walker, Simon J.; Makowski, Marcus R.; Botnar, Rene M.; Redwood, Simon R.; Shah, Ajay M.; Marber, Michael S.

2010-01-01

Aims Glycogen synthase kinase 3 (GSK-3) signalling is implicated in the growth of the heart during development and in response to stress. However, its precise role remains unclear. We set out to characterize developmental growth and response to chronic isoproterenol (ISO) stress in knockin (KI) mice lacking the critical N-terminal serines, 21 of GSK-3α and 9 of GSK-3β respectively, required for inactivation by upstream kinases. Methods and results Between 5 and 15 weeks, KI mice grew more rapidly, but normalized heart weight and contractile performance were similar to wild-type (WT) mice. Isolated hearts of both genotypes responded comparably to acute ISO infusion with increases in heart rate and contractility. In WT mice, chronic subcutaneous ISO infusion over 14 days resulted in cardiac hypertrophy, interstitial fibrosis, and impaired contractility, accompanied by foetal gene reactivation. These effects were all significantly attenuated in KI mice. Indeed, ISO-treated KI hearts demonstrated reversible physiological remodelling traits with increased stroke volume and a preserved contractile response to acute adrenergic stimulation. Furthermore, simultaneous pharmacological inhibition of GSK-3 in KI mice treated with chronic subcutaneous ISO recapitulated the adverse remodelling phenotype seen in WT hearts. Conclusion Expression of inactivation-resistant GSK-3α/β does not affect eutrophic myocardial growth but protects against pathological hypertrophy induced by chronic adrenergic stimulation, maintaining cardiac function and attenuating interstitial fibrosis. Accordingly, strategies to prevent phosphorylation of Ser-21/9, and consequent inactivation of GSK-3α/β, may enable a sustained cardiac response to chronic β-agonist stimulation while preventing pathological remodelling. PMID:20299330
D409H GBA1 mutation accelerates the progression of pathology in A53T α-synuclein transgenic mouse model.

PubMed

Kim, Donghoon; Hwang, Heehong; Choi, Seulah; Kwon, Sang Ho; Lee, Suhyun; Park, Jae Hong; Kim, SangMin; Ko, Han Seok

2018-04-27

Heterozygous mutations in glucocerebrosidase 1 (GBA1) are a major genetic risk factor for Parkinson's disease and Dementia with Lewy bodies. Mutations in GBA1 leads to GBA1 enzyme deficiency, and GBA1-associated parkinsonism has an earlier age of onset and more progressive parkinsonism. To investigate a potential influence of GBA1 deficiency caused by mutations in GBA1 on the disease progression of PD, GBA1 mice carrying D409H knock-in mutation were crossbred with the human A53T (hA53T) α-synuclein transgenic mice. Here, we show that GBA1 enzyme activity plays a significant role in the hA53T α-synuclein induced α-synucleinopathy. The expression of D409H GBA1 markedly shortens the lifespan of hA53T α-synuclein transgenic mice. Moreover, D409H GBA1 expression exacerbates the formation of insoluble aggregates of α-synuclein, glial activation, neuronal degeneration, and motor abnormalities in the hA53T α-synuclein transgenic mice. Interestingly, the expression of D409H GBA1 results in the loss of dopaminergic neurons in the substantia nigra pars compacta of hA53T transgenic mice. Taken together, these results indicate that GBA1 deficiency due to D409H mutation affects the disease onset and course in hA53T α-synuclein transgenic mice. Therefore, strategies aimed to maintain GBA1 enzyme activity could be employed to develop an effective novel therapy for GBA1 linked-PD and related α-synucleinopathies.
Mutation of the 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) Substrate-Docking Site in the Developing Brain Causes Microcephaly with Abnormal Brain Morphogenesis Independently of Akt, Leading to Impaired Cognition and Disruptive Behaviors

PubMed Central

Cordón-Barris, Lluís; Pascual-Guiral, Sònia; Yang, Shaobin; Giménez-Llort, Lydia; Lope-Piedrafita, Silvia; Niemeyer, Carlota; Claro, Enrique; Lizcano, Jose M.

2016-01-01

The phosphoinositide (PI) 3-kinase/Akt signaling pathway plays essential roles during neuronal development. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) coordinates the PI 3-kinase signals by activating 23 kinases of the AGC family, including Akt. Phosphorylation of a conserved docking site in the substrate is a requisite for PDK1 to recognize, phosphorylate, and activate most of these kinases, with the exception of Akt. We exploited this differential mechanism of regulation by generating neuron-specific conditional knock-in mice expressing a mutant form of PDK1, L155E, in which the substrate-docking site binding motif, termed the PIF pocket, was disrupted. As a consequence, activation of all the PDK1 substrates tested except Akt was abolished. The mice exhibited microcephaly, altered cortical layering, and reduced circuitry, leading to cognitive deficits and exacerbated disruptive behavior combined with diminished motivation. The abnormal patterning of the adult brain arises from the reduced ability of the embryonic neurons to polarize and extend their axons, highlighting the essential roles that the PDK1 signaling beyond Akt plays in mediating the neuronal responses that regulate brain development. PMID:27644329

Mice with a fra-1 knock-in into the c-fos locus show impaired spatial but regular contextual learning and normal LTP.

PubMed

Gass, Peter; Fleischmann, Alexander; Hvalby, Oivind; Jensen, Vidar; Zacher, Christiane; Strekalova, Tatyana; Kvello, Ane; Wagner, Erwin F; Sprengel, Rolf

2004-11-04

The immediate early gene c-fos is part of the AP-1 transcription factor complex, which is involved in molecular mechanisms underlying learning and memory. Mice that lack c-Fos in the brain show impairments in spatial reference and contextual learning, and also exhibit a reduced long-term potentiation of synaptic transmission (LTP) at CA3-to-CA1 synapses. In the present study, we investigated mice in which c-fos was deleted and replaced by fra-1 (c-fos(fra-1) mice) to determine whether other members of the c-fos gene family can substitute for the functions of the c-fos gene. In c-fos(fra-1) mice, both CA3-to-CA1 LTP and contextual learning in a Pavlovian fear conditioning task were similar to wild-type littermates, indicating that Fra-1 expression restored the impairments caused by brain-specific c-Fos depletion. However, c-Fos-mediated learning deficits in a reference memory task of the Morris watermaze were also present in c-fos(fra-1) mice. These findings suggest that different c-Fos target genes are involved in LTP, contextual learning, and spatial reference memory formation.
Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

PubMed Central

Wu, Xue; Yin, Jiani; Simpson, Jeremy; Kim, Kyoung-Han; Gu, Shengqing; Hong, Jenny H.; Bayliss, Peter; Backx, Peter H.

2012-01-01

Noonan syndrome (NS) is a relatively common autosomal dominant disorder characterized by congenital heart defects, short stature, and facial dysmorphia. NS is caused by germ line mutations in several components of the RAS–RAF–MEK–extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, including both kinase-activating and kinase-impaired alleles of RAF1 (∼3 to 5%), which encodes a serine-threonine kinase for MEK1/2. To investigate how kinase-impaired RAF1 mutants cause NS, we generated knock-in mice expressing Raf1D486N. Raf1D486N/+ (here D486N/+) female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1D486N-expressing cells compared with controls. RAF1D486N, as well as other kinase-impaired RAF1 mutants, showed increased heterodimerization with BRAF, which was necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also required heterodimerization to enhance MEK/ERK activation. Our results suggest that an increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations. PMID:22826437
TRPV1 variants impair intracellular Ca2+ signaling and may confer susceptibility to malignant hyperthermia.

PubMed

Abeele, Fabien Vanden; Lotteau, Sabine; Ducreux, Sylvie; Dubois, Charlotte; Monnier, Nicole; Hanna, Amy; Gkika, Dimitra; Romestaing, Caroline; Noyer, Lucile; Flourakis, Matthieu; Tessier, Nolwenn; Al-Mawla, Ribal; Chouabe, Christophe; Lefai, Etienne; Lunardi, Joël; Hamilton, Susan; Fauré, Julien; Van Coppenolle, Fabien; Prevarskaya, Natalia

2018-06-21

Malignant hyperthermia (MH) is a pharmacogenetic disorder arising from uncontrolled muscle calcium release due to an abnormality in the sarcoplasmic reticulum (SR) calcium-release mechanism triggered by halogenated inhalational anesthetics. However, the molecular mechanisms involved are still incomplete. We aimed to identify transient receptor potential vanilloid 1 (TRPV1) variants within the entire coding sequence in patients who developed sensitivity to MH of unknown etiology. In vitro and in vivo functional studies were performed in heterologous expression system, trpv1 -/- mice, and a murine model of human MH. We identified TRPV1 variants in two patients and their heterologous expression in muscles of trpv1 -/- mice strongly enhanced calcium release from SR upon halogenated anesthetic stimulation, suggesting they could be responsible for the MH phenotype. We confirmed the in vivo significance by using mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation associated with MH in humans. We showed that the TRPV1 antagonist capsazepine slows the heat-induced hypermetabolic response in this model. We propose that TRPV1 contributes to MH and could represent an actionable therapeutic target for prevention of the pathology and also be responsible for MH sensitivity when mutated.
Gain-of-function mutant of angiotensin II receptor, type 1A, causes hypertension and cardiovascular fibrosis in mice

PubMed Central

Billet, Sandrine; Bardin, Sabine; Verp, Sonia; Baudrie, Véronique; Michaud, Annie; Conchon, Sophie; Muffat-Joly, Martine; Escoubet, Brigitte; Souil, Evelyne; Hamard, Ghislaine; Bernstein, Kenneth E.; Gasc, Jean Marie; Elghozi, Jean-Luc; Corvol, Pierre; Clauser, Eric

2007-01-01

The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT1A), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT1A leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension. PMID:17607364
RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency.

PubMed

Song, Jun; Yang, Dongshan; Xu, Jie; Zhu, Tianqing; Chen, Y Eugene; Zhang, Jifeng

2016-01-28

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.
Enamelin Is Critical for Ameloblast Integrity and Enamel Ultrastructure Formation

PubMed Central

Hu, Jan C.-C.; Hu, Yuanyuan; Lu, Yuhe; Smith, Charles E.; Lertlam, Rangsiyakorn; Wright, John Timothy; Suggs, Cynthia; McKee, Marc D.; Beniash, Elia; Kabir, M. Enamul; Simmer, James P.

2014-01-01

Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam−/− mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam−/− mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using β-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing amelogenin, they were not able to create a well-defined enamel space or produce normal enamel crystals. When enamelin is present at half of the normal quantity, enamel was thinner with enamel rods not as tightly arranged as in wild type suggesting that a specific quantity of enamelin is critical for normal enamel formation. Enamelin dosage effect was further demonstrated in transgenic mouse lines over expressing enamelin. Introducing enamelin transgene at various expression levels into the Enam−/− background did not fully recover enamel formation while a medium expresser in the Enam+/− background did. Too much or too little enamelin abolishes the production of enamel crystals and prism structure. Enamelin is essential for ameloblast integrity and enamel formation. PMID:24603688
Ankyrin-B metabolic syndrome combines age-dependent adiposity with pancreatic β cell insufficiency.

PubMed

Lorenzo, Damaris N; Healy, Jane A; Hostettler, Janell; Davis, Jonathan; Yang, Jiayu; Wang, Chao; Hohmeier, Hans Ewald; Zhang, Mingjie; Bennett, Vann

2015-08-03

Rare functional variants of ankyrin-B have been implicated in human disease, including hereditary cardiac arrhythmia and type 2 diabetes (T2D). Here, we developed murine models to evaluate the metabolic consequences of these alterations in vivo. Specifically, we generated knockin mice that express either the human ankyrin-B variant R1788W, which is present in 0.3% of North Americans of mixed European descent and is associated with T2D, or L1622I, which is present in 7.5% of African Americans. Young AnkbR1788W/R1788W mice displayed primary pancreatic β cell insufficiency that was characterized by reduced insulin secretion in response to muscarinic agonists, combined with increased peripheral glucose uptake and concomitantly increased plasma membrane localization of glucose transporter 4 (GLUT4) in skeletal muscle and adipocytes. In contrast, older AnkbR1788W/R1788W and AnkbL1622I/L1622I mice developed increased adiposity, a phenotype that was reproduced in cultured adipocytes, and insulin resistance. GLUT4 trafficking was altered in animals expressing mutant forms of ankyrin-B, and we propose that increased cell surface expression of GLUT4 in skeletal muscle and fatty tissue of AnkbR1788W/R1788W mice leads to the observed age-dependent adiposity. Together, our data suggest that ankyrin-B deficiency results in a metabolic syndrome that combines primary pancreatic β cell insufficiency with peripheral insulin resistance and is directly relevant to the nearly one million North Americans bearing the R1788W ankyrin-B variant.
JAK2V617F expression in mice amplifies early hematopoietic cells and gives them a competitive advantage that is hampered by IFNα.

PubMed

Hasan, Salma; Lacout, Catherine; Marty, Caroline; Cuingnet, Marie; Solary, Eric; Vainchenker, William; Villeval, Jean-Luc

2013-08-22

The acquired gain-of-function V617F mutation in the Janus Kinase 2 (JAK2(V617F)) is the main mutation involved in BCR/ABL-negative myeloproliferative neoplasms (MPNs), but its effect on hematopoietic stem cells as a driver of disease emergence has been questioned. Therefore, we reinvestigated the role of endogenous expression of JAK2(V617F) on early steps of hematopoiesis as well as the effect of interferon-α (IFNα), which may target the JAK2(V617F) clone in humans by using knock-in mice with conditional expression of JAK2(V617F) in hematopoietic cells. These mice develop a MPN mimicking polycythemia vera with large amplification of myeloid mature and precursor cells, displaying erythroid endogenous growth and progressing to myelofibrosis. Interestingly, early hematopoietic compartments [Lin-, LSK, and SLAM (LSK/CD48-/CD150+)] increased with the age. Competitive repopulation assays demonstrated disease appearance and progressive overgrowth of myeloid, Lin-, LSK, and SLAM cells, but not lymphocytes, from a low number of engrafted JAK2(V617F) SLAM cells. Finally, IFNα treatment prevented disease development by specifically inhibiting JAK2(V617F) cells at an early stage of differentiation and eradicating disease-initiating cells. This study shows that JAK2(V617F) in mice amplifies not only late but also early hematopoietic cells, giving them a proliferative advantage through high cell cycling and low apoptosis that may sustain MPN emergence but is lost upon IFNα treatment.
Loss of corticostriatal and thalamostriatal synaptic terminals precedes striatal projection neuron pathology in heterozygous Q140 Huntington's disease mice.

PubMed

Deng, Y P; Wong, T; Bricker-Anthony, C; Deng, B; Reiner, A

2013-12-01

Motor slowing, forebrain white matter loss, and striatal shrinkage have been reported in premanifest Huntington's disease (HD) prior to overt striatal neuron loss. We carried out detailed LM and EM studies in a genetically precise HD mimic, heterozygous Q140 HD knock-in mice, to examine the possibility that loss of corticostriatal and thalamostriatal terminals prior to striatal neuron loss underlies these premanifest HD abnormalities. In our studies, we used VGLUT1 and VGLUT2 immunolabeling to detect corticostriatal and thalamostriatal (respectively) terminals in dorsolateral (motor) striatum over the first year of life, prior to striatal projection neuron pathology. VGLUT1+ axospinous corticostriatal terminals represented about 55% of all excitatory terminals in striatum, and VGLUT2+ axospinous thalamostriatal terminals represented about 35%, with VGLUT1+ and VGLUT2+ axodendritic terminals accounting for the remainder. In Q140 mice, a significant 40% shortfall in VGLUT2+ axodendritic thalamostriatal terminals and a 20% shortfall in axospinous thalamostriatal terminals were already observed at 1 month of age, but VGLUT1+ terminals were normal in abundance. The 20% deficiency in VGLUT2+ thalamostriatal axospinous terminals persisted at 4 and 12 months in Q140 mice, and an additional 30% loss of VGLUT1+ corticostriatal terminals was observed at 12 months. The early and persistent deficiency in thalamostriatal axospinous terminals in Q140 mice may reflect a development defect, and the impoverishment of this excitatory drive to striatum may help explain early motor defects in Q140 mice and in premanifest HD. The loss of corticostriatal terminals at 1 year in Q140 mice is consistent with prior evidence from other mouse models of corticostriatal disconnection early during progression, and can explain both the measurable bradykinesia and striatal white matter loss in late premanifest HD. © 2013.
Behavioral and Electrophysiological Characterization of Dyt1 Heterozygous Knockout Mice

PubMed Central

Yokoi, Fumiaki; Chen, Huan-Xin; Dang, Mai Tu; Cheetham, Chad C.; Campbell, Susan L.; Roper, Steven N.; Sweatt, J. David; Li, Yuqing

2015-01-01

DYT1 dystonia is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most of the patients have a trinucleotide deletion (ΔGAG) corresponding to a glutamic acid in the C-terminal region (torsinAΔE). Dyt1 ΔGAG heterozygous knock-in (KI) mice, which mimic ΔGAG mutation in the endogenous gene, exhibit motor deficits and deceased frequency of spontaneous excitatory post-synaptic currents (sEPSCs) and normal theta-burst-induced long-term potentiation (LTP) in the hippocampal CA1 region. Although Dyt1 KI mice show decreased hippocampal torsinA levels, it is not clear whether the decreased torsinA level itself affects the synaptic plasticity or torsinAΔE does it. To analyze the effect of partial torsinA loss on motor behaviors and synaptic transmission, Dyt1 heterozygous knock-out (KO) mice were examined as a model of a frame-shift DYT1 mutation in patients. Consistent with Dyt1 KI mice, Dyt1 heterozygous KO mice showed motor deficits in the beam-walking test. Dyt1 heterozygous KO mice showed decreased hippocampal torsinA levels lower than those in Dyt1 KI mice. Reduced sEPSCs and normal miniature excitatory post-synaptic currents (mEPSCs) were also observed in the acute hippocampal brain slices from Dyt1 heterozygous KO mice, suggesting that the partial loss of torsinA function in Dyt1 KI mice causes action potential-dependent neurotransmitter release deficits. On the other hand, Dyt1 heterozygous KO mice showed enhanced hippocampal LTP, normal input-output relations and paired pulse ratios in the extracellular field recordings. The results suggest that maintaining an appropriate torsinA level is important to sustain normal motor performance, synaptic transmission and plasticity. Developing therapeutics to restore a normal torsinA level may help to prevent and treat the symptoms in DYT1 dystonia. PMID:25799505
Behavioral and electrophysiological characterization of Dyt1 heterozygous knockout mice.

PubMed

Yokoi, Fumiaki; Chen, Huan-Xin; Dang, Mai Tu; Cheetham, Chad C; Campbell, Susan L; Roper, Steven N; Sweatt, J David; Li, Yuqing

2015-01-01

DYT1 dystonia is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most of the patients have a trinucleotide deletion (ΔGAG) corresponding to a glutamic acid in the C-terminal region (torsinA(ΔE)). Dyt1 ΔGAG heterozygous knock-in (KI) mice, which mimic ΔGAG mutation in the endogenous gene, exhibit motor deficits and deceased frequency of spontaneous excitatory post-synaptic currents (sEPSCs) and normal theta-burst-induced long-term potentiation (LTP) in the hippocampal CA1 region. Although Dyt1 KI mice show decreased hippocampal torsinA levels, it is not clear whether the decreased torsinA level itself affects the synaptic plasticity or torsinA(ΔE) does it. To analyze the effect of partial torsinA loss on motor behaviors and synaptic transmission, Dyt1 heterozygous knock-out (KO) mice were examined as a model of a frame-shift DYT1 mutation in patients. Consistent with Dyt1 KI mice, Dyt1 heterozygous KO mice showed motor deficits in the beam-walking test. Dyt1 heterozygous KO mice showed decreased hippocampal torsinA levels lower than those in Dyt1 KI mice. Reduced sEPSCs and normal miniature excitatory post-synaptic currents (mEPSCs) were also observed in the acute hippocampal brain slices from Dyt1 heterozygous KO mice, suggesting that the partial loss of torsinA function in Dyt1 KI mice causes action potential-dependent neurotransmitter release deficits. On the other hand, Dyt1 heterozygous KO mice showed enhanced hippocampal LTP, normal input-output relations and paired pulse ratios in the extracellular field recordings. The results suggest that maintaining an appropriate torsinA level is important to sustain normal motor performance, synaptic transmission and plasticity. Developing therapeutics to restore a normal torsinA level may help to prevent and treat the symptoms in DYT1 dystonia.
Early life environmental and pharmacological stressors result in persistent dysregulations of the serotonergic system

PubMed Central

Wong, Peiyan; Sze, Ying; Gray, Laura Jane; Chang, Cecilia Chin Roei; Cai, Shiwei; Zhang, Xiaodong

2015-01-01

Dysregulations in the brain serotonergic system and exposure to environmental stressors have been implicated in the development of major depressive disorder. Here, we investigate the interactions between the stress and serotonergic systems by characterizing the behavioral and biochemical effects of chronic stress applied during early-life or adulthood in wild type (WT) mice and mice with deficient tryptophan hydroxylase 2 (TPH2) function. We showed that chronic mild stress applied in adulthood did not affect the behaviors and serotonin levels of WT and TPH2 knock-in (KI) mice. Whereas, maternal separation (MS) stress increased anxiety- and depressive-like behaviors of WT mice, with no detectable behavioral changes in TPH2 KI mice. Biochemically, we found that MS WT mice had reduced brain serotonin levels, which was attributed to increased expression of monoamine oxidase A (MAO A). The increased MAO A expression was detected in MS WT mice at 4 weeks old and adulthood. No change in TPH2 expression was detected. To determine whether a pharmacological stressor, dexamethasone (Dex), will result in similar biochemical results obtained from MS, we used an in vitro system, SH-SY5Y cells, and found that Dex treatment resulted in increased MAO A expression levels. We then treated WT mice with Dex for 5 days, either during postnatal days 7–11 or adulthood. Both groups of Dex treated WT mice had reduced basal corticosterone and glucocorticoid receptors expression levels. However, only Dex treatment during PND7–11 resulted in reduced serotonin levels and increased MAO A expression. Just as with MS WT mice, TPH2 expression in PND7–11 Dex-treated WT mice was unaffected. Taken together, our findings suggest that both environmental and pharmacological stressors affect the expression of MAO A, and not TPH2, when applied during the critical postnatal period. This leads to long-lasting perturbations in the serotonergic system, and results in anxiety- and depressive-like behaviors. PMID:25964750
Increased mtDNA mutations with aging promotes amyloid accumulation and brain atrophy in the APP/Ld transgenic mouse model of Alzheimer’s disease

PubMed Central

2014-01-01

Background The role of mitochondrial dysfunction has long been implicated in age-related brain pathology, including Alzheimer’s disease (AD). However, the mechanism by which mitochondrial dysfunction may cause neurodegeneration in AD is unclear. To model mitochondrial dysfunction in vivo, we utilized mice that harbor a knockin mutation that inactivates the proofreading function of mitochondrial DNA polymerase γ (PolgA D257A), so that these mice accumulate mitochondrial DNA mutations with age. PolgA D257A mice develop a myriad of mitochondrial bioenergetic defects and physical phenotypes that mimic premature ageing, with subsequent death around one year of age. Results We crossed the D257A mice with a well-established transgenic AD mouse model (APP/Ld) that develops amyloid plaques. We hypothesized that mitochondrial dysfunction would affect Aβ synthesis and/or clearance, thus contributing to amyloidogenesis and triggering neurodegeneration. Initially, we discovered that Aβ42 levels along with Aβ42 plaque density were increased in D257A; APP/Ld bigenic mice compared to APP/Ld monogenic mice. Elevated Aβ production was not responsible for increased amyloid pathology, as levels of BACE1, PS1, C99, and C83 were unchanged in D257A; APP/Ld compared to APP/Ld mice. However, the levels of a major Aβ clearance enzyme, insulin degrading enzyme (IDE), were reduced in mice with the D257A mutation, suggesting this as mechanism for increased amyloid load. In the presence of the APP transgene, D257A mice also exhibited significant brain atrophy with apparent cortical thinning but no frank neuron loss. D257A; APP/Ld mice had increased levels of 17 kDa cleaved caspase-3 and p25, both indicative of neurodegeneration. Moreover, D257A; APP/Ld neurons appeared morphologically disrupted, with swollen and vacuolated nuclei. Conclusions Overall, our results implicate synergism between the effects of the PolgA D257A mutation and Aβ in causing neurodegeneration. These findings provide insight into mechanisms of mitochondrial dysfunction that may contribute to the pathogenesis of AD via decreased clearance of Aβ. PMID:24885175
Esrrb-Cre Excises loxP-Flanked Alleles in Early Four-Cell Embryos

PubMed Central

Kim, Suyeon; Shaffer, Benjamin; Simerly, Calvin R.; Richard Chaillet, J.; Barak, Yaacov

2015-01-01

Among transgenic mice with ubiquitous Cre recombinase activity, all strains to date excise loxP-flanked (floxed) alleles, either at or before the zygote stage or at nondescript stages of development. This manuscript describes a new mouse strain, in which Cre recombinase, integrated into the Esrrb locus, efficiently excises floxed alleles in pre-implantation embryos at the onset of the four-cell stage. By enabling inactivation of genes only after the embryo has undergone two cleavages, this strain should facilitate in vivo studies of genes with essential gametic or zygotic functions. In addition, this study describes a new, highly pluripotent hybrid C57BL/6J × 129S1/SvImJ mouse embryonic stem cell line, HYB12, in which this knock-in and additional targeted alleles have been generated. PMID:26663459
Apolipoprotein E4 causes age- and Tau-dependent impairment of GABAergic interneurons, leading to learning and memory deficits in mice.

PubMed

Andrews-Zwilling, Yaisa; Bien-Ly, Nga; Xu, Qin; Li, Gang; Bernardo, Aubrey; Yoon, Seo Yeon; Zwilling, Daniel; Yan, Tonya Xue; Chen, Ligong; Huang, Yadong

2010-10-13

Apolipoprotein E4 (apoE4) is the major genetic risk factor for Alzheimer's disease. However, the underlying mechanisms are unclear. We found that female apoE4 knock-in (KI) mice had an age-dependent decrease in hilar GABAergic interneurons that correlated with the extent of learning and memory deficits, as determined in the Morris water maze, in aged mice. Treating apoE4-KI mice with daily peritoneal injections of the GABA(A) receptor potentiator pentobarbital at 20 mg/kg for 4 weeks rescued the learning and memory deficits. In neurotoxic apoE4 fragment transgenic mice, hilar GABAergic interneuron loss was even more pronounced and also correlated with the extent of learning and memory deficits. Neurodegeneration and tauopathy occurred earliest in hilar interneurons in apoE4 fragment transgenic mice; eliminating endogenous Tau prevented hilar GABAergic interneuron loss and the learning and memory deficits. The GABA(A) receptor antagonist picrotoxin abolished this rescue, while pentobarbital rescued learning deficits in the presence of endogenous Tau. Thus, apoE4 causes age- and Tau-dependent impairment of hilar GABAergic interneurons, leading to learning and memory deficits in mice. Consequently, reducing Tau and enhancing GABA signaling are potential strategies to treat or prevent apoE4-related Alzheimer's disease.
RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency

PubMed Central

Song, Jun; Yang, Dongshan; Xu, Jie; Zhu, Tianqing; Chen, Y. Eugene; Zhang, Jifeng

2016-01-01

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells. PMID:26817820
Bitopic Sphingosine 1-Phosphate Receptor 3 (S1P3) Antagonist Rescue from Complete Heart Block: Pharmacological and Genetic Evidence for Direct S1P3 Regulation of Mouse Cardiac Conduction.

PubMed

Sanna, M Germana; Vincent, Kevin P; Repetto, Emanuela; Nguyen, Nhan; Brown, Steven J; Abgaryan, Lusine; Riley, Sean W; Leaf, Nora B; Cahalan, Stuart M; Kiosses, William B; Kohno, Yasushi; Brown, Joan Heller; McCulloch, Andrew D; Rosen, Hugh; Gonzalez-Cabrera, Pedro J

2016-01-01

The molecular pharmacology of the G protein-coupled receptors for sphingosine 1-phosphate (S1P) provides important insight into established and new therapeutic targets. A new, potent bitopic S1P3 antagonist, SPM-354, with in vivo activity, has been used, together with S1P3-knockin and S1P3-knockout mice to define the spatial and functional properties of S1P3 in regulating cardiac conduction. We show that S1P3 is a key direct regulator of cardiac rhythm both in vivo and in isolated perfused hearts. 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol in vivo and S1P in isolated hearts induced a spectrum of cardiac effects, ranging from sinus bradycardia to complete heart block, as measured by a surface electrocardiogram in anesthetized mice and in volume-conducted Langendorff preparations. The agonist effects on complete heart block are absent in S1P3-knockout mice and are reversed in wild-type mice with SPM-354, as characterized and described here. Homologous knockin of S1P3-mCherry is fully functional pharmacologically and is strongly expressed by immunohistochemistry confocal microscopy in Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4)-positive atrioventricular node and His-Purkinje fibers, with relative less expression in the HCN4-positive sinoatrial node. In Langendorff studies, at constant pressure, SPM-354 restored sinus rhythm in S1P-induced complete heart block and fully reversed S1P-mediated bradycardia. S1P3 distribution and function in the mouse ventricular cardiac conduction system suggest a direct mechanism for heart block risk that should be further studied in humans. A richer understanding of receptor and ligand usage in the pacemaker cells of the cardiac system is likely to be useful in understanding ventricular conduction in health, disease, and pharmacology. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
Apolipoprotein E4 Causes Age- and Sex-Dependent Impairments of Hilar GABAergic Interneurons and Learning and Memory Deficits in Mice

PubMed Central

Leung, Laura; Andrews-Zwilling, Yaisa; Yoon, Seo Yeon; Jain, Sachi; Ring, Karen; Dai, Jessica; Wang, Max Mu; Tong, Leslie; Walker, David; Huang, Yadong

2012-01-01

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease (AD). ApoE4 has sex-dependent effects, whereby the risk of developing AD is higher in apoE4-expressing females than males. However, the mechanism underlying the sex difference, in relation to apoE4, is unknown. Previous findings indicate that apoE4 causes age-dependent impairments of hilar GABAergic interneurons in female mice, leading to learning and memory deficits. Here, we investigate whether the detrimental effects of apoE4 on hilar GABAergic interneurons are sex-dependent using apoE knock-in (KI) mice across different ages. We found that in female apoE-KI mice, there was an age-dependent depletion of hilar GABAergic interneurons, whereby GAD67- or somatostatin-positive–but not NPY- or parvalbumin-positive–interneuron loss was exacerbated by apoE4. Loss of these neuronal populations was correlated with the severity of spatial learning deficits at 16 months of age in female apoE4-KI mice; however, this effect was not observed in female apoE3-KI mice. In contrast, we found an increase in the numbers of hilar GABAergic interneurons with advancing age in male apoE-KI mice, regardless of apoE genotype. Moreover, male apoE-KI mice showed a consistent ratio of hilar inhibitory GABAergic interneurons to excitatory mossy cells approximating 1.5 that is independent of apoE genotype and age, whereas female apoE-KI mice exhibited an age-dependent decrease in this ratio, which was exacerbated by apoE4. Interestingly, there are no apoE genotype effects on GABAergic interneurons in the CA1 and CA3 subregions of the hippocampus as well as the entorhinal and auditory cortexes. These findings suggest that the sex-dependent effects of apoE4 on developing AD is in part attributable to inherent sex-based differences in the numbers of hilar GABAergic interneurons, which is further modulated by apoE genotype. PMID:23300939
Mice expressing T4826I-RYR1 are viable but exhibit sex- and genotype-dependent susceptibility to malignant hyperthermia and muscle damage

PubMed Central

Yuen, Benjamin; Boncompagni, Simona; Feng, Wei; Yang, Tianzhong; Lopez, Jose R.; Matthaei, Klaus I.; Goth, Samuel R.; Protasi, Feliciano; Franzini-Armstrong, Clara; Allen, Paul D.; Pessah, Isaac N.

2012-01-01

Mutation T4825I in the type 1 ryanodine receptor (RYR1T4825I/+) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1T4826I/+ (Het) or homozygous RYR1T4826I/T4826I (Hom) mice are fully viable under typical rearing conditions but exhibit genotype- and sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca2+]rest, and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy.—Yuen, B., Boncompagni, S., Feng, W., Yang, T., Lopez, J. R., Matthaei, K. I., Goth, S. R., Protasi, F., Franzini-Armstrong, C., Allen, P. D., Pessah, I. N. Mice expressing T4826I-RYR1 are viable but exhibit sex- and genotype-dependent susceptibility to malignant hyperthermia and muscle damage. PMID:22131268
MEK-ERK pathway modulation ameliorates disease phenotypes in a mouse model of Noonan syndrome associated with the Raf1L613V mutation

PubMed Central

Wu, Xue; Simpson, Jeremy; Hong, Jenny H.; Kim, Kyoung-Han; Thavarajah, Nirusha K.; Backx, Peter H.; Neel, Benjamin G.; Araki, Toshiyuki

2011-01-01

Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden death in children and young adults. Abnormalities in several signaling pathways are implicated in the pathogenesis of HCM, but the role of the RAS-RAF-MEK-ERK MAPK pathway has been controversial. Noonan syndrome (NS) is one of several autosomal-dominant conditions known as RASopathies, which are caused by mutations in different components of this pathway. Germline mutations in RAF1 (which encodes the serine-threonine kinase RAF1) account for approximately 3%–5% of cases of NS. Unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with HCM. To explore the pathogenesis of such mutations, we generated knockin mice expressing the NS-associated Raf1L613V mutation. Like NS patients, mice heterozygous for this mutation (referred to herein as L613V/+ mice) had short stature, craniofacial dysmorphia, and hematologic abnormalities. Valvuloseptal development was normal, but L613V/+ mice exhibited eccentric cardiac hypertrophy and aberrant cardiac fetal gene expression, and decompensated following pressure overload. Agonist-evoked MEK-ERK activation was enhanced in multiple cell types, and postnatal MEK inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice. These data show that different NS genes have intrinsically distinct pathological effects, demonstrate that enhanced MEK-ERK activity is critical for causing HCM and other RAF1-mutant NS phenotypes, and suggest a mutation-specific approach to the treatment of RASopathies. PMID:21339642

Mutated CaV2.1 channels dysregulate CASK/P2X3 signaling in mouse trigeminal sensory neurons of R192Q Cacna1a knock-in mice

PubMed Central

2013-01-01

Background ATP-gated P2X3 receptors of sensory ganglion neurons are important transducers of pain as they adapt their expression and function in response to acute and chronic nociceptive signals. The present study investigated the role of calcium/calmodulin-dependent serine protein kinase (CASK) in controlling P2X3 receptor expression and function in trigeminal ganglia from Cacna1a R192Q-mutated knock-in (KI) mice, a genetic model for familial hemiplegic migraine type-1. Results KI ganglion neurons showed more abundant CASK/P2X3 receptor complex at membrane level, a result that likely originated from gain-of-function effects of R192Q-mutated CaV2.1 channels and downstream enhanced CaMKII activity. The selective CaV2.1 channel blocker ω-Agatoxin IVA and the CaMKII inhibitor KN-93 were sufficient to return CASK/P2X3 co-expression to WT levels. After CASK silencing, P2X3 receptor expression was decreased in both WT and KI ganglia, supporting the role of CASK in P2X3 receptor stabilization. This process was functionally observed as reduced P2X3 receptor currents. Conclusions We propose that, in trigeminal sensory neurons, the CASK/P2X3 complex has a dynamic nature depending on intracellular calcium and related signaling, that are enhanced in a transgenic mouse model of genetic hemiplegic migraine. PMID:24294842
A Knock-in Mouse Model of Human PHD2 Gene-associated Erythrocytosis Establishes a Haploinsufficiency Mechanism*

PubMed Central

Arsenault, Patrick R.; Pei, Fei; Lee, Rebecca; Kerestes, Heddy; Percy, Melanie J.; Keith, Brian; Simon, M. Celeste; Lappin, Terence R. J.; Khurana, Tejvir S.; Lee, Frank S.

2013-01-01

The central pathway for controlling red cell mass is the PHD (prolyl hydroxylase domain protein):hypoxia-inducible factor (HIF) pathway. HIF, which is negatively regulated by PHD, activates numerous genes, including ones involved in erythropoiesis, such as the ERYTHROPOIETIN (EPO) gene. Recent studies have implicated PHD2 as the key PHD isoform regulating red cell mass. Studies of humans have identified erythrocytosis-associated, heterozygous point mutations in the PHD2 gene. A key question concerns the mechanism by which human mutations lead to phenotypes. In the present report, we generated and characterized a mouse line in which a P294R knock-in mutation has been introduced into the mouse Phd2 locus to model the first reported human PHD2 mutation (P317R). Phd2P294R/+ mice display a degree of erythrocytosis equivalent to that seen in Phd2+/− mice. The Phd2P294R/+-associated erythrocytosis is reversed in a Hif2a+/−, but not a Hif1a+/− background. Additional studies using various conditional knock-outs of Phd2 reveal that erythrocytosis can be induced by homozygous and heterozygous knock-out of Phd2 in renal cortical interstitial cells using a Pax3-Cre transgene or by homozygous knock-out of Phd2 in hematopoietic progenitors driven by a Vav1-Cre transgene. These studies formally prove that a missense mutation in PHD2 is the cause of the erythrocytosis, show that this occurs through haploinsufficiency, and point to multifactorial control of red cell mass by PHD2. PMID:24121508
Tissue-specific deletion of c-Jun in the pancreas has limited effects on pancreas formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamamoto, Kaoru; Miyatsuka, Takeshi; Tanaka, Ayako

2007-11-30

It is well known that activating protein-1 (AP-1) is involved in a variety of cellular functions such as proliferation, differentiation, apoptosis, and oncogenesis. AP-1 is a dimer complex consisting of different subunits, and c-Jun is known to be one of its major components. In addition, it has been shown that mice lacking c-Jun are embryonic lethal and that c-Jun is essential for liver and heart development. However, the role of c-Jun in the pancreas is not well known. The aim of this study was to examine the possible role of c-Jun in the pancreas. First, c-Jun was strongly expressed inmore » pancreatic duct-like structures at an embryonic stage, while a lower level of expression was observed in some part of the adult pancreas, implying that c-Jun might play a role during pancreas development. Second, to address this point, we generated pancreas-specific c-Jun knock-out mice (Ptf1a-Cre; c-Jun{sup flox/flox} mice) by crossing Ptf1a-Cre knock-in mice with c-Jun floxed mice. Ptf1a is a pancreatic transcription factor and its expression is confined to pancreatic stem/progenitor cells, which give rise to all three types of pancreatic tissue: endocrine, exocrine, and duct. Contrary to our expectation, however, there was no morphological difference in the pancreas between Ptf1a-Cre; c-Jun{sup flox/flox} and control mice. In addition, there was no difference in body weight, pancreas weight, and the expression of various pancreas-related factors (insulin, glucagon, cytokeratin, and amylase) between the two groups. Furthermore, there was no difference in glucose tolerance between Ptf1a-Cre; c-Jun{sup flox/flox} and control mice. Taken together, although we cannot exclude the possibility that c-Jun ablation is compensated by some unknown factors, c-Jun appears to be dispensable for pancreas development at least after ptf1a gene promoter is activated.« less
Thymidine kinase 2 deficiency-induced mitochondrial DNA depletion causes abnormal development of adipose tissues and adipokine levels in mice.

PubMed

Villarroya, Joan; Dorado, Beatriz; Vilà, Maya R; Garcia-Arumí, Elena; Domingo, Pere; Giralt, Marta; Hirano, Michio; Villarroya, Francesc

2011-01-01

Mammal adipose tissues require mitochondrial activity for proper development and differentiation. The components of the mitochondrial respiratory chain/oxidative phosphorylation system (OXPHOS) are encoded by both mitochondrial and nuclear genomes. The maintenance of mitochondrial DNA (mtDNA) is a key element for a functional mitochondrial oxidative activity in mammalian cells. To ascertain the role of mtDNA levels in adipose tissue, we have analyzed the alterations in white (WAT) and brown (BAT) adipose tissues in thymidine kinase 2 (Tk2) H126N knockin mice, a model of TK2 deficiency-induced mtDNA depletion. We observed respectively severe and moderate mtDNA depletion in TK2-deficient BAT and WAT, showing both tissues moderate hypotrophy and reduced fat accumulation. Electron microscopy revealed altered mitochondrial morphology in brown but not in white adipocytes from TK2-deficient mice. Although significant reduction in mtDNA-encoded transcripts was observed both in WAT and BAT, protein levels from distinct OXPHOS complexes were significantly reduced only in TK2-deficient BAT. Accordingly, the activity of cytochrome c oxidase was significantly lowered only in BAT from TK2-deficient mice. The analysis of transcripts encoding up to fourteen components of specific adipose tissue functions revealed that, in both TK2-deficient WAT and BAT, there was a consistent reduction of thermogenesis related gene expression and a severe reduction in leptin mRNA. Reduced levels of resistin mRNA were found in BAT from TK2-deficient mice. Analysis of serum indicated a dramatic reduction in circulating levels of leptin and resistin. In summary, our present study establishes that mtDNA depletion leads to a moderate impairment in mitochondrial respiratory function, especially in BAT, causes substantial alterations in WAT and BAT development, and has a profound impact in the endocrine properties of adipose tissues. © 2011 Villarroya et al.
Thymidine Kinase 2 Deficiency-Induced Mitochondrial DNA Depletion Causes Abnormal Development of Adipose Tissues and Adipokine Levels in Mice

PubMed Central

Villarroya, Joan; Dorado, Beatriz; Vilà, Maya R.; Garcia-Arumí, Elena; Domingo, Pere; Giralt, Marta; Hirano, Michio; Villarroya, Francesc

2011-01-01

Mammal adipose tissues require mitochondrial activity for proper development and differentiation. The components of the mitochondrial respiratory chain/oxidative phosphorylation system (OXPHOS) are encoded by both mitochondrial and nuclear genomes. The maintenance of mitochondrial DNA (mtDNA) is a key element for a functional mitochondrial oxidative activity in mammalian cells. To ascertain the role of mtDNA levels in adipose tissue, we have analyzed the alterations in white (WAT) and brown (BAT) adipose tissues in thymidine kinase 2 (Tk2) H126N knockin mice, a model of TK2 deficiency-induced mtDNA depletion. We observed respectively severe and moderate mtDNA depletion in TK2-deficient BAT and WAT, showing both tissues moderate hypotrophy and reduced fat accumulation. Electron microscopy revealed altered mitochondrial morphology in brown but not in white adipocytes from TK2-deficient mice. Although significant reduction in mtDNA-encoded transcripts was observed both in WAT and BAT, protein levels from distinct OXPHOS complexes were significantly reduced only in TK2-deficient BAT. Accordingly, the activity of cytochrome c oxidase was significantly lowered only in BAT from TK2-deficient mice. The analysis of transcripts encoding up to fourteen components of specific adipose tissue functions revealed that, in both TK2-deficient WAT and BAT, there was a consistent reduction of thermogenesis related gene expression and a severe reduction in leptin mRNA. Reduced levels of resistin mRNA were found in BAT from TK2-deficient mice. Analysis of serum indicated a dramatic reduction in circulating levels of leptin and resistin. In summary, our present study establishes that mtDNA depletion leads to a moderate impairment in mitochondrial respiratory function, especially in BAT, causes substantial alterations in WAT and BAT development, and has a profound impact in the endocrine properties of adipose tissues. PMID:22216345
Dental abnormalities in a mouse model for craniometaphyseal dysplasia.

PubMed

Dutra, E H; Chen, I-P; Reichenberger, E J

2013-02-01

Mice carrying a knock-in mutation (Phe377del) in the Ank gene replicate many skeletal characteristics of human craniometaphyseal dysplasia, including hyperostotic mandibles. Ank (KI/KI) mice have normal morphology of erupted molars and incisors but excessive cementum deposition with increased numbers of Ibsp- and Dmp1-positive cells on root surfaces. The cervical loops of adult Ank (KI/KI) lower incisors are at the level of the third molars, while they are close to the mandibular foramen in Ank (+/+) mice. Furthermore, Ank (KI/KI) incisors show decreased eruption rates, decreased proliferation of odontoblast precursors, and increased cell apoptosis in the stellate reticulum. However, their capability for continuous elongation is not compromised. Quantification of TRAP-positive cells in the apical ends of Ank (KI/KI) incisors revealed decreased osteoclast numbers and osteoclast surfaces. Bisphosphonate injections in Ank (+/+) mice replicate the Ank (KI/KI) incisor phenotype. These results and a comparison with the dental phenotype of Ank loss-of-function mouse models suggest that increased cementum thickness may be caused by decreased extracellular PPi levels and that the incisor phenotype is likely due to hyperostosis of mandibles, which distinguishes Ank (KI/KI) mice from the other Ank mouse models.
Danish dementia mice suggest that loss of function and not the amyloid cascade causes synaptic plasticity and memory deficits

PubMed Central

Tamayev, Robert; Matsuda, Shuji; Fà, Mauro; Arancio, Ottavio; D’Adamio, Luciano

2010-01-01

According to the prevailing “amyloid cascade hypothesis,” genetic dementias such as Alzheimer’s disease and familial Danish dementia (FDD) are caused by amyloid deposits that trigger tauopathy, neurodegeneration, and behavioral/cognitive alterations. To efficiently reproduce amyloid lesions, murine models of human dementias invariably use transgenic expression systems. However, recent FDD transgenic models showed that Danish amyloidosis does not cause memory defects, suggesting that other mechanisms cause Danish dementia. We studied an animal knock-in model of FDD (FDDKI/+) genetically congruous with human cases. FDDKI/+ mice present reduced Bri2 levels, impaired synaptic plasticity and severe hippocampal memory deficits. These animals show no cerebral lesions that are reputed characteristics of human dementia, such as tangles or amyloid plaques. Bri2+/− mice exhibit synaptic and memory deficits similar to FDDKI/+ mice, and memory loss of FDDKI/+ mice is prevented by expression of WT BRI2, indicating that Danish dementia is caused by loss of BRI2 function. Together, the data suggest that clinical dementia in Danish patients occurs via a loss of function mechanism and not as a result of amyloidosis and tauopathy. PMID:21098268
Danish dementia mice suggest that loss of function and not the amyloid cascade causes synaptic plasticity and memory deficits.

PubMed

Tamayev, Robert; Matsuda, Shuji; Fà, Mauro; Arancio, Ottavio; D'Adamio, Luciano

2010-11-30

According to the prevailing "amyloid cascade hypothesis," genetic dementias such as Alzheimer's disease and familial Danish dementia (FDD) are caused by amyloid deposits that trigger tauopathy, neurodegeneration, and behavioral/cognitive alterations. To efficiently reproduce amyloid lesions, murine models of human dementias invariably use transgenic expression systems. However, recent FDD transgenic models showed that Danish amyloidosis does not cause memory defects, suggesting that other mechanisms cause Danish dementia. We studied an animal knock-in model of FDD (FDD(KI/+)) genetically congruous with human cases. FDD(KI/+) mice present reduced Bri2 levels, impaired synaptic plasticity and severe hippocampal memory deficits. These animals show no cerebral lesions that are reputed characteristics of human dementia, such as tangles or amyloid plaques. Bri2(+/-) mice exhibit synaptic and memory deficits similar to FDD(KI/+) mice, and memory loss of FDD(KI/+) mice is prevented by expression of WT BRI2, indicating that Danish dementia is caused by loss of BRI2 function. Together, the data suggest that clinical dementia in Danish patients occurs via a loss of function mechanism and not as a result of amyloidosis and tauopathy.
DNA polymerase ι functions in the generation of tandem mutations during somatic hypermutation of antibody genes.

PubMed

Maul, Robert W; MacCarthy, Thomas; Frank, Ekaterina G; Donigan, Katherine A; McLenigan, Mary P; Yang, William; Saribasak, Huseyin; Huston, Donald E; Lange, Sabine S; Woodgate, Roger; Gearhart, Patricia J

2016-08-22

DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol ι, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol ι, and knock-in mice that express full-length Pol ι that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol ι did not change overall mutagenesis. We next examined if Pol ι affected tandem mutations generated by another error-prone polymerase, Pol ζ. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol ι-compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol ι occasionally accesses the replication fork to generate a first mutation, and Pol ζ extends the mismatch with a second mutation. @2016.
DNA polymerase ι functions in the generation of tandem mutations during somatic hypermutation of antibody genes

PubMed Central

Donigan, Katherine A.; Huston, Donald E.; Lange, Sabine S.

2016-01-01

DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol ι, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol ι, and knock-in mice that express full-length Pol ι that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol ι did not change overall mutagenesis. We next examined if Pol ι affected tandem mutations generated by another error-prone polymerase, Pol ζ. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol ι–compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol ι occasionally accesses the replication fork to generate a first mutation, and Pol ζ extends the mismatch with a second mutation. PMID:27455952
Mutation of the inhibitory ethanol site in GABAA ρ1 receptors promotes tolerance to ethanol-induced motor incoordination.

PubMed

Blednov, Yuri A; Borghese, Cecilia M; Ruiz, Carlos I; Cullins, Madeline A; Da Costa, Adriana; Osterndorff-Kahanek, Elizabeth A; Homanics, Gregg E; Harris, R Adron

2017-09-01

Genes encoding the ρ1/2 subunits of GABA A receptors have been associated with alcohol (ethanol) dependence in humans, and ρ1 was also shown to regulate some of the behavioral effects of ethanol in animal models. Ethanol inhibits GABA-mediated responses in wild-type (WT) ρ1, but not ρ1(T6'Y) mutant receptors expressed in Xenopus laevis oocytes, indicating the presence of an inhibitory site for ethanol in the second transmembrane helix. In this study, we found that ρ1(T6'Y) receptors expressed in oocytes display overall normal responses to GABA, the endogenous GABA modulator (zinc), and partial agonists (β-alanine and taurine). We generated ρ1 (T6'Y) knockin (KI) mice using CRISPR/Cas9 to test the behavioral importance of the inhibitory actions of ethanol on this receptor. Both ρ1 KI and knockout (KO) mice showed faster recovery from acute ethanol-induced motor incoordination compared to WT mice. Both KI and KO mutant strains also showed increased tolerance to motor impairment produced by ethanol. The KI mice did not differ from WT mice in other behavioral actions, including ethanol intake and preference, conditioned taste aversion to ethanol, and duration of ethanol-induced loss of righting reflex. WT and KI mice did not differ in levels of ρ1 or ρ2 mRNA in cerebellum or in ethanol clearance. Our findings indicate that the inhibitory site for ethanol in GABA A ρ1 receptors regulates acute functional tolerance to moderate ethanol intoxication. We note that low sensitivity to alcohol intoxication has been linked to risk for development of alcohol dependence in humans. Copyright © 2017 Elsevier Ltd. All rights reserved.
Glycine Receptors Containing α2 or α3 Subunits Regulate Specific Ethanol-Mediated Behaviors

PubMed Central

Blednov, Yuri A.; Benavidez, Jillian M.; Black, Mendy; Leiter, Courtney R.; Osterndorff-Kahanek, Elizabeth

2015-01-01

Glycine receptors (GlyRs) are broadly expressed in the central nervous system. Ethanol enhances the function of brain GlyRs, and the GlyRα1 subunit is associated with some of the behavioral actions of ethanol, such as loss of righting reflex. The in vivo role of GlyRα2 and α3 subunits in alcohol responses has not been characterized despite high expression levels in the nucleus accumbens and amygdala, areas that are important for the rewarding properties of drugs of abuse. We used an extensive panel of behavioral tests to examine ethanol actions in mice lacking Glra2 (the gene encoding the glycine receptor alpha 2 subunit) or Glra3 (the gene encoding the glycine receptor alpha 3 subunit). Deletion of Glra2 or Glra3 alters specific ethanol-induced behaviors. Glra2 knockout mice demonstrate reduced ethanol intake and preference in the 24-hour two-bottle choice test and increased initial aversive responses to ethanol and lithium chloride. In contrast, Glra3 knockout mice show increased ethanol intake and preference in the 24-hour intermittent access test and increased development of conditioned taste aversion to ethanol. Mutants and wild-type mice consumed similar amounts of ethanol in the limited access drinking in the dark test. Other ethanol effects, such as anxiolysis, motor incoordination, loss of righting reflex, and acoustic startle response, were not altered in the mutants. The behavioral changes in mice lacking GlyRα2 or α3 subunits were distinct from effects previously observed in mice with knock-in mutations in the α1 subunit. We provide evidence that GlyRα2 and α3 subunits may regulate ethanol consumption and the aversive response to ethanol. PMID:25678534
Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture

PubMed Central

New, Laura A.; Martin, Claire E.; Scott, Rizaldy P.; Platt, Mathew J.; Keyvani Chahi, Ava; Stringer, Colin D.; Lu, Peihua; Samborska, Bozena; Eremina, Vera; Takano, Tomoko; Simpson, Jeremy A.; Quaggin, Susan E.

2016-01-01

Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin–rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphonephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt nephrin tyrosine phosphorylation and Nck1/2 binding (nephrinY3F/Y3F mice). Homozygous nephrinY3F/Y3F mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, nephrinY3F/Y3F mice displayed delayed recovery in podocyte injury models. Profiling of nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton. PMID:26802179
Eosinophil Activities Modulate the Immune/Inflammatory Character of Allergic Respiratory Responses in Mice

PubMed Central

Jacobsen, Elizabeth A.; LeSuer, William E.; Willetts, Lian; Zellner, Katie R.; Mazzolini, Kirea; Antonios, Nathalie; Beck, Brandon; Protheroe, Cheryl; Ochkur, Sergei I.; Colbert, Dana; Lacy, Paige; Moqbel, Redwan; Appleton, Judith; Lee, Nancy A.; Lee, James J.

2014-01-01

Background The importance and specific role(s) of eosinophils in modulating the immune/inflammatory phenotype of allergic pulmonary disease remain to be defined. Established animals models assessing the role(s) of eosinophils as contributors and/or causative agents of disease have relied on congenitally deficient mice where the developmental consequences of eosinophil depletion are unknown. Methods We developed a novel conditional eosinophil-deficient strain of mice (iPHIL) through a gene knock-in strategy inserting the human diphtheria toxin (DT) receptor (DTR) into the endogenous eosinophil peroxidase genomic locus. Results Expression of DTR rendered resistant mouse eosinophil progenitors sensitive to DT without affecting any other cell types. The presence of eosinophils was shown to be unnecessary during the sensitization phase of either ovalbumin (OVA) or house dust mite (HDM) acute asthma models. However, eosinophil ablation during airway challenge led to a predominantly neutrophilic phenotype (>15% neutrophils) accompanied by allergen-induced histopathologies and airway hyperresponsiveness in response to methacholine indistinguishable from eosinophilic wild type mice. Moreover, the iPHIL neutrophilic airway phenotype was shown to be a steroid-resistant allergic respiratory variant that was reversible upon restoration of peripheral eosinophils. Conclusions Eosinophil contributions to allergic immune/inflammatory responses appear to be limited to the airway challenge and not the sensitization phase of allergen provocation models. The reversible steroid-resistant character of the iPHIL neutrophilic airway variant suggests underappreciated mechanisms by which eosinophils shape the character of allergic respiratory responses. PMID:24266710
Adrenergic Receptors in Individual Ventricular Myocytes: The Beta-1 and Alpha-1B Are in All Cells, the Alpha-1A Is in a Subpopulation, and the Beta-2 and Beta-3 Are Mostly Absent.

PubMed

Myagmar, Bat-Erdene; Flynn, James M; Cowley, Patrick M; Swigart, Philip M; Montgomery, Megan D; Thai, Kevin; Nair, Divya; Gupta, Rumita; Deng, David X; Hosoda, Chihiro; Melov, Simon; Baker, Anthony J; Simpson, Paul C

2017-03-31

It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), β1, β2, β3, α1A, and α1B. The β1 and β2 are thought to be the dominant myocyte ARs. Quantify the 5 cardiac ARs in individual ventricular myocytes. We studied ventricular myocytes from wild-type mice, mice with α1A and α1B knockin reporters, and β1 and β2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal-regulated kinase and phospholamban), and contraction. We found that the β1 and α1B were present in all myocytes. The α1A was present in 60%, with high levels in 20%. The β2 and β3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total β-ARs were β2 and 20% were β3, both mainly in nonmyocytes. The dominant ventricular myocyte ARs present in all cells are the β1 and α1B. The β2 and β3 are mostly absent in myocytes but are abundant in nonmyocytes. The α1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with β1 and α1B only; 60% that also have the α1A; and 5% each that also have the β2 or β3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms. © 2017 American Heart Association, Inc.
Treadmill exercise delays the onset of non-motor behaviors and striatal pathology in the CAG140 knock-in mouse model of Huntington's disease.

PubMed

Stefanko, D P; Shah, V D; Yamasaki, W K; Petzinger, G M; Jakowec, M W

2017-09-01

Depression, cognitive impairments, and other neuropsychiatric disturbances are common during the prodromal phase of Huntington's disease (HD) well before the onset of classical motor symptoms of this degenerative disorder. The purpose of this study was to examine the potential impact of physical activity in the form of exercise on a motorized treadmill on non-motor behavioral features including depression-like behavior and cognition in the CAG 140 knock-in (KI) mouse model of HD. The CAG 140 KI mouse model has a long lifespan compared to other HD rodent models with HD motor deficits emerging after 12months of age and thus provides the opportunity to investigate early life interventions such as exercise on disease progression. Motorized treadmill running was initiated at 4weeks of age (1h per session, 3 times per week) and continued for 6months. Non-motor behaviors were assessed up to 6months of age and included analysis of depression-like behavior (using the tail-suspension and forced-swim tests) and cognition (using the T-maze and object recognition tests). At both 4 and 6months of age, CAG 140 KI mice displayed significant depression-like behavior in the forced swim and tail suspension tests and cognitive impairment by deficits in reversal relearning in the T-maze test. These deficits were not evident in mice engaged in treadmill running. In addition, exercise restored striatal dopamine D2 receptor expression and dopamine neurotransmitter levels both reduced in sedentary HD mice. Finally, we examined the pattern of striatal expression of mutant huntingtin (mHTT) protein and showed that the number and intensity of immunohistochemical staining patterns of intranuclear aggregates were significantly reduced with exercise. Altogether these findings begin to address the potential impact of lifestyle and early intervention such as exercise on modifying HD progression. Copyright © 2017 Elsevier Inc. All rights reserved.
DNA damage checkpoint pathway modulates the regulation of skeletal growth and osteoblastic bone formation by parathyroid hormone-related peptide.

PubMed

Zhang, Ying; Chen, Guangpei; Gu, Zhen; Sun, Haijian; Karaplis, Andrew; Goltzman, David; Miao, Dengshun

2018-01-01

We previously demonstrated that parathyroid hormone-related peptide (PTHrP) 1-84 knockin ( Pthrp KI) mice, which lacked a PTHrP nuclear localization sequence (NLS) and C-terminus, displayed early senescence, defective osteoblastic bone formation, and skeletal growth retardation. However, the mechanism of action of the PTHrP NLS and C-terminus in regulating development of skeleton is still unclear. In this study, we examined alterations of oxidative stress and DNA damage response-related molecules in Pthrp KI skeletal tissue. We found that ROS levels, protein expression levels of γ-H2AX, a DNA damage marker, and the DNA damage response markers p-Chk2 and p53 were up-regulated, whereas gene expression levels of anti-oxidative enzymes were down-regulated significantly. We therefore further disrupted the DNA damage response pathway by deleting the Chk2 in Pthrp KI (Chk2 -/- KI) mice and did comparison with WT, Chk2 -/- and Pthrp KI littermates. The Pthrp KI mice with Chk2 deletion exhibited a longer lifespan, improvement in osteoblastic bone formation and skeletal growth including width of growth plates and length of long bones, trabecular and epiphyseal bone volume, BMD, osteoblast numbers, type I collagen and ALP positive bone areas, the numbers of total colony-forming unit fibroblasts (CFU-f), ALP + CFU-f and the expression levels of osteogenic genes. In addition, the genes associated with anti-oxidative enzymes were up-regulated significantly, whereas the tumor suppressor genes related to senescence were down-regulated in Chk2 -/- KI mice compared to Pthrp KI mice. Our results suggest that Chk2 deletion in Pthrp KI mice can somewhat rescue defects in osteoblastic bone formation and skeletal growth by enhancing endochondral bone formation and osteogenesis. These studies therefore indicate that the DNA damage checkpoint pathway may be a target for the nuclear action of PTHrP to regulate skeletal development and growth.
Mutation of the key residue for extraribosomal function of ribosomal protein S19 cause increased grooming behaviors in mice.

PubMed

Chen, Jun; Kaitsuka, Taku; Fujino, Rika; Araki, Kimi; Tomizawa, Kazuhito; Yamamoto, Tetsuro

2016-08-26

Ribosomal protein S19 (RP S19) possesses ribosomal function as RP S19 monomer and extraribosomal function as cross-linked RP S19 oligomers which function as a ligand of the complement 5a (C5a) receptor (CD88). We have generated a Gln137Glu-RP S19 knock-in (KI) mouse, which is shown to possess the weakened extraribosomal function of RP S19. Because whether the extraribosomal function of RP S19 has a role in brain function had been unclear, we performed behavioral analysis on these mice and demonstrated that KI mice displayed an increased grooming behavior during open-field test and elevated plus maze test and an enhanced freezing behavior in contextual fear conditioning test. These results suggest an involvement of RP S19 oligomers in some anxiety-like behavior, especially grooming behavior. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
Modeling recent human evolution in mice by expression of a selected EDAR variant

PubMed Central

Kamberov, Yana G.; Wang, Sijia; Tan, Jingze; Gerbault, Pascale; Wark, Abigail; Tan, Longzhi; Yang, Yajun; Li, Shilin; Tang, Kun; Chen, Hua; Powell, Adam; Itan, Yuval; Fuller, Dorian; Lohmueller, Jason; Mao, Junhao; Schachar, Asa; Paymer, Madeline; Hostetter, Elizabeth; Byrne, Elizabeth; Burnett, Melissa; McMahon, Andrew P.; Thomas, Mark G.; Lieberman, Daniel E.; Jin, Li; Tabin, Clifford J.; Morgan, Bruce A.; Sabeti, Pardis C.

2013-01-01

Summary An adaptive variant of the human Ectodysplasin receptor, EDARV370A, is one of the strongest candidates of recent positive selection from genome-wide scans. We have modeled EDAR370A in mice and characterized its phenotype and evolutionary origins in humans. Our computational analysis suggests the allele arose in Central China approximately 30,000 years ago. Although EDAR370A has been associated with increased scalp hair thickness and changed tooth morphology in humans, its direct biological significance and potential adaptive role remain unclear. We generated a knock-in mouse model and find that, as in humans, hair thickness is increased in EDAR370A mice. We identify novel biological targets affected by the mutation, including mammary and eccrine glands. Building on these results, we find that EDAR370A is associated with an increased number of active eccrine glands in the Han Chinese. This interdisciplinary approach yields unique insight into the generation of adaptive variation among modern humans. PMID:23415220
4-aminopyridine reverses ataxia and cerebellar firing deficiency in a mouse model of spinocerebellar ataxia type 6

PubMed Central

Jayabal, Sriram; Chang, Hui Ho Vanessa; Cullen, Kathleen E.; Watt, Alanna J.

2016-01-01

Spinocerebellar ataxia type 6 (SCA6) is a devastating midlife-onset autosomal dominant motor control disease with no known treatment. Using a hyper-expanded polyglutamine (84Q) knock-in mouse, we found that cerebellar Purkinje cell firing precision was degraded in heterozygous (SCA684Q/+) mice at 19 months when motor deficits are observed. Similar alterations in firing precision and motor control were observed at disease onset at 7 months in homozygous (SCA684Q/84Q) mice, as well as a reduction in firing rate. We further found that chronic administration of the FDA-approved drug 4-aminopyridine (4-AP), which targets potassium channels, alleviated motor coordination deficits and restored cerebellar Purkinje cell firing precision to wildtype (WT) levels in SCA684Q/84Q mice both in acute slices and in vivo. These results provide a novel therapeutic approach for treating ataxic symptoms associated with SCA6. PMID:27381005

4-aminopyridine reverses ataxia and cerebellar firing deficiency in a mouse model of spinocerebellar ataxia type 6.

PubMed

Jayabal, Sriram; Chang, Hui Ho Vanessa; Cullen, Kathleen E; Watt, Alanna J

2016-07-06

Spinocerebellar ataxia type 6 (SCA6) is a devastating midlife-onset autosomal dominant motor control disease with no known treatment. Using a hyper-expanded polyglutamine (84Q) knock-in mouse, we found that cerebellar Purkinje cell firing precision was degraded in heterozygous (SCA6(84Q/+)) mice at 19 months when motor deficits are observed. Similar alterations in firing precision and motor control were observed at disease onset at 7 months in homozygous (SCA6(84Q/84Q)) mice, as well as a reduction in firing rate. We further found that chronic administration of the FDA-approved drug 4-aminopyridine (4-AP), which targets potassium channels, alleviated motor coordination deficits and restored cerebellar Purkinje cell firing precision to wildtype (WT) levels in SCA6(84Q/84Q) mice both in acute slices and in vivo. These results provide a novel therapeutic approach for treating ataxic symptoms associated with SCA6.
Estrogen Receptor Alpha Binding to ERE is Required for Full Tlr7- and Tlr9-Induced Inflammation

PubMed Central

Cunningham, Melissa A; Wirth, Jena R; Naga, Osama; Eudaly, Jackie; Gilkeson, Gary S

2014-01-01

We previously found that a maximum innate inflammatory response induced by stimulation of Toll-like receptors (TLRs) 3, 7 and 9 requires ERα, but does not require estrogen in multiple cell types from both control and lupus-prone mice. Given the estrogen-independence, we hypothesized that ERα mediates TLR signaling by tethering to, and enhancing, the activity of downstream transcription factors such as NFκB, rather than acting classically by binding EREs on target genes. To investigate the mechanism of ERα impact on TLR signaling, we utilized mice with a knock-in ERα mutant that is unable to bind ERE. After stimulation with TLR ligands, both ex vivo spleen cells and bone marrow-derived dendritic cells (BM-DCs) isolated from mutant ERα (“KIKO”) mice produced significantly less IL-6 compared with cells from wild-type (WT) littermates. These results suggest that ERα modulation of TLR signaling does indeed require ERE binding for its effect on the innate immune response. PMID:25061615
Mutations in the Katnb1 gene cause left-right asymmetry and heart defects.

PubMed

Furtado, Milena B; Merriner, D Jo; Berger, Silke; Rhodes, Danielle; Jamsai, Duangporn; O'Bryan, Moira K

2017-12-01

The microtubule-severing protein complex katanin is composed two subunits, the ATPase subunit, KATNA1, and the noncatalytic regulatory subunit, KATNB1. Recently, the Katnb1 gene has been linked to infertility, regulation of centriole and cilia formation in fish and mammals, as well as neocortical brain development. KATNB1 protein is expressed in germ cells in humans and mouse, mitotic/meiotic spindles and cilia, although the full expression pattern of the Katnb1 gene has not been described. Using a knockin-knockout mouse model of Katnb1 dysfunction we demonstrate that Katnb1 is ubiquitously expressed during embryonic development, although a stronger expression is seen in the crown cells of the gastrulation organizer, the murine node. Furthermore, null and hypomorphic Katnb1 gene mutations show a novel correlation between Katnb1 dysregulation and the development of impaired left-right signaling, including cardiac malformations. Katanin function is a critical regulator of heart development in mice. These findings are potentially relevant to human cardiac development. Developmental Dynamics 246:1027-1035, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis.

PubMed

Wu, Haibo; Wang, Yongsheng; Zhang, Yan; Yang, Mingqi; Lv, Jiaxing; Liu, Jun; Zhang, Yong

2015-03-31

Transcription activator-like effector nuclease (TALEN)-mediated genome modification has been applied successfully to create transgenic animals in various species, such as mouse, pig, and even monkey. However, transgenic cattle with gene knockin have yet to be created using TALENs. Here, we report site-specific knockin of the transcription activator-like effector (TALE) nickase-mediated SP110 nuclear body protein gene (SP110) via homologous recombination to produce tuberculosis-resistant cattle. In vitro and in vivo challenge and transmission experiments proved that the transgenic cattle are able to control the growth and multiplication of Mycobacterium bovis, turn on the apoptotic pathway of cell death instead of necrosis after infection, and efficiently resist the low dose of M. bovis transmitted from tuberculous cattle in nature. In this study, we developed TALE nickases to modify the genome of Holstein-Friesian cattle, thereby engineering a heritable genome modification that facilitates resistance to tuberculosis.
TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis

PubMed Central

Wu, Haibo; Wang, Yongsheng; Zhang, Yan; Yang, Mingqi; Lv, Jiaxing; Liu, Jun; Zhang, Yong

2015-01-01

Transcription activator-like effector nuclease (TALEN)-mediated genome modification has been applied successfully to create transgenic animals in various species, such as mouse, pig, and even monkey. However, transgenic cattle with gene knockin have yet to be created using TALENs. Here, we report site-specific knockin of the transcription activator-like effector (TALE) nickase-mediated SP110 nuclear body protein gene (SP110) via homologous recombination to produce tuberculosis-resistant cattle. In vitro and in vivo challenge and transmission experiments proved that the transgenic cattle are able to control the growth and multiplication of Mycobacterium bovis, turn on the apoptotic pathway of cell death instead of necrosis after infection, and efficiently resist the low dose of M. bovis transmitted from tuberculous cattle in nature. In this study, we developed TALE nickases to modify the genome of Holstein–Friesian cattle, thereby engineering a heritable genome modification that facilitates resistance to tuberculosis. PMID:25733846
Next-generation mammalian genetics toward organism-level systems biology.

PubMed

Susaki, Etsuo A; Ukai, Hideki; Ueda, Hiroki R

2017-01-01

Organism-level systems biology in mammals aims to identify, analyze, control, and design molecular and cellular networks executing various biological functions in mammals. In particular, system-level identification and analysis of molecular and cellular networks can be accelerated by next-generation mammalian genetics. Mammalian genetics without crossing, where all production and phenotyping studies of genome-edited animals are completed within a single generation drastically reduce the time, space, and effort of conducting the systems research. Next-generation mammalian genetics is based on recent technological advancements in genome editing and developmental engineering. The process begins with introduction of double-strand breaks into genomic DNA by using site-specific endonucleases, which results in highly efficient genome editing in mammalian zygotes or embryonic stem cells. By using nuclease-mediated genome editing in zygotes, or ~100% embryonic stem cell-derived mouse technology, whole-body knock-out and knock-in mice can be produced within a single generation. These emerging technologies allow us to produce multiple knock-out or knock-in strains in high-throughput manner. In this review, we discuss the basic concepts and related technologies as well as current challenges and future opportunities for next-generation mammalian genetics in organism-level systems biology.
Exercise induces autophagy in peripheral tissues and in the brain.

PubMed

He, Congcong; Sumpter, Rhea; Levine, Beth

2012-10-01

We recently identified physical exercise as a newly defined inducer of autophagy in vivo. Exercise induced autophagy in multiple organs involved in metabolic regulation, such as muscle, liver, pancreas and adipose tissue. To study the physiological role of exercise-induced autophagy, we generated mice with a knock-in nonphosphorylatable mutation in BCL2 (Thr69Ala, Ser70Ala and Ser84Ala) (BCL2 AAA) that are defective in exercise- and starvation-induced autophagy but not in basal autophagy. We found that BCL2 AAA mice could not run on a treadmill as long as wild-type mice, and did not undergo exercise-mediated increases in skeletal glucose muscle uptake. Unlike wild-type mice, the BCL2 AAA mice failed to reverse high-fat diet-induced glucose intolerance after 8 weeks of exercise training, possibly due to defects in signaling pathways that regulate muscle glucose uptake and metabolism during exercise. Together, these findings suggested a hitherto unknown important role of autophagy in mediating exercise-induced metabolic benefits. In the present addendum, we show that treadmill exercise also induces autophagy in the cerebral cortex of adult mice. This observation raises the intriguing question of whether autophagy may in part mediate the beneficial effects of exercise in neurodegeneration, adult neurogenesis and improved cognitive function.
Anxiolytic effect of music exposure on BDNFMet/Met transgenic mice.

PubMed

Li, Wen-Jing; Yu, Hui; Yang, Jian-Min; Gao, Jing; Jiang, Hong; Feng, Min; Zhao, Yu-Xia; Chen, Zhe-Yu

2010-08-06

Brain-derived neurotrophic factor (BDNF) has been reported to play important roles in the modulation of anxiety, mood stabilizers, and pathophysiology of affective disorders. Recently, a single nucleotide polymorphism (SNP) in the BDNF gene (Val66Met) has been found to be associated with depression and anxiety disorders. The humanized BDNF(Met/Met) knock-in transgenic mice exhibited increased anxiety-related behaviors that were unresponsive to serotonin reuptake inhibitors, fluoxetine. Music is known to be able to elicit emotional changes, including anxiolytic effects. In this study, we found that music treatment could significantly decrease anxiety state in BDNF(Met/Met) mice, but not in BDNF(+/)(-), mice compared with white noise exposure in open field and elevated plus maze test. Moreover, in contrast to white noise exposure, BDNF expression levels in the prefrontal cortex (PFC), amygdala and hippocampus were significantly increased in music-exposed adult BDNF(Met/Met) mice. However, music treatment could not upregulate BDNF levels in the PFC, amygdala, and hippocampus in BDNF(+/)(-) mice, which suggests the essential role of BDNF in the anxiolytic effect of music. Together, our results imply that music may provide an effective therapeutic intervention for anxiety disorders in humans with this genetic BDNF(Met) variant. Copyright 2010 Elsevier B.V. All rights reserved.
Physiological Levels of Pik3ca H1047R Mutation in the Mouse Mammary Gland Results in Ductal Hyperplasia and Formation of ERα-Positive Tumors

PubMed Central

Tikoo, Anjali; Roh, Vincent; Montgomery, Karen G.; Ivetac, Ivan; Waring, Paul; Pelzer, Rebecca; Hare, Lauren; Shackleton, Mark; Humbert, Patrick; Phillips, Wayne A.

2012-01-01

PIK3CA, the gene coding for the p110α subunit of phosphoinositide 3-kinase, is frequently mutated in a variety of human tumors including breast cancers. To better understand the role of mutant PIK3CA in the initiation and/or progression of breast cancer, we have generated mice with a conditional knock-in of the common activating mutation, Pik3caH1047R, into one allele of the endogenous gene in the mammary gland. These mice developed a ductal anaplasia and hyperplasia by 6 weeks of age characterized by multi-layering of the epithelial lining of the mammary ducts and expansion of the luminal progenitor (Lin−; CD29lo; CD24+; CD61+) cell population. The Pik3caH1047R expressing mice eventually develop mammary tumors with 100% penetrance but with a long latency (>12 months). This is significantly longer than has been reported for transgenic models where expression of the mutant Pik3ca is driven by an exogenous promoter. Histological analysis of the tumors formed revealed predominantly ERα-positive fibroadenomas, carcinosarcomas and sarcomas. In vitro induction of Pik3caH1047R in immortalized mammary epithelial cells also resulted in tumor formation when injected into the mammary fat pad of immunodeficient recipient mice. This novel model, which reproduces the scenario of a heterozygous somatic mutation occurring in the endogenous PIK3CA gene, will thus be a valuable tool for investigating the role of Pik3caH1047R mutation in mammary tumorigenesis both in vivo and in vitro. PMID:22666336
Effect of Early-Life Fluoxetine on Anxiety-Like Behaviors in BDNF Val66Met Mice.

PubMed

Dincheva, Iva; Yang, Jianmin; Li, Anfei; Marinic, Tina; Freilingsdorf, Helena; Huang, Chienchun; Casey, B J; Hempstead, Barbara; Glatt, Charles E; Lee, Francis S; Bath, Kevin G; Jing, Deqiang

2017-12-01

Adolescence is a developmental stage in which the incidence of psychiatric disorders, such as anxiety disorders, peaks. Selective serotonin reuptake inhibitors (SSRIs) are the main class of agents used to treat anxiety disorders. However, the impact of SSRIs on the developing brain during adolescence remains unknown. The authors assessed the impact of developmentally timed SSRI administration in a genetic mouse model displaying elevated anxiety-like behaviors. Knock-in mice containing a common human single-nucleotide polymorphism (Val66Met; rs6265) in brain-derived neurotrophic factor (BDNF), a growth factor implicated in the mechanism of action of SSRIs, were studied based on their established phenotype of increased anxiety-like behavior. Timed administration of fluoxetine was delivered during one of three developmental periods (postnatal days 21-42, 40-61, or 60-81), spanning the transition from childhood to adulthood. Neurochemical and anxiety-like behavioral analyses were performed. We identified a "sensitive period" during periadolescence (postnatal days 21-42) in which developmentally timed fluoxetine administration rescued anxiety-like phenotypes in BDNF Val66Met mice in adulthood. Compared with littermate controls, BDNF Met/Met mice exhibited diminished maturation of serotonergic fibers projecting particularly to the prefrontal cortex, as well as decreased expression of the serotonergic trophic factor S100B in the dorsal raphe. Interestingly, deficient serotonergic innervation, as well as S100B levels, were rescued with fluoxetine administration during periadolescence. These findings suggest that SSRI administration during a "sensitive period" during periadolescence leads to long-lasting anxiolytic effects in a genetic mouse model of elevated anxiety-like behaviors. These persistent effects highlight the role of BDNF in the maturation of the serotonin system and the capacity to enhance its development through a pharmacological intervention.
Upk3b is dispensable for development and integrity of urothelium and mesothelium.

PubMed

Rudat, Carsten; Grieskamp, Thomas; Röhr, Christian; Airik, Rannar; Wrede, Christoph; Hegermann, Jan; Herrmann, Bernhard G; Schuster-Gossler, Karin; Kispert, Andreas

2014-01-01

The mesothelium, the lining of the coelomic cavities, and the urothelium, the inner lining of the urinary drainage system, are highly specialized epithelia that protect the underlying tissues from mechanical stress and seal them from the overlying fluid space. The development of these epithelia from simple precursors and the molecular characteristics of the mature tissues are poorly analyzed. Here, we show that uroplakin 3B (Upk3b), which encodes an integral membrane protein of the tetraspanin superfamily, is specifically expressed both in development as well as under homeostatic conditions in adult mice in the mesothelia of the body cavities, i.e., the epicardium and pericardium, the pleura and the peritoneum, and in the urothelium of the urinary tract. To analyze Upk3b function, we generated a creERT2 knock-in allele by homologous recombination in embryonic stem cells. We show that Upk3bcreERT2 represents a null allele despite the lack of creERT2 expression from the mutated locus. Morphological, histological and molecular analyses of Upk3b-deficient mice did not detect changes in differentiation or integrity of the urothelium and the mesothelia that cover internal organs. Upk3b is coexpressed with the closely related Upk3a gene in the urothelium but not in the mesothelium, leaving the possibility of a functional redundancy between the two genes in the urothelium only.
Pharmacological Amelioration of Cone Survival and Vision in a Mouse Model for Leber Congenital Amaurosis

PubMed Central

Li, Songhua; Samardzija, Marijana; Yang, Zhihui; Grimm, Christian

2016-01-01

RPE65, an abundant membrane-associate protein in the retinal pigment epithelium (RPE), is a key retinoid isomerase of the visual cycle necessary for generating 11-cis-retinal that functions not only as a molecular switch for activating cone and rod visual pigments in response to light stimulation, but also as a chaperone for normal trafficking of cone opsins to the outer segments. Many mutations in RPE65 are associated with Leber congenital amaurosis (LCA). A R91W substitution, the most frequent LCA-associated mutation, results in a severe decrease in protein level and enzymatic activity of RPE65, causing cone opsin mislocalization and early cone degeneration in the mutation knock-in mouse model of LCA. Here we show that R91W RPE65 undergoes ubiquitination-dependent proteasomal degradation in the knock-in mouse RPE due to misfolding. The 26S proteasome non-ATPase regulatory subunit 13 mediated degradation specifically of misfolded R91W RPE65. The mutation disrupted membrane-association and colocalization of RPE65 with lecithin:retinol acyltransferase (LRAT) that provides the hydrophobic substrate for RPE65. Systemic administration of sodium 4-phenylbutyrate (PBA), a chemical chaperone, increased protein stability, enzymatic activity, membrane-association, and colocalization of R91W RPE65 with LRAT. This rescue effect increased synthesis of 11-cis-retinal and 9-cis-retinal, a functional iso-chromophore of the visual pigments, led to alleviation of S-opsin mislocalization and cone degeneration in the knock-in mice. Importantly, PBA-treatment also improved cone-mediated vision in the mutant mice. These results indicate that PBA, a U.S. Food and Drug Administration-approved safe oral medication, may provide a noninvasive therapeutic intervention that delays daylight vision loss in patients with RPE65 mutations. SIGNIFICANCE STATEMENT LCA is a severe early onset retinal dystrophy. Recent clinical trials of gene therapy have implicated the need of an alternative or combination therapy to improve cone survival and function in patients with LCA caused by RPE65 mutations. Using a mouse model carrying the most frequent LCA-associated mutation (R91W), we found that the mutant RPE65 underwent ubiquitination-dependent proteasomal degradation due to misfolding. Treatment of the mice with a chemical chaperone partially corrected stability, enzymatic activity, and subcellular localization of R91W RPE65, which was also accompanied by improvement of cone survival and vision. These findings identify an in vivo molecular pathogenic mechanism for R91W mutation and provide a feasible pharmacological approach that can delay vision loss in patients with RPE65 mutations. PMID:27225770
Immature doublecortin-positive hippocampal neurons are important for learning but not for remembering.

PubMed

Vukovic, Jana; Borlikova, Gilyana G; Ruitenberg, Marc J; Robinson, Gregory J; Sullivan, Robert K P; Walker, Tara L; Bartlett, Perry F

2013-04-10

It is now widely accepted that hippocampal neurogenesis underpins critical cognitive functions, such as learning and memory. To assess the behavioral importance of adult-born neurons, we developed a novel knock-in mouse model that allowed us to specifically and reversibly ablate hippocampal neurons at an immature stage. In these mice, the diphtheria toxin receptor (DTR) is expressed under control of the doublecortin (DCX) promoter, which allows for specific ablation of immature DCX-expressing neurons after administration of diphtheria toxin while leaving the neural precursor pool intact. Using a spatially challenging behavioral test (a modified version of the active place avoidance test), we present direct evidence that immature DCX-expressing neurons are required for successful acquisition of spatial learning, as well as reversal learning, but are not necessary for the retrieval of stored long-term memories. Importantly, the observed learning deficits were rescued as newly generated immature neurons repopulated the granule cell layer upon termination of the toxin treatment. Repeat (or cyclic) depletion of immature neurons reinstated behavioral deficits if the mice were challenged with a novel task. Together, these findings highlight the potential of stimulating neurogenesis as a means to enhance learning.
Dysfunctional Coq9 protein causes predominant encephalomyopathy associated with CoQ deficiency.

PubMed

García-Corzo, Laura; Luna-Sánchez, Marta; Doerrier, Carolina; García, José A; Guarás, Adela; Acín-Pérez, Rebeca; Bullejos-Peregrín, Javier; López, Ana; Escames, Germaine; Enríquez, José A; Acuña-Castroviejo, Darío; López, Luis C

2013-03-15

Coenzyme Q10 (CoQ(10)) or ubiquinone is a well-known component of the mitochondrial respiratory chain. In humans, CoQ(10) deficiency causes a mitochondrial syndrome with an unexplained variability in the clinical presentations. To try to understand this heterogeneity in the clinical phenotypes, we have generated a Coq9 Knockin (R239X) mouse model. The lack of a functional Coq9 protein in homozygous Coq9 mutant (Coq9(X/X)) mice causes a severe reduction in the Coq7 protein and, as consequence, a widespread CoQ deficiency and accumulation of demethoxyubiquinone. The deficit in CoQ induces a brain-specific impairment of mitochondrial bioenergetics performance, a reduction in respiratory control ratio, ATP levels and ATP/ADP ratio and specific loss of respiratory complex I. These effects lead to neuronal death and demyelinization with severe vacuolization and astrogliosis in the brain of Coq9(X/X) mice that consequently die between 3 and 6 months of age. These results suggest that the instability of mitochondrial complex I in the brain, as a primary event, triggers the development of mitochondrial encephalomyopathy associated with CoQ deficiency.
Animal models of pituitary neoplasia

PubMed Central

Lines, K.E.; Stevenson, M.; Thakker, R.V.

2016-01-01

Pituitary neoplasias can occur as part of a complex inherited disorder, or more commonly as sporadic (non-familial) disease. Studies of the molecular and genetic mechanisms causing such pituitary tumours have identified dysregulation of >35 genes, with many revealed by studies in mice, rats and zebrafish. Strategies used to generate these animal models have included gene knockout, gene knockin and transgenic over-expression, as well as chemical mutagenesis and drug induction. These animal models provide an important resource for investigation of tissue-specific tumourigenic mechanisms, and evaluations of novel therapies, illustrated by studies into multiple endocrine neoplasia type 1 (MEN1), a hereditary syndrome in which ∼30% of patients develop pituitary adenomas. This review describes animal models of pituitary neoplasia that have been generated, together with some recent advances in gene editing technologies, and an illustration of the use of the Men1 mouse as a pre clinical model for evaluating novel therapies. PMID:26320859
K-RasG12D–induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to γ-secretase inhibitors

PubMed Central

Cornejo, Melanie G.; Scholl, Claudia; Liu, Jianing; Leeman, Dena S.; Haydu, J. Erika; Fröhling, Stefan; Lee, Benjamin H.; Gilliland, D. Gary

2008-01-01

To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-RasG12D murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to γ-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways. PMID:18663146
Activated Alk triggers prolonged neurogenesis and Ret upregulation providing a therapeutic target in ALK-mutated neuroblastoma

PubMed Central

Cazes, Alex; Lopez-Delisle, Lucille; Tsarovina, Konstantina; Pierre-Eugène, Cécile; De Preter, Katleen; Peuchmaur, Michel; Nicolas, André; Provost, Claire; Louis-Brennetot, Caroline; Daveau, Romain; Kumps, Candy; Cascone, Ilaria; Schleiermacher, Gudrun; Prignon, Aurélie; Speleman, Frank; Rohrer, Hermann; Delattre, Olivier; Janoueix-Lerosey, Isabelle

2014-01-01

Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in AlkF1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI AlkR1279Q and MYCN/KI AlkF1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma. PMID:24811913
Endothelial Fcγ Receptor IIB Activation Blunts Insulin Delivery to Skeletal Muscle to Cause Insulin Resistance in Mice

PubMed Central

Tanigaki, Keiji; Chambliss, Ken L.; Yuhanna, Ivan S.; Sacharidou, Anastasia; Ahmed, Mohamed; Atochin, Dmitriy N.; Huang, Paul L.

2016-01-01

Modest elevations in C-reactive protein (CRP) are associated with type 2 diabetes. We previously revealed in mice that increased CRP causes insulin resistance and mice globally deficient in the CRP receptor Fcγ receptor IIB (FcγRIIB) were protected from the disorder. FcγRIIB is expressed in numerous cell types including endothelium and B lymphocytes. Here we investigated how endothelial FcγRIIB influences glucose homeostasis, using mice with elevated CRP expressing or lacking endothelial FcγRIIB. Whereas increased CRP caused insulin resistance in mice expressing endothelial FcγRIIB, mice deficient in the endothelial receptor were protected. The insulin resistance with endothelial FcγRIIB activation was due to impaired skeletal muscle glucose uptake caused by attenuated insulin delivery, and it was associated with blunted endothelial nitric oxide synthase (eNOS) activation in skeletal muscle. In culture, CRP suppressed endothelial cell insulin transcytosis via FcγRIIB activation and eNOS antagonism. Furthermore, in knock-in mice harboring constitutively active eNOS, elevated CRP did not invoke insulin resistance. Collectively these findings reveal that by inhibiting eNOS, endothelial FcγRIIB activation by CRP blunts insulin delivery to skeletal muscle to cause insulin resistance. Thus, a series of mechanisms in endothelium that impairs insulin movement has been identified that may contribute to type 2 diabetes pathogenesis. PMID:27207525
Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.

PubMed

Kimura, Yukiko; Hisano, Yu; Kawahara, Atsuo; Higashijima, Shin-ichi

2014-10-08

The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish.
Amyloid-β₂₅₋₃₅ induces impairment of cognitive function and long-term potentiation through phosphorylation of collapsin response mediator protein 2.

PubMed

Isono, Toshinari; Yamashita, Naoya; Obara, Masami; Araki, Tomomi; Nakamura, Fumio; Kamiya, Yoshinori; Alkam, Tursun; Nitta, Atsumi; Nabeshima, Toshitaka; Mikoshiba, Katsuhiko; Ohshima, Toshio; Goshima, Yoshio

2013-11-01

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) protein and tau deposition in the brain. Numerous studies have reported a central role of Aβ in the development of AD, but the pathogenesis is not well understood. Collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. To gain insight into the role of CRMP2 phosphorylation in AD pathogenesis, we investigated the effects of Aβ neurotoxicity in CRMP2 phosphorylation-deficient knock-in (crmp2(ki/ki)) mice, in which the serine residue at 522 was replaced with alanine. Intracerebroventricular (i.c.v.) injection of Aβ₂₅₋₃₅ peptide, a neurotoxic fragment of Aβ protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of Aβ₂₅₋₃₅ peptide caused impairment of novel object recognition in wt mice, while the same peptide did not in crmp2(ki/ki) mice. In electrophysiological recording, wt and crmp2(ki/ki) mice have similar input-output basal synaptic transmission and paired-pulse ratios. However, long-term potentiation was impaired in hippocampal slices of Aβ₂₅₋₃₅ peptide-treated wt but not those of crmp2(ki/ki). Our findings indicate that CRMP2 phosphorylation is required for Aβ-induced impairment of cognitive memory and synaptic plasticity. Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Alternative splicing in the C-terminal tail of Cav2.1 is essential for preventing a neurological disease in mice.

PubMed

Aikawa, Tomonori; Watanabe, Takaki; Miyazaki, Taisuke; Mikuni, Takayasu; Wakamori, Minoru; Sakurai, Miyano; Aizawa, Hidenori; Ishizu, Nobutaka; Watanabe, Masahiko; Kano, Masanobu; Mizusawa, Hidehiro; Watase, Kei

2017-08-15

Alternative splicing (AS) that occurs at the final coding exon (exon 47) of the Cav2.1 voltage-gated calcium channel (VGCC) gene produces two major isoforms in the brain, MPI and MPc. These isoforms differ in their splice acceptor sites; human MPI is translated into a polyglutamine tract associated with spinocerebellar ataxia type 6 (SCA6), whereas MPc splices to an immediate stop codon, resulting in a shorter cytoplasmic tail. To gain insight into the functional role of the AS in vivo and whether modulating the splice patterns at this locus can be a potential therapeutic strategy for SCA6, here we created knockin mice that exclusively express MPc by inserting the splice-site mutation. The resultant Cacna1aCtmKO/CtmKO mice developed non-progressive neurological phenotypes, featuring early-onset ataxia and absence seizure without significant alterations in the basic properties of the channel. Interactions of Cav2.1 with Cavβ4 and Rimbp2 were significantly reduced while those with GABAB2 were enhanced in the cerebellum of Cacna1aCtmKO/CtmKO mice. Treatment with the GABAB antagonist CGP35348 partially rescued the motor impairments seen in Cacna1aCtmKO/CtmKO mice. These results suggest that the carboxyl-terminal domain of Cav2.1 is not essential for maintaining the basic properties of the channel in the cerebellar Purkinje neurons but is involved in multiple interactions of Cav2.1 with other proteins, and plays an essential role in preventing a complex neurological disease. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
The Psen1-L166P-knock-in mutation leads to amyloid deposition in human wild-type amyloid precursor protein YAC transgenic mice

PubMed Central

Vidal, Ruben; Sammeta, Neeraja; Garringer, Holly J.; Sambamurti, Kumar; Miravalle, Leticia; Lamb, Bruce T.; Ghetti, Bernardino

2012-01-01

Genetically engineered mice have been generated to model cerebral β-amyloidosis, one of the hallmarks of Alzheimer disease (AD) pathology, based on the overexpression of a mutated cDNA of the amyloid-β precursor protein (AβPP) or by knock-in of the murine Aβpp gene alone or with presenilin1 mutations. Here we describe the generation and initial characterization of a new mouse line based on the presence of 2 copies of the human genomic region encoding the wild-type AβPP and the L166P presenilin 1 mutation. At ∼6 mo of age, double-mutant mice develop amyloid pathology, with signs of neuritic dystrophy, intracellular Aβ accumulation, and glial inflammation, an increase in AβPP C-terminal fragments, and an 8 times increase in Aβ42 levels with a 40% decrease in Aβ40 levels, leading to a significant increase (14 times) of Aβ42/Aβ40 ratios, with minimal effects on presenilin or the Notch1 pathway in the brain. We conclude that in mice, neither mutations in AβPP nor overexpression of an AβPP isoform are a prerequisite for Aβ pathology. This model will allow the study of AD pathogenesis and testing of therapeutic strategies in a more relevant environment without experimental artifacts due to the overexpression of a single-mutant AβPP isoform using exogenous promoters.—Vidal, R., Sammeta, N., Garringer, H. J., Sambamurti, K., Miravalle, L., Lamb B. T., Ghetti, B. The Psen1-L166P-knock-in mutation leads to amyloid deposition in human wild-type amyloid precursor protein YAC transgenic mice. PMID:22459153
A Longitudinal Motor Characterisation of the HdhQ111 Mouse Model of Huntington's Disease.

PubMed

Yhnell, Emma; Dunnett, Stephen B; Brooks, Simon P

2016-05-31

Huntington's disease (HD) is a rare, incurable neurodegenerative disorder caused by a CAG trinucleotide expansion with the first exon of the huntingtin gene. Numerous knock-in mouse models are currently available for modelling HD. However, before their use in scientific research, these models must be characterised to determine their face and predictive validity as models of the disease and their reliability in recapitulating HD symptoms. Manifest HD is currently diagnosed upon the onset of motor symptoms, thus we sought to longitudinally characterise the progression and severity of motor signs in the HdhQ111 knock-in mouse model of HD, in heterozygous mice. An extensive battery of motor tests including: rotarod, inverted lid test, balance beam, spontaneous locomotor activity and gait analysis were applied longitudinally to a cohort of HdhQ111 heterozygous mice in order to progressively assess motor function. A progressive failure to gain body weight was demonstrated from 11 months of age and motor problems in all measures of balance beam performance were shown in HdhQ111 heterozygous animals in comparison to wild type control animals from 9 months of age. A decreased latency to fall from the rotarod was demonstrated in HdhQ111 heterozygous animals in comparison to wild type animals, although this was not progressive with time. No genotype specific differences were demonstrated in any of the other motor tests included in the test battery. The HdhQ111 heterozygous mouse demonstrates a subtle and progressive motor phenotype that begins at 9 months of age. This mouse model represents an early disease stage and would be ideal for testing therapeutic strategies that require elongated lead-in times, such as viral gene therapies or striatal transplantation.
High resolution time-course mapping of early transcriptomic, molecular and cellular phenotypes in Huntington's disease CAG knock-in mice across multiple genetic backgrounds.

PubMed

Ament, Seth A; Pearl, Jocelynn R; Grindeland, Andrea; St Claire, Jason; Earls, John C; Kovalenko, Marina; Gillis, Tammy; Mysore, Jayalakshmi; Gusella, James F; Lee, Jong-Min; Kwak, Seung; Howland, David; Lee, Min Young; Baxter, David; Scherler, Kelsey; Wang, Kai; Geman, Donald; Carroll, Jeffrey B; MacDonald, Marcy E; Carlson, George; Wheeler, Vanessa C; Price, Nathan D; Hood, Leroy E

2017-03-01

Huntington's disease is a dominantly inherited neurodegenerative disease caused by the expansion of a CAG repeat in the HTT gene. In addition to the length of the CAG expansion, factors such as genetic background have been shown to contribute to the age at onset of neurological symptoms. A central challenge in understanding the disease progression that leads from the HD mutation to massive cell death in the striatum is the ability to characterize the subtle and early functional consequences of the CAG expansion longitudinally. We used dense time course sampling between 4 and 20 postnatal weeks to characterize early transcriptomic, molecular and cellular phenotypes in the striatum of six distinct knock-in mouse models of the HD mutation. We studied the effects of the HttQ111 allele on the C57BL/6J, CD-1, FVB/NCr1, and 129S2/SvPasCrl genetic backgrounds, and of two additional alleles, HttQ92 and HttQ50, on the C57BL/6J background. We describe the emergence of a transcriptomic signature in HttQ111/+ mice involving hundreds of differentially expressed genes and changes in diverse molecular pathways. We also show that this time course spanned the onset of mutant huntingtin nuclear localization phenotypes and somatic CAG-length instability in the striatum. Genetic background strongly influenced the magnitude and age at onset of these effects. This work provides a foundation for understanding the earliest transcriptional and molecular changes contributing to HD pathogenesis. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Microenvironment-derived HGF overcomes genetically determined sensitivity to anti-MET drugs.

PubMed

Pennacchietti, Selma; Cazzanti, Manuela; Bertotti, Andrea; Rideout, William M; Han, May; Gyuris, Jeno; Perera, Timothy; Comoglio, Paolo M; Trusolino, Livio; Michieli, Paolo

2014-11-15

Cell-based drug screenings indicate that tumors displaying c-MET gene amplification are "addicted" to MET signaling and therefore are very sensitive to MET-targeted agents. However, these screenings were conducted in the absence of the MET ligand, hepatocyte growth factor (HGF), which is abundant in the tumor microenvironment. Sensitivity of six MET-addicted human tumor cells to three MET kinase inhibitors (JNJ-38877605, PHA-665752, crizotinib) and one antagonistic anti-MET antibody (DN30 Fab) was analyzed in the absence or presence of HGF, in a stroma-tumor coculture system, and by combining anti-MET drugs with an HGF neutralizing antibody (ficlatuzumab) in human HGF knock-in mice bearing c-MET-amplified tumors. In all models examined, HGF promoted resistance to MET-targeted agents, affecting both their potency and efficacy. HGF-induced resistance was due to restoration of physiologic GAB1-mediated PI3K activation that compensated for loss of aberrant HER3-dependent PI3K signaling. Ficlatuzumab restored sensitivity to MET-targeted agents in coculture systems and overcame resistance to JNJ-38877605, crizotinib, and DN30 Fab in human HGF knock-in mice. These data suggest that c-MET-amplified tumor cells-which normally exhibit ligand-independent, constitutive MET activation-become dependent on HGF for survival upon pharmacologic MET inhibition. Because HGF is frequently overexpressed in human cancer, this mechanism may represent a major cause of resistance to anti-MET therapies. The ability of ficlatuzumab to overcome HGF-mediated resistance generates proof of principle that vertical inhibition of both a tyrosine kinase receptor and its ligand can be therapeutically beneficial and opens new perspectives for the treatment of MET-dependent tumors. ©2014 American Association for Cancer Research.
Boosting CNS axon regeneration by harnessing antagonistic effects of GSK3 activity.

PubMed

Leibinger, Marco; Andreadaki, Anastasia; Golla, Renate; Levin, Evgeny; Hilla, Alexander M; Diekmann, Heike; Fischer, Dietmar

2017-07-03

Implications of GSK3 activity for axon regeneration are often inconsistent, if not controversial. Sustained GSK3 activity in GSK3 S/A knock-in mice reportedly accelerates peripheral nerve regeneration via increased MAP1B phosphorylation and concomitantly reduces microtubule detyrosination. In contrast, the current study shows that lens injury-stimulated optic nerve regeneration was significantly compromised in these knock-in mice. Phosphorylation of MAP1B and CRMP2 was expectedly increased in retinal ganglion cell (RGC) axons upon enhanced GSK3 activity, but, surprisingly, no GSK3-mediated CRMP2 inhibition was detected in sciatic nerves, thus revealing a fundamental difference between central and peripheral axons. Conversely, genetic or shRNA-mediated conditional KO/knockdown of GSK3β reduced inhibitory phosphorylation of CRMP2 in RGCs and improved optic nerve regeneration. Accordingly, GSK3β KO-mediated neurite growth promotion and myelin disinhibition were abrogated by CRMP2 inhibition and largely mimicked in WT neurons upon expression of constitutively active CRMP2 (CRMP2 T/A ). These results underscore the prevalent requirement of active CRMP2 for optic nerve regeneration. Strikingly, expression of CRMP2 T/A in GSK3 S/A RGCs further boosted optic nerve regeneration, with axons reaching the optic chiasm within 3 wk. Thus, active GSK3 can also markedly promote axonal growth in central nerves if CRMP2 concurrently remains active. Similar to peripheral nerves, GSK3-mediated MAP1B phosphorylation/activation and the reduction of microtubule detyrosination contributed to this effect. Overall, these findings reconcile conflicting data on GSK3-mediated axon regeneration. In addition, the concept of complementary modulation of normally antagonistically targeted GSK3 substrates offers a therapeutically applicable approach to potentiate the regenerative outcome in the injured CNS.
Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor signaling.

PubMed

Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

2016-09-01

Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of AMPA receptor signaling

PubMed Central

Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

2016-01-01

Objectives Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. Methods In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Results Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. Conclusions These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. PMID:27687706
The granin VGF promotes genesis of secretory vesicles, and regulates circulating catecholamine levels and blood pressure

PubMed Central

Fargali, Samira; Garcia, Angelo L.; Sadahiro, Masato; Jiang, Cheng; Janssen, William G.; Lin, Wei-Jye; Cogliani, Valeria; Elste, Alice; Mortillo, Steven; Cero, Cheryl; Veitenheimer, Britta; Graiani, Gallia; Pasinetti, Giulio M.; Mahata, Sushil K.; Osborn, John W.; Huntley, George W.; Phillips, Greg R.; Benson, Deanna L.; Bartolomucci, Alessandro; Salton, Stephen R.

2014-01-01

Secretion of proteins and neurotransmitters from large dense core vesicles (LDCVs) is a highly regulated process. Adrenal LDCV formation involves the granin proteins chromogranin A (CgA) and chromogranin B (CgB); CgA- and CgB-derived peptides regulate catecholamine levels and blood pressure. We investigated function of the granin VGF (nonacronymic) in LDCV formation and the regulation of catecholamine levels and blood pressure. Expression of exogenous VGF in nonendocrine NIH 3T3 fibroblasts resulted in the formation of LDCV-like structures and depolarization-induced VGF secretion. Analysis of germline VGF-knockout mouse adrenal medulla revealed decreased LDCV size in noradrenergic chromaffin cells, increased adrenal norepinephrine and epinephrine content and circulating plasma epinephrine, and decreased adrenal CgB. These neurochemical changes in VGF-knockout mice were associated with hypertension. Germline knock-in of human VGF1–615 into the mouse Vgf locus rescued the hypertensive knockout phenotype, while knock-in of a truncated human VGF1–524 that lacks several C-terminal peptides, including TLQP-21, resulted in a small but significant increase in systolic blood pressure compared to hVGF1–615 mice. Finally, acute and chronic administration of the VGF-derived peptide TLQP-21 to rodents decreased blood pressure. Our studies establish a role for VGF in adrenal LDCV formation and the regulation of catecholamine levels and blood pressure.—Fargali, S., Garcia, A. L., Sadahiro, M., Jiang, C., Janssen, W. G., Lin, W.-J., Cogliani, V., Elste, A., Mortillo, S., Cero, C., Veitenheimer, B., Graiani, G., Pasinetti, G. M., Mahata, S. K., Osborn, J. W., Huntley, G. W., Phillips, G. R., Benson, D. L., Bartolomucci, A., Salton, S. R. The granin VGF promotes genesis of secretory vesicles, and regulates circulating catecholamine levels and blood pressure. PMID:24497580
High-resolution respirometry of fine-needle muscle biopsies in pre-manifest Huntington's disease expansion mutation carriers shows normal mitochondrial respiratory function.

PubMed

Buck, Eva; Zügel, Martina; Schumann, Uwe; Merz, Tamara; Gumpp, Anja M; Witting, Anke; Steinacker, Jürgen M; Landwehrmeyer, G Bernhard; Weydt, Patrick; Calzia, Enrico; Lindenberg, Katrin S

2017-01-01

Alterations in mitochondrial respiration are an important hallmark of Huntington's disease (HD), one of the most common monogenetic causes of neurodegeneration. The ubiquitous expression of the disease causing mutant huntingtin gene raises the prospect that mitochondrial respiratory deficits can be detected in skeletal muscle. While this tissue is readily accessible in humans, transgenic animal models offer the opportunity to cross-validate findings and allow for comparisons across organs, including the brain. The integrated respiratory chain function of the human vastus lateralis muscle was measured by high-resolution respirometry (HRR) in freshly taken fine-needle biopsies from seven pre-manifest HD expansion mutation carriers and nine controls. The respiratory parameters were unaffected. For comparison skeletal muscle isolated from HD knock-in mice (HdhQ111) as well as a broader spectrum of tissues including cortex, liver and heart muscle were examined by HRR. Significant changes of mitochondrial respiration in the HdhQ knock-in mouse model were restricted to the liver and the cortex. Mitochondrial mass as quantified by mitochondrial DNA copy number and citrate synthase activity was stable in murine HD-model tissue compared to control. mRNA levels of key enzymes were determined to characterize mitochondrial metabolic pathways in HdhQ mice. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that alterations in respiratory parameters of pre-manifest human muscle biopsies are rather limited and mirrored by a similar absence of marked alterations in HdhQ skeletal muscle. In contrast, the HdhQ111 murine cortex and liver did show respiratory alterations highlighting the tissue specific nature of mutant huntingtin effects on respiration.
Cholinergic Neurons Excite Cortically Projecting Basal Forebrain GABAergic Neurons

PubMed Central

Yang, Chun; McKenna, James T.; Zant, Janneke C.; Winston, Stuart; Basheer, Radhika

2014-01-01

The basal forebrain (BF) plays an important role in the control of cortical activation and attention. Understanding the modulation of BF neuronal activity is a prerequisite to treat disorders of cortical activation involving BF dysfunction, such as Alzheimer's disease. Here we reveal the interaction between cholinergic neurons and cortically projecting BF GABAergic neurons using immunohistochemistry and whole-cell recordings in vitro. In GAD67-GFP knock-in mice, BF cholinergic (choline acetyltransferase-positive) neurons were intermingled with GABAergic (GFP+) neurons. Immunohistochemistry for the vesicular acetylcholine transporter showed that cholinergic fibers apposed putative cortically projecting GABAergic neurons containing parvalbumin (PV). In coronal BF slices from GAD67-GFP knock-in or PV-tdTomato mice, pharmacological activation of cholinergic receptors with bath application of carbachol increased the firing rate of large (>20 μm diameter) BF GFP+ and PV (tdTomato+) neurons, which exhibited the intrinsic membrane properties of cortically projecting neurons. The excitatory effect of carbachol was blocked by antagonists of M1 and M3 muscarinic receptors in two subpopulations of BF GABAergic neurons [large hyperpolarization-activated cation current (Ih) and small Ih, respectively]. Ion substitution experiments and reversal potential measurements suggested that the carbachol-induced inward current was mediated mainly by sodium-permeable cation channels. Carbachol also increased the frequency of spontaneous excitatory and inhibitory synaptic currents. Furthermore, optogenetic stimulation of cholinergic neurons/fibers caused a mecamylamine- and atropine-sensitive inward current in putative GABAergic neurons. Thus, cortically projecting, BF GABAergic/PV neurons are excited by neighboring BF and/or brainstem cholinergic neurons. Loss of cholinergic neurons in Alzheimer's disease may impair cortical activation, in part, through disfacilitation of BF cortically projecting GABAergic/PV neurons. PMID:24553925
Nitroglycerin fails to lower blood pressure in redox-dead Cys42Ser PKG1α knock-in mouse.

PubMed

Rudyk, Olena; Prysyazhna, Oleksandra; Burgoyne, Joseph R; Eaton, Philip

2012-07-17

Although nitroglycerin has remained in clinical use since 1879, the mechanism by which it relaxes blood vessels to lower blood pressure remains incompletely understood. Nitroglycerin undergoes metabolism that generates several reaction products, including oxidants, and this bioactivation process is essential for vasodilation. Protein kinase G (PKG) mediates classic nitric oxide-dependent vasorelaxation, but the 1α isoform is also independently activated by oxidation that involves interprotein disulfide formation within this homodimeric protein complex. We hypothesized that nitroglycerin-induced vasodilation is mediated by disulfide activation of PKG1α. Treating smooth muscle cells or isolated blood vessels with nitroglycerin caused PKG1α disulfide dimerization. PKG1α disulfide formation was increased in wild-type mouse aortas by in vivo nitroglycerin treatment, but this oxidation was lost as tolerance developed. To establish whether kinase oxidation underlies nitroglycerin-induced vasodilation in vivo, we used a Cys42Ser PKG1α knock-in mouse that cannot transduce oxidant signals because it does not contain the vital redox-sensing thiol. This redox-dead knock-in mouse was substantively deficient in hypotensive response to nitroglycerin compared with wild-type littermates as measured in vivo by radiotelemetry. Resistance blood vessels from knock-ins were markedly less sensitive to nitroglycerin-induced vasodilation (EC(50)=39.2 ± 10.7 μmol/L) than wild-types (EC(50)=12.1 ± 2.9 μmol/L). Furthermore, after ≈24 hours of treatment, wild-type controls stopped vasodilating to nitroglycerin, and the vascular sensitivity to nitroglycerin was decreased, whereas this tolerance phenomenon, which routinely hampers the management of hypertensive patients, was absent in knock-ins. PKG1α disulfide formation is a significant mediator of nitroglycerin-induced vasodilation, and tolerance to nitroglycerin is associated with loss of kinase oxidation.
Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system.

PubMed

Katoh, Yohei; Michisaka, Saki; Nozaki, Shohei; Funabashi, Teruki; Hirano, Tomoaki; Takei, Ryota; Nakayama, Kazuhisa

2017-04-01

The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore the use of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved in ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and increase the versatility of our knock-in system, we constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability. © 2017 Katoh et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
CRISPR/Cas9 Allows Efficient and Complete Knock-In of a Destabilization Domain-Tagged Essential Protein in a Human Cell Line, Allowing Rapid Knockdown of Protein Function

PubMed Central

Park, Arnold; Won, Sohui T.; Pentecost, Mickey; Bartkowski, Wojciech; Lee, Benhur

2014-01-01

Although modulation of protein levels is an important tool for study of protein function, it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes, a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach, which confers instability to the tagged protein in the absence of the compound Shield-1, has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies, partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles, this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles. The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles, we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent, and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein, and that destabilization of DD-TCOF1 resulted in impaired cell growth, as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to achieve rapid modulation of protein levels in mammalian cells. PMID:24743236
CRISPR/Cas9 allows efficient and complete knock-in of a destabilization domain-tagged essential protein in a human cell line, allowing rapid knockdown of protein function.

PubMed

Park, Arnold; Won, Sohui T; Pentecost, Mickey; Bartkowski, Wojciech; Lee, Benhur

2014-01-01

Although modulation of protein levels is an important tool for study of protein function, it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes, a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach, which confers instability to the tagged protein in the absence of the compound Shield-1, has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies, partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles, this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles. The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles, we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent, and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein, and that destabilization of DD-TCOF1 resulted in impaired cell growth, as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to achieve rapid modulation of protein levels in mammalian cells.
Pathogenesis of depression- and anxiety-like behavior in an animal model of hypertrophic cardiomyopathy

PubMed Central

Dossat, Amanda M.; Sanchez-Gonzalez, Marcos A.; Koutnik, Andrew P.; Leitner, Stefano; Ruiz, Edda L.; Griffin, Brittany; Rosenberg, Jens T.; Grant, Samuel C.; Fincham, Francis D.; Pinto, Jose R.; Kabbaj, Mohamed

2017-01-01

Cardiovascular dysfunction is highly comorbid with mood disorders, such as anxiety and depression. However, the mechanisms linking cardiovascular dysfunction with the core behavioral features of mood disorder remain poorly understood. In this study, we used mice bearing a knock-in sarcomeric mutation, which is exhibited in human hypertrophic cardiomyopathy (HCM), to investigate the influence of HCM over the development of anxiety and depression. We employed behavioral, MRI, and biochemical techniques in young (3–4 mo) and aged adult (7–8 mo) female mice to examine the effects of HCM on the development of anxiety- and depression-like behaviors. We focused on females because in both humans and rodents, they experience a 2-fold increase in mood disorder prevalence vs. males. Our results showed that young and aged HCM mice displayed echocardiographic characteristics of the heart disease condition, yet only aged HCM females displayed anxiety- and depression-like behaviors. Electrocardiographic parameters of sympathetic nervous system activation were increased in aged HCM females vs. controls and correlated with mood disorder–related symptoms. In addition, when compared with controls, aged HCM females exhibited adrenal gland hypertrophy, reduced volume in mood-related brain regions, and reduced hippocampal signaling proteins, such as brain-derived neurotrophic factor and its downstream targets vs. controls. In conclusion, prolonged systemic HCM stress can lead to development of mood disorders, possibly through inducing structural and functional brain changes, and thus, mood disorders in patients with heart disease should not be considered solely a psychologic or situational condition.—Dossat, A. M., Sanchez-Gonzalez, M. A., Koutnik, A. P., Leitner, S., Ruiz, E. L., Griffin, B., Rosenberg, J. T., Grant, S. C., Fincham, F. D., Pinto, J. R. Kabbaj, M. Pathogenesis of depression- and anxiety-like behavior in an animal model of hypertrophic cardiomyopathy. PMID:28235781
Pathogenesis of depression- and anxiety-like behavior in an animal model of hypertrophic cardiomyopathy.

PubMed

Dossat, Amanda M; Sanchez-Gonzalez, Marcos A; Koutnik, Andrew P; Leitner, Stefano; Ruiz, Edda L; Griffin, Brittany; Rosenberg, Jens T; Grant, Samuel C; Fincham, Francis D; Pinto, Jose R; Kabbaj, Mohamed

2017-06-01

Cardiovascular dysfunction is highly comorbid with mood disorders, such as anxiety and depression. However, the mechanisms linking cardiovascular dysfunction with the core behavioral features of mood disorder remain poorly understood. In this study, we used mice bearing a knock-in sarcomeric mutation, which is exhibited in human hypertrophic cardiomyopathy (HCM), to investigate the influence of HCM over the development of anxiety and depression. We employed behavioral, MRI, and biochemical techniques in young (3-4 mo) and aged adult (7-8 mo) female mice to examine the effects of HCM on the development of anxiety- and depression-like behaviors. We focused on females because in both humans and rodents, they experience a 2-fold increase in mood disorder prevalence vs. males. Our results showed that young and aged HCM mice displayed echocardiographic characteristics of the heart disease condition, yet only aged HCM females displayed anxiety- and depression-like behaviors. Electrocardiographic parameters of sympathetic nervous system activation were increased in aged HCM females vs. controls and correlated with mood disorder-related symptoms. In addition, when compared with controls, aged HCM females exhibited adrenal gland hypertrophy, reduced volume in mood-related brain regions, and reduced hippocampal signaling proteins, such as brain-derived neurotrophic factor and its downstream targets vs. controls. In conclusion, prolonged systemic HCM stress can lead to development of mood disorders, possibly through inducing structural and functional brain changes, and thus, mood disorders in patients with heart disease should not be considered solely a psychologic or situational condition.-Dossat, A. M., Sanchez-Gonzalez, M. A., Koutnik, A. P., Leitner, S., Ruiz, E. L., Griffin, B., Rosenberg, J. T., Grant, S. C., Fincham, F. D., Pinto, J. R. Kabbaj, M. Pathogenesis of depression- and anxiety-like behavior in an animal model of hypertrophic cardiomyopathy. © FASEB.
K-Ras(V14I) -induced Noonan syndrome predisposes to tumour development in mice.

PubMed

Hernández-Porras, Isabel; Schuhmacher, Alberto J; Garcia-Medina, Raquel; Jiménez, Beatriz; Cañamero, Marta; de Martino, Alba; Guerra, Carmen

2016-06-01

The Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. A significant proportion of NS patients may also develop myeloproliferative disorders (MPDs), including juvenile myelomonocytic leukaemia (JMML). Surprisingly, scarce information is available in relation to other tumour types in these patients. We have previously developed and characterized a knock-in mouse model that carries one of the most frequent KRAS-NS-related mutations, the K-Ras(V14I) substitution, which recapitulates most of the alterations described in NS patients, including MPDs. The K-Ras(V14I) mutation is a mild activating K-Ras protein; thus, we have used this model to study tumour susceptibility in comparison with mice expressing the classical K-Ras(G12V) oncogene. Interestingly, our studies have shown that these mice display a generalized tumour predisposition and not just MPDs. In fact, we have observed that the K-Ras(V14I) mutation is capable of cooperating with the p16Ink4a/p19Arf and Trp53 tumour suppressors, as well as with other risk factors such as pancreatitis, thereby leading to a higher cancer incidence. In conclusion, our results illustrate that the K-Ras(V14I) activating protein is able to induce cancer, although at a much lower level than the classical K-Ras(G12V) oncogene, and that it can be significantly modulated by both genetic and non-genetic events. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
APP heterozygosity averts memory deficit in knockin mice expressing the Danish dementia BRI2 mutant.

PubMed

Tamayev, Robert; Matsuda, Shuji; Giliberto, Luca; Arancio, Ottavio; D'Adamio, Luciano

2011-05-17

An autosomal dominant mutation in the BRI2/ITM2B gene causes familial Danish dementia (FDD). Analysis of FDD(KI) mice, a mouse model of FDD genetically congruous to the human disease since they carry one mutant and one wild-type Bri2/Itm2b allele, has shown that the Danish mutation causes loss of Bri2 protein, synaptic plasticity and memory impairments. BRI2 is a physiological interactor of Aβ-precursor protein (APP), a gene associated with Alzheimer disease, which inhibits processing of APP. Here, we show that APP/Bri2 complexes are reduced in synaptic membranes of FDD(KI) mice. Consequently, APP metabolites derived from processing of APP by β-, α- and γ-secretases are increased in Danish dementia mice. APP haplodeficiency prevents memory and synaptic dysfunctions, consistent with a role for APP metabolites in the pathogenesis of memory and synaptic deficits. This genetic suppression provides compelling evidence that APP and BRI2 functionally interact, and that the neurological effects of the Danish form of BRI2 only occur when sufficient levels of APP are supplied by two alleles. This evidence establishes a pathogenic sameness between familial Danish and Alzheimer's dementias.
APP heterozygosity averts memory deficit in knockin mice expressing the Danish dementia BRI2 mutant

PubMed Central

Tamayev, Robert; Matsuda, Shuji; Giliberto, Luca; Arancio, Ottavio; D'Adamio, Luciano

2011-01-01

An autosomal dominant mutation in the BRI2/ITM2B gene causes familial Danish dementia (FDD). Analysis of FDDKI mice, a mouse model of FDD genetically congruous to the human disease since they carry one mutant and one wild-type Bri2/Itm2b allele, has shown that the Danish mutation causes loss of Bri2 protein, synaptic plasticity and memory impairments. BRI2 is a physiological interactor of Aβ-precursor protein (APP), a gene associated with Alzheimer disease, which inhibits processing of APP. Here, we show that APP/Bri2 complexes are reduced in synaptic membranes of FDDKI mice. Consequently, APP metabolites derived from processing of APP by β-, α- and γ-secretases are increased in Danish dementia mice. APP haplodeficiency prevents memory and synaptic dysfunctions, consistent with a role for APP metabolites in the pathogenesis of memory and synaptic deficits. This genetic suppression provides compelling evidence that APP and BRI2 functionally interact, and that the neurological effects of the Danish form of BRI2 only occur when sufficient levels of APP are supplied by two alleles. This evidence establishes a pathogenic sameness between familial Danish and Alzheimer's dementias. PMID:21587206

Early Detection of Apathetic Phenotypes in Huntington's Disease Knock-in Mice Using Open Source Tools.

PubMed

Minnig, Shawn; Bragg, Robert M; Tiwana, Hardeep S; Solem, Wes T; Hovander, William S; Vik, Eva-Mari S; Hamilton, Madeline; Legg, Samuel R W; Shuttleworth, Dominic D; Coffey, Sydney R; Cantle, Jeffrey P; Carroll, Jeffrey B

2018-02-02

Apathy is one of the most prevalent and progressive psychiatric symptoms in Huntington's disease (HD) patients. However, preclinical work in HD mouse models tends to focus on molecular and motor, rather than affective, phenotypes. Measuring behavior in mice often produces noisy data and requires large cohorts to detect phenotypic rescue with appropriate power. The operant equipment necessary for measuring affective phenotypes is typically expensive, proprietary to commercial entities, and bulky which can render adequately sized mouse cohorts as cost-prohibitive. Thus, we describe here a home-built, open-source alternative to commercial hardware that is reliable, scalable, and reproducible. Using off-the-shelf hardware, we adapted and built several of the rodent operant buckets (ROBucket) to test Htt Q111/+ mice for attention deficits in fixed ratio (FR) and progressive ratio (PR) tasks. We find that, despite normal performance in reward attainment in the FR task, Htt Q111/+ mice exhibit reduced PR performance at 9-11 months of age, suggesting motivational deficits. We replicated this in two independent cohorts, demonstrating the reliability and utility of both the apathetic phenotype, and these ROBuckets, for preclinical HD studies.
Activated Braf induces esophageal dilation and gastric epithelial hyperplasia in mice.

PubMed

Inoue, Shin-Ichi; Takahara, Shingo; Yoshikawa, Takeo; Niihori, Tetsuya; Yanai, Kazuhiko; Matsubara, Yoichi; Aoki, Yoko

2017-12-01

Germline mutations in BRAF are a major cause of cardio-facio-cutaneous (CFC) syndrome, which is characterized by heart defects, characteristic craniofacial dysmorphology and dermatologic abnormalities. Patients with CFC syndrome also commonly show gastrointestinal dysfunction, including feeding and swallowing difficulties and gastroesophageal reflux. We have previously found that knock-in mice expressing a Braf Q241R mutation exhibit CFC syndrome-related phenotypes, such as growth retardation, craniofacial dysmorphisms, congenital heart defects and learning deficits. However, it remains unclear whether BrafQ241R/+ mice exhibit gastrointestinal dysfunction. Here, we report that BrafQ241R/+ mice have neonatal feeding difficulties and esophageal dilation. The esophagus tissues from BrafQ241R/+ mice displayed incomplete replacement of smooth muscle with skeletal muscle and decreased contraction. Furthermore, the BrafQ241R/+ mice showed hyperkeratosis and a thickened muscle layer in the forestomach. Treatment with MEK inhibitors ameliorated the growth retardation, esophageal dilation, hyperkeratosis and thickened muscle layer in the forestomach in BrafQ241R/+ mice. The esophageal dilation with aberrant skeletal-smooth muscle boundary in BrafQ241R/+ mice were recovered after treatment with the histone H3K27 demethylase inhibitor GSK-J4. Our results provide clues to elucidate the pathogenesis and possible treatment of gastrointestinal dysfunction and failure to thrive in patients with CFC syndrome. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Cortical Spreading Depression Causes Unique Dysregulation of Inflammatory Pathways in a Transgenic Mouse Model of Migraine.

PubMed

Eising, Else; Shyti, Reinald; 't Hoen, Peter A C; Vijfhuizen, Lisanne S; Huisman, Sjoerd M H; Broos, Ludo A M; Mahfouz, Ahmed; Reinders, Marcel J T; Ferrari, Michel D; Tolner, Else A; de Vries, Boukje; van den Maagdenberg, Arn M J M

2017-05-01

Familial hemiplegic migraine type 1 (FHM1) is a rare monogenic subtype of migraine with aura caused by mutations in CACNA1A that encodes the α 1A subunit of voltage-gated Ca V 2.1 calcium channels. Transgenic knock-in mice that carry the human FHM1 R192Q missense mutation ('FHM1 R192Q mice') exhibit an increased susceptibility to cortical spreading depression (CSD), the mechanism underlying migraine aura. Here, we analysed gene expression profiles from isolated cortical tissue of FHM1 R192Q mice 24 h after experimentally induced CSD in order to identify molecular pathways affected by CSD. Gene expression profiles were generated using deep serial analysis of gene expression sequencing. Our data reveal a signature of inflammatory signalling upon CSD in the cortex of both mutant and wild-type mice. However, only in the brains of FHM1 R192Q mice specific genes are up-regulated in response to CSD that are implicated in interferon-related inflammatory signalling. Our findings show that CSD modulates inflammatory processes in both wild-type and mutant brains, but that an additional unique inflammatory signature becomes expressed after CSD in a relevant mouse model of migraine.
Allele compensation in tip60+/- mice rescues white adipose tissue function in vivo.

PubMed

Gao, Yuan; Hamers, Nicole; Rakhshandehroo, Maryam; Berger, Ruud; Lough, John; Kalkhoven, Eric

2014-01-01

Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis), a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/- mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/- mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/- displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/- mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice.
H63D mutation in hemochromatosis alters cholesterol metabolism and induces memory impairment.

PubMed

Ali-Rahmani, Fatima; Grigson, Patricia S; Lee, Sang; Neely, Elizabeth; Connor, James R; Schengrund, Cara-Lynne

2014-06-01

The H63D variant of the hemochromatosis (HFE) gene, when expressed in carriers of the apolipoprotein E4 allele, is implicated as a risk factor for earlier onset of Alzheimer's disease (AD). We tested the hypothesis that like expression of apolipoprotein E4, expression of H63D-HFE disrupts cholesterol metabolism contributing to an increase in neurodegeneration and memory deficits. Analysis of SH-SY5Y human neuroblastoma cells transfected to stably express either wild type- (WT) or H63D-HFE indicated about a 50% reduction in cholesterol content in cells expressing H63D-HFE. This was accompanied by a significant decrease in expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase, and a significant increase in expression of cholesterol 24-hydroxylase. Consistent with these studies, H67D-HFE (orthologous to human H63D-HFE) knock-in mice, showed a greater age dependent decline in brain cholesterol than WT-HFE animals and changes in expression of proteins regulating cholesterol metabolism. Brains of aged H67D-HFE mice also exhibited a significant decrease in expression of synapse proteins and a significant increase in caspase-3 expression relative to WT-HFE controls. H67D-HFE mice also had a greater reduction in brain volume and poorer recognition and spatial memory than WT-HFE mice, symptoms associated with AD. These results indicate that the alterations in cholesterol metabolism associated with expression of H63D-HFE may contribute to the development of AD. Copyright © 2014 Elsevier Inc. All rights reserved.
The novel selective PPARα modulator (SPPARMα) pemafibrate improves dyslipidemia, enhances reverse cholesterol transport and decreases inflammation and atherosclerosis.

PubMed

Hennuyer, Nathalie; Duplan, Isabelle; Paquet, Charlotte; Vanhoutte, Jonathan; Woitrain, Eloise; Touche, Véronique; Colin, Sophie; Vallez, Emmanuelle; Lestavel, Sophie; Lefebvre, Philippe; Staels, Bart

2016-06-01

Atherosclerosis is characterized by lipid accumulation and chronic inflammation in the arterial wall. Elevated levels of apolipoprotein (apo) B-containing lipoproteins are a risk factor for cardiovascular disease (CVD). By contrast, plasma levels of functional high-density lipoprotein (HDL) and apoA-I are protective against CVD by enhancing reverse cholesterol transport (RCT). Activation of peroxisome proliferator-activated receptor-α (PPARα), a ligand-activated transcription factor, controls lipid metabolism, cellular cholesterol trafficking in macrophages and influences inflammation. To study whether pharmacological activation of PPARα with a novel highly potent and selective PPARα modulator, pemafibrate, improves lipid metabolism, macrophage cholesterol efflux, inflammation and consequently atherosclerosis development in vitro and in vivo using human apolipoprotein E2 Knock-In (apoE2KI) and human apoA-I transgenic (hapoA-I tg) mice. Pemafibrate treatment decreases apoB secretion in chylomicrons by polarized Caco-2/TC7 intestinal epithelium cells and reduces triglyceride levels in apoE2KI mice. Pemafibrate treatment of hapoA-I tg mice increases plasma HDL cholesterol, apoA-I and stimulates RCT to feces. In primary human macrophages, pemafibrate promotes macrophage cholesterol efflux to HDL and exerts anti-inflammatory activities. Pemafibrate also reduces markers of inflammation and macrophages in the aortic crosses as well as aortic atherosclerotic lesion burden in western diet-fed apoE2KI mice. These results demonstrate that the novel selective PPARα modulator pemafibrate exerts beneficial effects on lipid metabolism, RCT and inflammation resulting in anti-atherogenic properties. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
Novel mouse models of oculopharyngeal muscular dystrophy (OPMD) reveal early onset mitochondrial defects and suggest loss of PABPN1 may contribute to pathology.

PubMed

Vest, Katherine E; Phillips, Brittany L; Banerjee, Ayan; Apponi, Luciano H; Dammer, Eric B; Xu, Weiting; Zheng, Dinghai; Yu, Julia; Tian, Bin; Pavlath, Grace K; Corbett, Anita H

2017-09-01

Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease caused by polyalanine expansion in the poly(A) binding protein nuclear 1 (PABPN1). Several mouse models have been generated to study OPMD; however, most of these models have employed transgenic overexpression of alanine-expanded PABPN1. These models do not recapitulate the OPMD patient genotype and PABPN1 overexpression could confound molecular phenotypes. We have developed a knock-in mouse model of OPMD (Pabpn1+/A17) that contains one alanine-expanded Pabpn1 allele under the control of the native promoter and one wild-type Pabpn1 allele. This mouse is the closest available genocopy of OPMD patients. We show that Pabpn1+/A17 mice have a mild myopathic phenotype in adult and aged animals. We examined early molecular and biochemical phenotypes associated with expressing native levels of A17-PABPN1 and detected shorter poly(A) tails, modest changes in poly(A) signal (PAS) usage, and evidence of mitochondrial damage in these mice. Recent studies have suggested that a loss of PABPN1 function could contribute to muscle pathology in OPMD. To investigate a loss of function model of pathology, we generated a heterozygous Pabpn1 knock-out mouse model (Pabpn1+/Δ). Like the Pabpn1+/A17 mice, Pabpn1+/Δ mice have mild histologic defects, shorter poly(A) tails, and evidence of mitochondrial damage. However, the phenotypes detected in Pabpn1+/Δ mice only partially overlap with those detected in Pabpn1+/A17 mice. These results suggest that loss of PABPN1 function could contribute to but may not completely explain the pathology detected in Pabpn1+/A17 mice. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Loss of Corticostriatal and Thalamostriatal Synaptic Terminals Precedes Striatal Projection Neuron Pathology in Heterozygous Q140 Huntington’s Disease Mice

PubMed Central

Deng, Y.P.; Wong, T.; Bricker-Anthony, C.; Deng, B.; Reiner, A.

2013-01-01

Motor slowing, forebrain white matter loss, and striatal shrinkage have been reported in premanifest Huntington’s disease (HD) prior to overt striatal neuron loss. We carried out detailed LM and EM studies in a genetically precise HD mimic, heterozygous Q140 HD knock-in mice, to examine the possibility that loss of corticostriatal and thalamostriatal terminals prior to striatal neuron loss underlies these premanifest HD abnormalities. In our studies, we used VGLUT1 and VGLUT2 immunolabeling to detect corticostriatal and thalamostriatal (respectively) terminals in dorsolateral (motor) striatum over the first year of life, prior to striatal projection neuron pathology. VGLUT1+ axospinous corticostriatal terminals represented about 55% of all excitatory terminals in striatum, and VGLUT2+ axospinous thalamostriatal terminals represented about 35%, with VGLUT1+ and VGLUT2+ axodendritic terminals accounting for the remainder. In Q140 mice, a significant 40% shortfall in VGLUT2+ axodendritic thalamostriatal terminals and a 20% shortfall in axospinous thalamostriatal terminals was already observed at 1 month of age, but VGLUT1+ terminals were normal in abundance. The 20% deficiency in VGLUT2+ thalamostriatal axospinous terminals persisted at 4 and 12 months in Q140 mice, and an additional 30% loss of VGLUT1+ corticostriatal terminals was observed at 12 months. The early and persistent deficiency in thalamostriatal axospinous terminals in Q140 mice may reflect a development defect, and the impoverishment of this excitatory drive to striatum may help explain early motor defects in Q140 mice and in premanifest HD. The loss of corticostriatal terminals at 1 year in Q140 mice is consistent with prior evidence from other mouse models of corticostriatal disconnection early during progression, and can explain both the measurable bradykinesia and striatal white matter loss in late premanifest HD. PMID:23969239
Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System.

PubMed

Li, Shenglan; Xue, Haipeng; Wu, Jianbo; Rao, Mahendra S; Kim, Dong H; Deng, Wenbin; Liu, Ying

2015-12-15

Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼ 33% correctly targeted clones) compared to conventional targeting protocol (∼ 3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations.
Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System

PubMed Central

Li, Shenglan; Xue, Haipeng; Wu, Jianbo; Rao, Mahendra S.; Kim, Dong H.; Deng, Wenbin

2015-01-01

Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼33% correctly targeted clones) compared to conventional targeting protocol (∼3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations. PMID:26414932
Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish.

PubMed

Albadri, Shahad; Del Bene, Filippo; Revenu, Céline

2017-05-15

With its variety of applications, the CRISPR/Cas9 genome editing technology has been rapidly evolving in the last few years. In the zebrafish community, knock-out reports are constantly increasing but insertion studies have been so far more challenging. With this review, we aim at giving an overview of the homologous directed repair (HDR)-based knock-in generation in zebrafish. We address the critical points and limitations of the procedure such as cutting efficiency of the chosen single guide RNA, use of cas9 mRNA or Cas9 protein, homology arm size etc. but also ways to circumvent encountered issues with HDR insertions by the development of non-homologous dependent strategies. While imprecise, these homology-independent mechanisms based on non-homologous-end-joining (NHEJ) repair have been employed in zebrafish to generate reporter lines or to accurately edit an open reading frame by the use of intron-targeting modifications. Therefore, with higher efficiency and insertion rate, NHEJ-based knock-in seems to be a promising approach to target endogenous loci and to circumvent the limitations of HDR whenever it is possible and appropriate. In this perspective, we propose new strategies to generate cDNA edited or tagged insertions, which once established will constitute a new and versatile toolbox for CRISPR/Cas9-based knock-ins in zebrafish. Copyright © 2017 Elsevier Inc. All rights reserved.
Fish Oil Accelerates Diet-Induced Entrainment of the Mouse Peripheral Clock via GPR120

PubMed Central

Itokawa, Misa; Nagahama, Hiroki; Ohtsu, Teiji; Furutani, Naoki; Kamagata, Mayo; Yang, Zhi-Hong; Hirasawa, Akira; Tahara, Yu; Shibata, Shigenobu

2015-01-01

The circadian peripheral clock is entrained by restricted feeding (RF) at a fixed time of day, and　insulin secretion regulates RF-induced entrainment of the peripheral clock in mice. Thus, carbohydrate-rich food may be ideal for facilitating RF-induced entrainment, although the role of dietary oils in insulin secretion and RF-induced entrainment has not been described. The soybean oil component of standard mouse chow was substituted with fish or soybean oil containing docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA). Tuna oil (high DHA/EPA), menhaden oil (standard), and DHA/EPA dissolved in soybean oil increased insulin secretion and facilitated RF-induced phase shifts of the liver clock as represented by the bioluminescence rhythms of PER2::LUCIFERASE knock-in mice. In this model, insulin depletion blocked the effect of tuna oil and fish oil had no effect on mice deficient for GPR120, a polyunsaturated fatty acid receptor. These results suggest food containing fish oil or DHA/EPA is ideal for adjusting the peripheral clock. PMID:26161796
A novel auditory ossicles membrane and the development of conductive hearing loss in Dmp1-null mice.

PubMed

Lv, Kun; Huang, Haiyang; Yi, Xing; Chertoff, Mark E; Li, Chaoyuan; Yuan, Baozhi; Hinton, Robert J; Feng, Jian Q

2017-10-01

Genetic mouse models are widely used for understanding human diseases but we know much less about the anatomical structure of the auditory ossicles in the mouse than we do about human ossicles. Furthermore, current studies have mainly focused on disease conditions such as osteomalacia and rickets in patients with hypophosphatemia rickets, although the reason that these patients develop late-onset hearing loss is unknown. In this study, we first analyzed Dmp1 lac Z knock-in auditory ossicles (in which the blue reporter is used to trace DMP1 expression in osteocytes) using X-gal staining and discovered a novel bony membrane surrounding the mouse malleus. This finding was further confirmed by 3-D micro-CT, X-ray, and alizarin red stained images. We speculate that this unique structure amplifies and facilitates sound wave transmissions in two ways: increasing the contact surface between the eardrum and malleus and accelerating the sound transmission due to its mineral content. Next, we documented a progressive deterioration in the Dmp1-null auditory ossicle structures using multiple imaging techniques. The auditory brainstem response test demonstrated a conductive hearing loss in the adult Dmp1-null mice. This finding may help to explain in part why patients with DMP1 mutations develop late-onset hearing loss, and supports the critical role of DMP1 in maintaining the integrity of the auditory ossicles and its bony membrane. Copyright © 2017 Elsevier Inc. All rights reserved.
Deacetylase activity of histone deacetylase 3 is required for productive VDJ recombination and B-cell development

PubMed Central

Stengel, Kristy R.; Barnett, Kelly R.; Wang, Jing; Liu, Qi; Hodges, Emily; Hiebert, Scott W.; Bhaskara, Srividya

2017-01-01

Histone deacetylase 3 (HDAC3) is the catalytic component of NCoR/SMRT corepressor complexes that mediate the actions of transcription factors implicated in the regulation of B-cell development and function. We crossed Hdac3 conditional knockout mice with Mb1-Cre knockin animals to delete Hdac3 in early progenitor B cells. The spleens of Hdac3F/−Mb1-Cre+/− mice were virtually devoid of mature B cells, and B220+CD43+ B-cell progenitors accumulated within the bone marrow. Quantitative deep sequencing of the Ig heavy chain locus from B220+CD43+ populations identified a defect in VHDJH recombination with a severe reduction in productive rearrangements, which directly corresponded to the loss of pre-B cells from Hdac3Δ/− bone marrow. For Hdac3Δ/− B cells that did show productive VDJ rearrangement, there was significant skewing toward the incorporation of proximal VH gene segments and a corresponding reduction in distal VH gene segment use. Although transcriptional effects within these loci were modest, Hdac3Δ/− progenitor cells displayed global changes in chromatin structure that likely hindered effective distal V-DJ recombination. Reintroduction of wild-type Hdac3 restored normal B-cell development, whereas an Hdac3 point mutant lacking deacetylase activity failed to complement this defect. Thus, the deacetylase activity of Hdac3 is required for the generation of mature B cells. PMID:28739911
Deletion of Mecom in mouse results in early-onset spinal deformity and osteopenia.

PubMed

Juneja, Subhash C; Vonica, Alin; Zeiss, Caroline; Lezon-Geyda, Kimberly; Yatsula, Bogdan; Sell, David R; Monnier, Vincent M; Lin, Sharon; Ardito, Thomas; Eyre, David; Reynolds, David; Yao, Zhenqiang; Awad, Hani A; Yu, Hongbo; Wilson, Michael; Honnons, Sylvie; Boyce, Brendan F; Xing, Lianping; Zhang, Yi; Perkins, Archibald S

2014-03-01

Recent studies have indicated a role for a MECOM allele in susceptibility to osteoporotic fractures in humans. We have generated a mutation in Mecom in mouse (termed ME(m1)) via lacZ knock-in into the upstream transcription start site for the gene, resulting in disruption of Mds1 and Mds1-Evi1 transcripts, but not of Evi1 transcripts. We demonstrate that ME(m1/m1) mice have severe kyphoscoliosis that is reminiscent of human congenital or primary kyphoscoliosis. ME(m1/m1) mice appear normal at birth, but by 2weeks, they exhibit a slight lumbar lordosis and narrowed intervertebral space. This progresses to severe lordosis with disc collapse and synostosis, together with kyphoscoliosis. Bone formation and strength testing show that ME(m1/m1) mice have normal bone formation and composition but are osteopenic. While endochondral bone development is normal, it is markedly dysplastic in its organization. Electron micrographs of the 1week postnatal intervertebral discs reveals marked disarray of collagen fibers, consistent with an inherent weakness in the non-osseous connective tissue associated with the spine. These findings indicate that lack of ME leads to a complex defect in both osseous and non-osseous musculoskeletal tissues, including a marked vertebral osteopenia, degeneration of the IVD, and disarray of connective tissues, which is likely due to an inherent inability to establish and/or maintain components of these tissues. Copyright © 2013 Elsevier Inc. All rights reserved.
CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development

PubMed Central

Kesavan, Gokul; Chekuru, Avinash; Machate, Anja; Brand, Michael

2017-01-01

The midbrain-hindbrain boundary (MHB) acts as an organizer and controls the fate of neighboring cells to develop into either mesencephalic (midbrain) or metencephalic (hindbrain) cells by secreting signaling molecules like Wnt1 and Fgf8. The zebrafish is an excellent vertebrate model for studying MHB development due to the ease of gene manipulation and the possibility of following cellular dynamics and morphogenetic processes using live imaging. Currently, only very few reporter and/or Cre-driver lines are available to study gene expression at the MHB, hampering the understanding of MHB development, and traditional transgenic technologies using promoter/enhancer fragments or bacterial artificial chromosome (BAC)-mediated transgenesis often do not faithfully recapitulate endogenous expression patterns. In contrast, CRISPR/Cas9-mediated genome editing technology now provides a great opportunity to efficiently knock-in or knock-out genes. We have generated four CRISPR/Cas9-based knock-in fluorescent reporter lines for two crucial genes involved in MHB development, namely otx2 and pax2a. The coding sequences of the reporters were knocked-in upstream of the corresponding ATG and are, thus, under the control of the endogenous promoter/enhancer elements. Interestingly, this strategy does not disturb endogenous gene expression. Using the fast maturing fluorescent protein reporter, Venus, enabled us to follow MHB development using cell tracking and live imaging. In addition, we show that these reporter lines label various neuronal and glial cell types in the adult zebrafish brain, making them highly suitable for investigating embryonic and adult midbrain, hindbrain, and MHB development. PMID:28713249
CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development.

PubMed

Kesavan, Gokul; Chekuru, Avinash; Machate, Anja; Brand, Michael

2017-01-01

The midbrain-hindbrain boundary (MHB) acts as an organizer and controls the fate of neighboring cells to develop into either mesencephalic (midbrain) or metencephalic (hindbrain) cells by secreting signaling molecules like Wnt1 and Fgf8. The zebrafish is an excellent vertebrate model for studying MHB development due to the ease of gene manipulation and the possibility of following cellular dynamics and morphogenetic processes using live imaging. Currently, only very few reporter and/or Cre-driver lines are available to study gene expression at the MHB, hampering the understanding of MHB development, and traditional transgenic technologies using promoter/enhancer fragments or bacterial artificial chromosome (BAC)-mediated transgenesis often do not faithfully recapitulate endogenous expression patterns. In contrast, CRISPR/Cas9-mediated genome editing technology now provides a great opportunity to efficiently knock-in or knock-out genes. We have generated four CRISPR/Cas9-based knock-in fluorescent reporter lines for two crucial genes involved in MHB development, namely otx2 and pax2a . The coding sequences of the reporters were knocked-in upstream of the corresponding ATG and are, thus, under the control of the endogenous promoter/enhancer elements. Interestingly, this strategy does not disturb endogenous gene expression. Using the fast maturing fluorescent protein reporter, Venus, enabled us to follow MHB development using cell tracking and live imaging. In addition, we show that these reporter lines label various neuronal and glial cell types in the adult zebrafish brain, making them highly suitable for investigating embryonic and adult midbrain, hindbrain, and MHB development.
Quantitative Electroencephalographic Analysis Provides an Early-Stage Indicator of Disease Onset and Progression in the zQ175 Knock-In Mouse Model of Huntington's Disease.

PubMed

Fisher, Simon P; Schwartz, Michael D; Wurts-Black, Sarah; Thomas, Alexia M; Chen, Tsui-Ming; Miller, Michael A; Palmerston, Jeremiah B; Kilduff, Thomas S; Morairty, Stephen R

2016-02-01

Patients with Huntington's disease (HD) show a high prevalence of sleep disorders that typically occur prior to the onset of motoric symptoms and neurodegeneration. Our understanding of the pathophysiological alterations in premanifest HD is limited, hindering the ability to measure disease modification in response to treatment. We used a full-length knock-in HD model to determine early changes in the electroencephalogram (EEG) and sleep that may predict the onset and progression of the disease. A 10-month longitudinal study was designed to determine the effect of the HD mutation on the EEG and sleep/wake changes in heterozygous (HET) and homozygous (HOM) zQ175 mice and wild-type (WT) littermates from 8 to 48 w of age. Mice were instrumented with tethered headmounts to record EEG/electromyography signals. Telemeters were implanted to continuously measure locomotor activity (LMA) and body temperature (Tb). Sleep deprivation (SDep) was performed at 8, 12, 16, 24, 32, and 48 w of age. The HD mutation disrupted the EEG field potential from 8-12 w in an age- and mutant huntington dose-dependent manner, prior to changes in sleep/wake states, LMA, and Tb. Prominent effects of the HD mutation on the EEG included a progressive reduction in low frequency power, a slowing of rapid eye movement peak theta frequency, and the emergence of state-dependent beta/gamma oscillations. There was no effect of genotype on the relative increase in nonrapid eye movement delta power or sleep time in response to SDep. The expression of the Huntington's disease (HD) mutation results in complex EEG alterations that occur prior to deficits in behavioral measures and are one of the earliest phenotypes uncovered in this mouse model. Despite these EEG changes, homeostatic responses to sleep loss were preserved in HET and HOM zQ175 mice. Greater insight into the localization and response of these EEG alterations to novel therapies may enable early intervention and improve outcomes for patients with HD. © 2016 Associated Professional Sleep Societies, LLC.
Blockade of the IL-6 trans-signalling/STAT3 axis suppresses cachexia in Kras-induced lung adenocarcinoma.

PubMed

Miller, A; McLeod, L; Alhayyani, S; Szczepny, A; Watkins, D N; Chen, W; Enriori, P; Ferlin, W; Ruwanpura, S; Jenkins, B J

2017-05-25

Lung cancer is the leading cause of cancer death worldwide, and is frequently associated with the devastating paraneoplastic syndrome of cachexia. The potent immunomodulatory cytokine interleukin (IL)-6 has been linked with the development of lung cancer as well as cachexia; however, the mechanisms by which IL-6 promotes muscle wasting in lung cancer cachexia are ill-defined. In this study, we report that the gp130 F/F knock-in mouse model displaying hyperactivation of the latent transcription factor STAT3 via the common IL-6 cytokine family signalling receptor, gp130, develops cachexia during Kras-driven lung carcinogenesis. Specifically, exacerbated weight loss, early mortality and reduced muscle and adipose tissue mass were features of the gp130 F/F :Kras G12D model, but not parental Kras G12D mice in which STAT3 was not hyperactivated. Gene expression profiling of muscle tissue in cachectic gp130 F/F :Kras G12D mice revealed the upregulation of IL-6 and STAT3-target genes compared with Kras G12D muscle tissue. These cachectic features of gp130 F/F :Kras G12D mice were abrogated upon the genetic normalization of STAT3 activation or ablation of IL-6 in gp130 F/F :Kras G12D :Stat3 -/+ or gp130 F/F :Kras G12D :Il6 -/- mice, respectively. Furthermore, protein levels of the soluble IL-6 receptor (sIL-6R), which is the central facilitator of IL-6 trans-signalling, were elevated in cachectic muscle from gp130 F/F :Kras G12D mice, and the specific blockade of IL-6 trans-signalling, but not classical signalling, with an anti-IL-6R antibody ameliorated cachexia-related characteristics in gp130 F/F :Kras G12D mice. Collectively, these preclinical findings identify trans-signalling via STAT3 as the signalling modality by which IL-6 promotes muscle wasting in lung cancer cachexia, and therefore support the clinical evaluation of the IL-6 trans-signalling/STAT3 axis as a therapeutic target in advanced lung cancer patients presenting with cachexia.
Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes.

PubMed

Wang, Bangmei; Li, Kunyu; Wang, Amy; Reiser, Michelle; Saunders, Thom; Lockey, Richard F; Wang, Jia-Wang

2015-10-01

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems.

PubMed

Sakuma, Tetsushi; Nakade, Shota; Sakane, Yuto; Suzuki, Ken-Ichi T; Yamamoto, Takashi

2016-01-01

Programmable nucleases enable engineering of the genome by utilizing endogenous DNA double-strand break (DSB) repair pathways. Although homologous recombination (HR)-mediated gene knock-in is well established, it cannot necessarily be applied in every cell type and organism because of variable HR frequencies. We recently reported an alternative method of gene knock-in, named the PITCh (Precise Integration into Target Chromosome) system, assisted by microhomology-mediated end-joining (MMEJ). MMEJ harnesses independent machinery from HR, and it requires an extremely short homologous sequence (5-25 bp) for DSB repair, resulting in precise gene knock-in with a more easily constructed donor vector. Here we describe a streamlined protocol for PITCh knock-in, including the design and construction of the PITCh vectors, and their delivery to either human cell lines by transfection or to frog embryos by microinjection. The construction of the PITCh vectors requires only a few days, and the entire process takes ∼ 1.5 months to establish knocked-in cells or ∼ 1 week from injection to early genotyping in frog embryos.
Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems.

PubMed

Kawabe, Yoshinori; Komatsu, Shinya; Komatsu, Shodai; Murakami, Mai; Ito, Akira; Sakuma, Tetsushi; Nakamura, Takahiro; Yamamoto, Takashi; Kamihira, Masamichi

2018-05-01

Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Building Cre Knockin Rat Lines Using CRISPR/Cas9.

PubMed

Ma, Yuanwu; Zhang, Lianfeng; Huang, Xingxu

2017-01-01

Conditional gene inactivation strategy helps researchers to study the gene functions that are critical in embryogenesis or in defined tissues of adulthood. The Cre/loxP system is widely used for conditional gene inactivation/activation in cells or organisms. Cre knockin animal lines are essential for gene expression or inactivation in a spatially and temporally restricted manner. However, to generate a Cre knockin line by traditional approach is laborious. Recently, the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) has been proven as a simple and efficient genome-editing tool. We have used CRISPR/Cas9 system to generate rat strains that carry Cre genes in different targeted gene loci by direct delivery of gRNAs/Cas9/donors into fertilized eggs. Here, we described a stepwise procedure for the generation of Cre knockin rat, including target site selection, RNA preparation, the construction of the template donor, pronuclear injection, and the genotyping of precise Cre insertion in F 0 rats. Taken together, the establishment of Cre knockin line can be achieved within 6 weeks.
A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging.

PubMed

Patterson, Victoria L; Thompson, Brian S; Cherry, Catherine; Wang, Shao-Bin; Chen, Bo; Hoh, Josephine

2016-07-14

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.
Genetic and pharmacological evidence that G2019S LRRK2 confers a hyperkinetic phenotype, resistant to motor decline associated with aging

PubMed Central

Longo, Francesco; Russo, Isabella; Shimshek, Derya R.; Greggio, Elisa; Morari, Michele

2014-01-01

The leucine-rich repeat kinase 2 mutation G2019S in the kinase-domain is the most common genetic cause of Parkinson's disease. To investigate the impact of the G2019S mutation on motor activity in vivo, a longitudinal phenotyping approach was developed in knock-in (KI) mice bearing this kinase-enhancing mutation. Two cohorts of G2019S KI mice and wild-type littermates (WT) were subjected to behavioral tests, specific for akinesia, bradykinesia and overall gait ability, at different ages (3, 6, 10, 15 and 19 months). The motor performance of G2019S KI mice remained stable up to the age of 19 months and did not show the typical age-related decline in immobility time and stepping activity of WT. Several lines of evidence suggest that enhanced LRRK2 kinase activity is the main contributor to the observed hyperkinetic phenotype of G2019S KI mice: i) KI mice carrying a LRRK2 kinase-dead mutation (D1994S KD) showed a similar progressive motor decline as WT; ii) two LRRK2 kinase inhibitors, H-1152 and Nov-LRRK2-11, acutely reversed the hyperkinetic phenotype of G2019S KI mice, while being ineffective in WT or D1994S KD animals. LRRK2 target engagement in vivo was further substantiated by reduction of LRRK2 phosphorylation at Ser935 in the striatum and cortex at efficacious doses of Nov-LRRK2-11, and in the striatum at efficacious doses of H-1152. In summary, expression of the G2019S mutation in the mouse LRRK2 gene confers a hyperkinetic phenotype that is resistant to age-related motor decline, likely via enhancement of LRRK2 kinase activity. This study provides an in vivo model to investigate the effects of LRRK2 inhibitors on motor function. PMID:25107341
Increased Notch3 Activity Mediates pathological Changes in Structure of Cerebral arteries

PubMed Central

Baron-Menguy, Celine; Domenga-Denier, Valérie; Ghezali, Lamia; Faraci, Frank; Joutel, Anne

2016-01-01

CADASIL, the most frequent genetic cause of stroke and vascular dementia, is caused by highly stereotyped mutations in the NOTCH3 receptor, which is predominantly expressed in vascular smooth muscle. The well-established TgNotch3R169C mouse model develops characteristic features of the human disease, with deposition of NOTCH3 and other proteins, including TIMP3 (tissue inhibitor of metalloproteinase 3), on brain vessels as well as reduced maximal dilation, and attenuated myogenic tone of cerebral arteries, but without elevated blood pressure. Increased TIMP3 levels were recently shown to be a major determinant of altered myogenic tone. In this study, we investigated the contribution of TIMP3 and Notch3 signaling to the impairment of maximal vasodilator capacity caused by the archetypal R169C mutation. Maximally dilated cerebral arteries in TgNotch3R169C mice exhibited a decrease in lumen diameter over a range of physiological pressures that occurred prior to myogenic tone deficits. This defect was not prevented by genetic reduction of TIMP3 in TgNotch3R169C mice and was not observed in mice overexpressing TIMP3. Knock-in mice with the R169C mutation (Notch3R170C/R170C) exhibited similar reductions in arterial lumen, and both TgNotch3R169C and Notch3R170C/R170C mice showed increased cerebral artery expression of Notch3 target genes. Reduced maximal vasodilation was prevented by conditional reduction of Notch activity in smooth muscle of TgNotch3R169C mice and mimicked by conditional activation of Notch3 in smooth muscle, an effect that was blood pressure-independent. We conclude that increased Notch3 activity mediates reduction in maximal dilator capacity of cerebral arteries in CADASIL and may contribute to reductions in cerebral blood flow. PMID:27821617
Increased Notch3 Activity Mediates Pathological Changes in Structure of Cerebral Arteries.

PubMed

Baron-Menguy, Celine; Domenga-Denier, Valérie; Ghezali, Lamia; Faraci, Frank M; Joutel, Anne

2017-01-01

CADASIL (Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy), the most frequent genetic cause of stroke and vascular dementia, is caused by highly stereotyped mutations in the NOTCH3 receptor, which is predominantly expressed in vascular smooth muscle. The well-established TgNotch3 R169C mouse model develops characteristic features of the human disease, with deposition of NOTCH3 and other proteins, including TIMP3 (tissue inhibitor of metalloproteinase 3), on brain vessels, as well as reduced maximal dilation, and attenuated myogenic tone of cerebral arteries, but without elevated blood pressure. Increased TIMP3 levels were recently shown to be a major determinant of altered myogenic tone. In this study, we investigated the contribution of TIMP3 and Notch3 signaling to the impairment of maximal vasodilator capacity caused by the archetypal R169C mutation. Maximally dilated cerebral arteries in TgNotch3 R169C mice exhibited a decrease in lumen diameter over a range of physiological pressures that occurred before myogenic tone deficits. This defect was not prevented by genetic reduction of TIMP3 in TgNotch3 R169C mice and was not observed in mice overexpressing TIMP3. Knock-in mice with the R169C mutation (Notch3 R170C/R170C ) exhibited similar reductions in arterial lumen, and both TgNotch3 R169C and Notch3 R170C/R170C mice showed increased cerebral artery expression of Notch3 target genes. Reduced maximal vasodilation was prevented by conditional reduction of Notch activity in smooth muscle of TgNotch3 R169C mice and mimicked by conditional activation of Notch3 in smooth muscle, an effect that was blood pressure-independent. We conclude that increased Notch3 activity mediates reduction in maximal dilator capacity of cerebral arteries in CADASIL and may contribute to reductions in cerebral blood flow. © 2016 American Heart Association, Inc.
Physiological Expression of AMPKγ2RG Mutation Causes Wolff-Parkinson-White Syndrome and Induces Kidney Injury in Mice*

PubMed Central

Yang, Xiaodong; Mudgett, John; Bou-About, Ghina; Champy, Marie-France; Jacobs, Hugues; Monassier, Laurent; Pavlovic, Guillaume; Sorg, Tania; Herault, Yann; Petit-Demoulière, Benoit; Lu, Ku; Feng, Wen; Wang, Hongwu; Ma, Li-Jun; Askew, Roger; Erion, Mark D.; Kelley, David E.; Myers, Robert W.; Li, Cai

2016-01-01

Mutations of the AMP-activated kinase gamma 2 subunit (AMPKγ2), N488I (AMPKγ2NI) and R531G (AMPKγ2RG), are associated with Wolff-Parkinson-White (WPW) syndrome, a cardiac disorder characterized by ventricular pre-excitation in humans. Cardiac-specific transgenic overexpression of human AMPKγ2NI or AMPKγ2RG leads to constitutive AMPK activation and the WPW phenotype in mice. However, overexpression of these mutant proteins also caused profound, non-physiological increase in cardiac glycogen, which might abnormally alter the true phenotype. To investigate whether physiological levels of AMPKγ2NI or AMPKγ2RG mutation cause WPW syndrome and metabolic changes in other organs, we generated two knock-in mouse lines on the C57BL/6N background harboring mutations of human AMPKγ2NI and AMPKγ2RG, respectively. Similar to the reported phenotypes of mice overexpressing AMPKγ2NI or AMPKγ2RG in the heart, both lines developed WPW syndrome and cardiac hypertrophy; however, these effects were independent of cardiac glycogen accumulation. Compared with AMPKγ2WT mice, AMPKγ2NI and AMPKγ2RG mice exhibited reduced body weight, fat mass, and liver steatosis when fed with a high fat diet (HFD). Surprisingly, AMPKγ2RG but not AMPKγ2NI mice fed with an HFD exhibited severe kidney injury characterized by glycogen accumulation, inflammation, apoptosis, cyst formation, and impaired renal function. These results demonstrate that expression of AMPKγ2NI and AMPKγ2RG mutations at physiological levels can induce beneficial metabolic effects but that this is accompanied by WPW syndrome. Our data also reveal an unexpected effect of AMPKγ2RG in the kidney, linking lifelong constitutive activation of AMPK to a potential risk for kidney dysfunction in the context of an HFD. PMID:27621313
Physiological Expression of AMPKγ2RG Mutation Causes Wolff-Parkinson-White Syndrome and Induces Kidney Injury in Mice.

PubMed

Yang, Xiaodong; Mudgett, John; Bou-About, Ghina; Champy, Marie-France; Jacobs, Hugues; Monassier, Laurent; Pavlovic, Guillaume; Sorg, Tania; Herault, Yann; Petit-Demoulière, Benoit; Lu, Ku; Feng, Wen; Wang, Hongwu; Ma, Li-Jun; Askew, Roger; Erion, Mark D; Kelley, David E; Myers, Robert W; Li, Cai; Guan, Hong-Ping

2016-11-04

Mutations of the AMP-activated kinase gamma 2 subunit (AMPKγ2), N488I (AMPKγ2 NI ) and R531G (AMPKγ2 RG ), are associated with Wolff-Parkinson-White (WPW) syndrome, a cardiac disorder characterized by ventricular pre-excitation in humans. Cardiac-specific transgenic overexpression of human AMPKγ2 NI or AMPKγ2 RG leads to constitutive AMPK activation and the WPW phenotype in mice. However, overexpression of these mutant proteins also caused profound, non-physiological increase in cardiac glycogen, which might abnormally alter the true phenotype. To investigate whether physiological levels of AMPKγ2 NI or AMPKγ2 RG mutation cause WPW syndrome and metabolic changes in other organs, we generated two knock-in mouse lines on the C57BL/6N background harboring mutations of human AMPKγ2 NI and AMPKγ2 RG , respectively. Similar to the reported phenotypes of mice overexpressing AMPKγ2 NI or AMPKγ2 RG in the heart, both lines developed WPW syndrome and cardiac hypertrophy; however, these effects were independent of cardiac glycogen accumulation. Compared with AMPKγ2 WT mice, AMPKγ2 NI and AMPKγ2 RG mice exhibited reduced body weight, fat mass, and liver steatosis when fed with a high fat diet (HFD). Surprisingly, AMPKγ2 RG but not AMPKγ2 NI mice fed with an HFD exhibited severe kidney injury characterized by glycogen accumulation, inflammation, apoptosis, cyst formation, and impaired renal function. These results demonstrate that expression of AMPKγ2 NI and AMPKγ2 RG mutations at physiological levels can induce beneficial metabolic effects but that this is accompanied by WPW syndrome. Our data also reveal an unexpected effect of AMPKγ2 RG in the kidney, linking lifelong constitutive activation of AMPK to a potential risk for kidney dysfunction in the context of an HFD. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Human CD45 is an F-component-specific receptor for the staphylococcal toxin Panton-Valentine leukocidin.

PubMed

Tromp, Angelino T; Van Gent, Michiel; Abrial, Pauline; Martin, Amandine; Jansen, Joris P; De Haas, Carla J C; Van Kessel, Kok P M; Bardoel, Bart W; Kruse, Elisabeth; Bourdonnay, Emilie; Boettcher, Michael; McManus, Michael T; Day, Christopher J; Jennings, Michael P; Lina, Gérard; Vandenesch, François; Van Strijp, Jos A G; Jan Lebbink, Robert; Haas, Pieter-Jan A; Henry, Thomas; Spaan, András N

2018-05-07

The staphylococcal bi-component leukocidins Panton-Valentine leukocidin (PVL) and γ-haemolysin CB (HlgCB) target human phagocytes. Binding of the toxins' S-components to human complement C5a receptor 1 (C5aR1) contributes to cellular tropism and human specificity of PVL and HlgCB. To investigate the role of both leukocidins during infection, we developed a human C5aR1 knock-in (hC5aR1 KI ) mouse model. HlgCB, but unexpectedly not PVL, contributed to increased bacterial loads in tissues of hC5aR1 KI mice. Compared to humans, murine hC5aR1 KI neutrophils showed a reduced sensitivity to PVL, which was mediated by the toxin's F-component LukF-PV. By performing a genome-wide CRISPR-Cas9 screen, we identified CD45 as a receptor for LukF-PV. The human-specific interaction between LukF-PV and CD45 provides a molecular explanation for resistance of hC5aR1 KI mouse neutrophils to PVL and probably contributes to the lack of a PVL-mediated phenotype during infection in these mice. This study demonstrates an unsuspected role of the F-component in driving the sensitivity of human phagocytes to PVL.
Loss of Cbl and Cbl-b ubiquitin ligases abrogates hematopoietic stem cell quiescence and sensitizes leukemic disease to chemotherapy

PubMed Central

An, Wei; Nadeau, Scott A.; Mohapatra, Bhopal C.; Feng, Dan; Zutshi, Neha; Storck, Matthew D.; Arya, Priyanka; Talmadge, James E.; Meza, Jane L.; Band, Vimla; Band, Hamid

2015-01-01

Cbl and Cbl-b are tyrosine kinase-directed RING finger type ubiquitin ligases (E3s) that negatively regulate cellular activation pathways. E3 activity-disrupting human Cbl mutations are associated with myeloproliferative disorders (MPD) that are reproduced in mice with Cbl RING finger mutant knock-in or hematopoietic Cbl and Cbl-b double knockout. However, the role of Cbl proteins in hematopoietic stem cell (HSC) homeostasis, especially in the context of MPD is unclear. Here we demonstrate that HSC expansion and MPD development upon combined Cbl and Cbl-b deletion are dependent on HSCs. Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion. We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion. In vivo, treatment with 5-fluorouracil hastens DKO HSC exhaustion and protects mice from death due to MPD. Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling. PMID:25871390
Genome Editing in Rats Using TALE Nucleases.

PubMed

Tesson, Laurent; Remy, Séverine; Ménoret, Séverine; Usal, Claire; Thinard, Reynald; Savignard, Chloé; De Cian, Anne; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio

2016-01-01

The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.
Fgfr1 regulates development through the combinatorial use of signaling proteins.

PubMed

Brewer, J Richard; Molotkov, Andrei; Mazot, Pierre; Hoch, Renée V; Soriano, Philippe

2015-09-01

Fibroblast growth factor (Fgf) signaling governs multiple processes important in development and disease. Many lines of evidence have implicated Erk1/2 signaling induced through Frs2 as the predominant effector pathway downstream from Fgf receptors (Fgfrs), but these receptors can also signal through other mechanisms. To explore the functional significance of the full range of signaling downstream from Fgfrs in mice, we engineered an allelic series of knock-in point mutations designed to disrupt Fgfr1 signaling functions individually and in combination. Analysis of each mutant indicates that Frs2 binding to Fgfr1 has the most pleiotropic functions in development but also that the receptor uses multiple proteins additively in vivo. In addition to Frs2, Crk proteins and Plcγ also contribute to Erk1/2 activation, affecting axis elongation and craniofacial and limb development and providing a biochemical mechanism for additive signaling requirements. Disruption of all known signaling functions diminished Erk1/2 and Plcγ activation but did not recapitulate the peri-implantation Fgfr1-null phenotype. This suggests that Erk1/2-independent signaling pathways are functionally important for Fgf signaling in vivo. © 2015 Brewer et al.; Published by Cold Spring Harbor Laboratory Press.
Specification of spatial identities of cerebellar neuron progenitors by ptf1a and atoh1 for proper production of GABAergic and glutamatergic neurons.

PubMed

Yamada, Mayumi; Seto, Yusuke; Taya, Shinichiro; Owa, Tomoo; Inoue, Yukiko U; Inoue, Takayoshi; Kawaguchi, Yoshiya; Nabeshima, Yo-Ichi; Hoshino, Mikio

2014-04-02

In the cerebellum, the bHLH transcription factors Ptf1a and Atoh1 are expressed in distinct neuroepithelial regions, the ventricular zone (VZ) and the rhombic lip (RL), and are required for producing GABAergic and glutamatergic neurons, respectively. However, it is unclear whether Ptf1a or Atoh1 is sufficient for specifying GABAergic or glutamatergic neuronal fates. To test this, we generated two novel knock-in mouse lines, Ptf1a(Atoh1) and Atoh1(Ptf1a), that are designed to express Atoh1 and Ptf1a ectopically in the VZ and RL, respectively. In Ptf1a(Atoh1) embryos, ectopically Atoh1-expressing VZ cells produced glutamatergic neurons, including granule cells and deep cerebellar nuclei neurons. Correspondingly, in Atoh1(Ptf1a) animals, ectopically Ptf1a-expressing RL cells produced GABAergic populations, such as Purkinje cells and GABAergic interneurons. Consistent results were also obtained from in utero electroporation of Ptf1a or Atoh1 into embryonic cerebella, suggesting that Ptf1a and Atoh1 are essential and sufficient for GABAergic versus glutamatergic specification in the neuroepithelium. Furthermore, birthdating analyses with BrdU in the knock-in mice or with electroporation studies showed that ectopically produced fate-changed neuronal types were generated at temporal schedules closely simulating those of the wild-type RL and VZ, suggesting that the VZ and RL share common temporal information. Observations of knock-in brains as well as electroporated brains revealed that Ptf1a and Atoh1 mutually negatively regulate their expression, probably contributing to formation of non-overlapping neuroepithelial domains. These findings suggest that Ptf1a and Atoh1 specify spatial identities of cerebellar neuron progenitors in the neuroepithelium, leading to appropriate production of GABAergic and glutamatergic neurons, respectively.
Enzymatic characteristics of I213T mutant presenilin-1/gamma-secretase in cell models and knock-in mouse brains: familial Alzheimer disease-linked mutation impairs gamma-site cleavage of amyloid precursor protein C-terminal fragment beta.

PubMed

Shimojo, Masafumi; Sahara, Naruhiko; Mizoroki, Tatsuya; Funamoto, Satoru; Morishima-Kawashima, Maho; Kudo, Takashi; Takeda, Masatoshi; Ihara, Yasuo; Ichinose, Hiroshi; Takashima, Akihiko

2008-06-13

Presenilin (PS)/gamma-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid beta (Abeta) peptides in which Abeta species of different lengths are generated through multiple cleavages at the gamma-, zeta-, and epsilon-sites. An increased Abeta42/Abeta40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased Abeta42/Abeta40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of gamma-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS(-/-)) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo Abeta generation, suggesting that mutation impairs the total catalytic rate of gamma-secretase. Analysis of each Abeta species revealed that the FAD mutation specifically reduced Abeta40 levels more drastically than Abeta42 levels, leading to an increased Abeta42/Abeta40 ratio. By contrast, the FAD mutation increased the generation of longer Abeta species such as Abeta43, Abeta45, and >Abeta46. These results were confirmed by analyses of gamma-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo Abeta generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased Abeta42/Abeta40 ratio observed in cases of FAD mutations is related to the differential inhibition of gamma-site cleavage reactions, in which the reaction producing Abeta40 is subject to more inhibition than that producing Abeta42. Our results also provide novel insight into how enhancing the generation of longer Abetas may contribute to Alzheimer disease onset.
Mutation-linked defective interdomain interactions within ryanodine receptor cause aberrant Ca²⁺release leading to catecholaminergic polymorphic ventricular tachycardia.

PubMed

Suetomi, Takeshi; Yano, Masafumi; Uchinoumi, Hitoshi; Fukuda, Masakazu; Hino, Akihiro; Ono, Makoto; Xu, Xiaojuan; Tateishi, Hiroki; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Ikeda, Yasuhiro; Yamamoto, Takeshi; Ikemoto, Noriaki; Matsuzaki, Masunori

2011-08-09

The molecular mechanism by which catecholaminergic polymorphic ventricular tachycardia is induced by single amino acid mutations within the cardiac ryanodine receptor (RyR2) remains elusive. In the present study, we investigated mutation-induced conformational defects of RyR2 using a knockin mouse model expressing the human catecholaminergic polymorphic ventricular tachycardia-associated RyR2 mutant (S2246L; serine to leucine mutation at the residue 2246). All knockin mice we examined produced ventricular tachycardia after exercise on a treadmill. cAMP-dependent increase in the frequency of Ca²⁺ sparks was more pronounced in saponin-permeabilized knockin cardiomyocytes than in wild-type cardiomyocytes. Site-directed fluorescent labeling and quartz microbalance assays of the specific binding of DP2246 (a peptide corresponding to the 2232 to 2266 region: the 2246 domain) showed that DP2246 binds with the K201-binding sequence of RyR2 (1741 to 2270). Introduction of S2246L mutation into the DP2246 increased the affinity of peptide binding. Fluorescence quench assays of interdomain interactions within RyR2 showed that tight interaction of the 2246 domain/K201-binding domain is coupled with domain unzipping of the N-terminal (1 to 600)/central (2000 to 2500) domain pair in an allosteric manner. Dantrolene corrected the mutation-caused domain unzipping of the domain switch and stopped the exercise-induced ventricular tachycardia. The catecholaminergic polymorphic ventricular tachycardia-linked mutation of RyR2, S2246L, causes an abnormally tight local subdomain-subdomain interaction within the central domain involving the mutation site, which induces defective interaction between the N-terminal and central domains. This results in an erroneous activation of Ca²⁺ channel in a diastolic state reflecting on the increased Ca²⁺ spark frequency, which then leads to lethal arrhythmia.
The PNPLA3 variant associated with fatty liver disease (I148M) accumulates on lipid droplets by evading ubiquitylation.

PubMed

BasuRay, Soumik; Smagris, Eriks; Cohen, Jonathan C; Hobbs, Helen H

2017-10-01

A sequence variation (I148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) is strongly associated with fatty liver disease, but the underlying mechanism remains obscure. In this study, we used knock-in (KI) mice (Pnpla3 148M/M ) to examine the mechanism responsible for accumulation of triglyceride (TG) and PNPLA3 in hepatic lipid droplets (LDs). No differences were found between Pnpla3 148M/M and Pnpla3 +/+ mice in hepatic TG synthesis, utilization, or secretion. These results are consistent with TG accumulation in the Pnpla3 148M/M mice being caused by impaired TG mobilization from LDs. Sucrose feeding, which is required to elicit fatty liver in KI mice, led to a much larger and more persistent increase in PNPLA3 protein in the KI mice than in wild-type (WT) mice. Inhibition of the proteasome (bortezomib), but not macroautophagy (3-methyladenine), markedly increased PNPLA3 levels in WT mice, coincident with the appearance of ubiquitylated forms of the protein. Bortezomib did not increase PNPLA3 levels in Pnpla3 148M/M mice, and only trace amounts of ubiquitylated PNPLA3 were seen in these animals. These results are consistent with the notion that the 148M variant disrupts ubiquitylation and proteasomal degradation of PNPLA3, resulting in accumulation of PNPLA3-148M and impaired mobilization of TG from LDs. (Hepatology 2017;66:1111-1124). © 2017 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.
Variant BDNF-Val66Met Polymorphism is Associated with Layer-Specific Alterations in GABAergic Innervation of Pyramidal Neurons, Elevated Anxiety and Reduced Vulnerability of Adolescent Male Mice to Activity-Based Anorexia.

PubMed

Chen, Yi-Wen; Surgent, Olivia; Rana, Barkha S; Lee, Francis; Aoki, Chiye

2017-08-01

Previously, we determined that rodents' vulnerability to food restriction (FR)-evoked wheel running during adolescence (activity-based anorexia, ABA) is associated with failures to increase GABAergic innervation of hippocampal and medial prefrontal pyramidal neurons. Since brain-derived neurotrophic factor (BDNF) promotes GABAergic synaptogenesis, we hypothesized that individual differences in this vulnerability may arise from differences in the link between BDNF bioavailability and FR-evoked wheel running. We tested this hypothesis in male BDNF-Val66Met knock-in mice (BDNFMet/Met), known for reduction in the activity-dependent BDNF secretion and elevated anxiety-like behaviors. We found that 1) in the absence of FR or a wheel (i.e., control), BDNFMet/Met mice are more anxious than wild-type (WT) littermates, 2) electron microscopically verified GABAergic innervations of pyramidal neurons of BDNFMet/Met mice are reduced at distal dendrites in hippocampal CA1 and medial prefrontal cortex, 3) following ABA, WT mice exhibit anxiety equal to those of the BDNFMet/Met mice and have lost GABAergic innervation along distal dendrites, 4) BDNFMet/Met mice show blunted ABA vulnerability, and 5) unexpectedly, GABAergic innervation is higher at somata of BDNFMet/Met mice than of WT. We conclude that lamina-specific GABAergic inhibition is important for regulating anxiety, whether arising from environmental stress, such as food deprivation, or genetically, such as BDNFMet/Met single nucleotide polymorphism. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Brain-Derived Neurotrophic Factor Val66Met Human Polymorphism Impairs the Beneficial Exercise-Induced Neurobiological Changes in Mice.

PubMed

Ieraci, Alessandro; Madaio, Alessandro I; Mallei, Alessandra; Lee, Francis S; Popoli, Maurizio

2016-12-01

Several studies have shown that exercise improves cognitive functions and emotional behaviors. Positive effects of exercise have been associated with enhanced brain plasticity, adult hippocampal neurogenesis, and increased levels of brain-derived neurotrophic factor (BDNF). However, a substantial variability of individual response to exercise has been described, which may be accounted for by individual genetic variants. Here, we have assessed whether and how the common human BDNF Val66Met polymorphism influences the neurobiological effects modulated by exercise in BDNF Val66Met knock-in male mice. Wild-type (BDNF Val/Val ) and homozygous BDNF Val66Met (BDNF Met/Met ) male mice were housed in cages equipped with or without running wheels for 4 weeks. Changes in behavioral phenotype, hippocampal adult neurogenesis, and gene expression were evaluated in exercised and sedentary control mice. We found that exercise reduced the latency to feed in the novelty suppressed feeding and the immobility time in the forced swimming test in BDNF Val/Val but not in BDNF Met/Met mice. Hippocampal neurogenesis was reduced in BDNF Met/Met mice compared with BDNF Val/Val mice. BDNF Met/Met mice had lower basal BDNF protein levels in the hippocampus, which was not recovered following exercise. Moreover, exercise-induced expression of total BDNF, BDNF splice variants 1, 2, 4, 6 and fibronectin type III domain-containing protein 5 (FNDC5) mRNA levels were absent or reduced in the dentate gyrus of BDNF Met/Met mice. Exercise failed to enhance PGC-1α and FNDC5 mRNA levels in the BDNF Met/Met muscle. Overall these results indicate that, in adult male mice, the BDNF Val66Met polymorphism impairs the beneficial behavioral and neuroplasticity effects induced by physical exercise.
Brain-Derived Neurotrophic Factor Val66Met Human Polymorphism Impairs the Beneficial Exercise-Induced Neurobiological Changes in Mice

PubMed Central

Ieraci, Alessandro; Madaio, Alessandro I; Mallei, Alessandra; Lee, Francis S; Popoli, Maurizio

2016-01-01

Several studies have shown that exercise improves cognitive functions and emotional behaviors. Positive effects of exercise have been associated with enhanced brain plasticity, adult hippocampal neurogenesis, and increased levels of brain-derived neurotrophic factor (BDNF). However, a substantial variability of individual response to exercise has been described, which may be accounted for by individual genetic variants. Here, we have assessed whether and how the common human BDNF Val66Met polymorphism influences the neurobiological effects modulated by exercise in BDNF Val66Met knock-in male mice. Wild-type (BDNFVal/Val) and homozygous BDNF Val66Met (BDNFMet/Met) male mice were housed in cages equipped with or without running wheels for 4 weeks. Changes in behavioral phenotype, hippocampal adult neurogenesis, and gene expression were evaluated in exercised and sedentary control mice. We found that exercise reduced the latency to feed in the novelty suppressed feeding and the immobility time in the forced swimming test in BDNFVal/Val but not in BDNFMet/Met mice. Hippocampal neurogenesis was reduced in BDNFMet/Met mice compared with BDNFVal/Val mice. BDNFMet/Met mice had lower basal BDNF protein levels in the hippocampus, which was not recovered following exercise. Moreover, exercise-induced expression of total BDNF, BDNF splice variants 1, 2, 4, 6 and fibronectin type III domain-containing protein 5 (FNDC5) mRNA levels were absent or reduced in the dentate gyrus of BDNFMet/Met mice. Exercise failed to enhance PGC-1α and FNDC5 mRNA levels in the BDNFMet/Met muscle. Overall these results indicate that, in adult male mice, the BDNF Val66Met polymorphism impairs the beneficial behavioral and neuroplasticity effects induced by physical exercise. PMID:27388329

New Experimental Models of Diabetic Nephropathy in Mice Models of Type 2 Diabetes: Efforts to Replicate Human Nephropathy

PubMed Central

Soler, María José; Riera, Marta; Batlle, Daniel

2012-01-01

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. The use of experimental models of DN has provided valuable information regarding many aspects of DN, including pathophysiology, progression, implicated genes, and new therapeutic strategies. A large number of mouse models of diabetes have been identified and their kidney disease was characterized to various degrees. Most experimental models of type 2 DN are helpful in studying early stages of DN, but these models have not been able to reproduce the characteristic features of more advanced DN in humans such as nodules in the glomerular tuft or glomerulosclerosis. The generation of new experimental models of DN created by crossing, knockdown, or knockin of genes continues to provide improved tools for studying DN. These models provide an opportunity to search for new mechanisms involving the development of DN, but their shortcomings should be recognized as well. Moreover, it is important to recognize that the genetic background has a substantial effect on the susceptibility to diabetes and kidney disease development in the various models of diabetes. PMID:22461787
Nitroglycerin fails to lower blood pressure in redox-dead Cys42Ser PKG1α knock-in mouse

PubMed Central

Rudyk, Olena; Prysyazhna, Oleksandra; Burgoyne, Joseph R; Eaton, Philip

2013-01-01

Background Although nitroglycerin has remained in clinical use since 1879 the mechanism by which it relaxes blood vessels to lower blood pressure remains incompletely understood. Nitroglycerin undergoes metabolism generating several reaction products, including oxidants, and this ‘bioactivation’ process is essential for vasodilation. Protein kinase G (PKG) mediates classical nitric oxide-dependent vasorelaxation, but the 1α isoform is also independently activated by oxidation involving interprotein disulfide formation within this homodimeric protein complex. We hypothesised that nitroglycerin-induced vasodilation is mediated by disulfide activation of PKG1α. Methods and Results Treating smooth muscle cells or isolated blood vessels with nitroglycerin caused PKG1α disulfide dimerization. PKG1α disulfide formation was increased in wild-type mouse aortae by in vivo nitroglycerin treatment, but this oxidation was lost as tolerance developed. To establish whether kinase oxidation underlies nitroglycerin-induced vasodilation in vivo we employed a Cys42Ser PKG1α knock-in mouse that cannot transduce oxidant signals as it doesn’t contain the vital redox-sensing thiol. This ‘redox-dead’ knock-in mouse was substantively deficient in hypotensive response to nitroglycerin compared to wild-type littermates as measured in vivo using radiotelemetry. Resistance blood vessels from knock-ins were markedly less sensitive to nitroglycerin-induced vasodilation (EC50=39.2±10.7μM) than wild-types (EC50=12.1±2.9μM). Furthermore, after ~24 hours of treatment wild-type controls stopped vasodilating to nitroglycerin and the vascular sensitivity to nitroglycerin was decreased, whereas this ‘tolerance’ phenomenon that routinely hampers the management of hypertensive patients was absent in knock-ins. Conclusions PKG1α disulfide formation is a significant mediator of nitroglycerin-induced vasodilation and tolerance to nitroglycerin is associated with loss of kinase oxidation. PMID:22685118
Transcription factor scleraxis vitally contributes to progenitor lineage direction in wound healing of adult tendon in mice.

PubMed

Sakabe, Tomoya; Sakai, Keiko; Maeda, Toru; Sunaga, Ataru; Furuta, Nao; Schweitzer, Ronen; Sasaki, Takako; Sakai, Takao

2018-04-20

Tendon is a dense connective tissue that transmits high mechanical forces from skeletal muscle to bone. The transcription factor scleraxis (Scx) is a highly specific marker of both precursor and mature tendon cells (tenocytes). Mice lacking scx exhibit a specific and virtually complete loss of tendons during development. However, the functional contribution of Scx to wound healing in adult tendon has not yet been fully characterized. Here, using ScxGFP -tracking and loss-of-function systems, we show in an adult mouse model of Achilles tendon injury that paratenon cells, representing a stem cell antigen-1 (Sca-1)-positive and Scx-negative progenitor subpopulation, display Scx induction, migrate to the wound site, and produce extracellular matrix (ECM) to bridge the defect, whereas resident tenocytes exhibit a delayed response. Scx induction in the progenitors is initiated by transforming growth factor β (TGF-β) signaling. scx -deficient mice had migration of Sca-1-positive progenitor cell to the lesion site but impaired ECM assembly to bridge the defect. Mechanistically, scx -null progenitors displayed higher chondrogenic potential with up-regulation of SRY-box 9 (Sox9) coactivator PPAR-γ coactivator-1α (PGC-1α) in vitro , and knock-in analysis revealed that forced expression of full-length scx significantly inhibited Sox9 expression. Accordingly, scx -null wounds formed cartilage-like tissues that developed ectopic ossification. Our findings indicate a critical role of Scx in a progenitor-cell lineage in wound healing of adult mouse tendon. These progenitor cells could represent targets in strategies to facilitate tendon repair. We propose that this lineage-regulatory mechanism in tissue progenitors could apply to a broader set of tissues or biological systems in the body. © 2018 Sakabe et al.
Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.

PubMed

Merkle, Florian T; Neuhausser, Werner M; Santos, David; Valen, Eivind; Gagnon, James A; Maas, Kristi; Sandoe, Jackson; Schier, Alexander F; Eggan, Kevin

2015-05-12

The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background.

PubMed

Carstea, Ana Claudia; Pirity, Melinda K; Dinnyes, Andras

2009-12-31

In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression ("knock-out"), or to introduce mutant or modified alleles at the locus of interest ("knock-in"). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence germline competence of embryonic stem cell lines. Increasingly, evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell (ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly strain-dependent and it may also influence the germline transmission capability. Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential. There are a number of options for generating chimeras from ES cells (ES chimera mice); however, each method has its advantages and disadvantages. Recent developments in induced pluripotent stem (iPS) cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies. The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines. The most recent methods for generating specifically ES and iPS chimera mice, including the advantages and disadvantages of each method are also discussed.
Congenital heart defect causing mutation in Nkx2.5 displays in vivo functional deficit.

PubMed

Zakariyah, Abeer F; Rajgara, Rashida F; Veinot, John P; Skerjanc, Ilona S; Burgon, Patrick G

2017-04-01

The Nkx2.5 gene encodes a transcription factor that plays a critical role in heart development. In humans, heterozygous mutations in NKX2.5 result in congenital heart defects (CHDs). However, the molecular mechanisms by which these mutations cause the disease remain unknown. NKX2.5-R142C is a mutation that was reported to be associated with atrial septal defect (ASD) and atrioventricular (AV) block in 13-patients from one family. The R142C mutation is located within both the DNA-binding domain and the nuclear localization sequence of NKX2.5 protein. The pathogenesis of CHDs in humans with R142C point mutation is not well understood. To examine the functional deficit associated with this mutation in vivo, we generated and characterized a knock-in mouse that harbours the human mutation R142C. Systematic structural and functional examination of the embryonic, newborn, and adult mice revealed that the homozygous embryos Nkx2.5 R141C/R141C are developmentally arrested around E10.5 with delayed heart morphogenesis and downregulation of Nkx2.5 target genes, Anf, Mlc2v, Actc1 and Cx40. Histological examination of Nkx2.5 R141C/+ newborn hearts showed that 36% displayed ASD, with at least 80% 0f adult heterozygotes displaying a septal defect. Moreover, heterozygous Nkx2.5 R141C/+ newborn mice have downregulation of ion channel genes with 11/12 adult mice manifesting a prolonged PR interval that is indicative of 1st degree AV block. Collectively, the present study demonstrates that mice with the R141C point mutation in the Nkx2.5 allele phenocopies humans with the NKX2.5 R142C point mutation. Copyright © 2017 Elsevier Ltd. All rights reserved.
Remodeling of Hepatic Metabolism and Hyperaminoacidemia in Mice Deficient in Proglucagon-Derived Peptides

PubMed Central

Watanabe, Chika; Seino, Yusuke; Miyahira, Hiroki; Yamamoto, Michiyo; Fukami, Ayako; Ozaki, Nobuaki; Takagishi, Yoshiko; Sato, Jun; Fukuwatari, Tsutomu; Shibata, Katsumi; Oiso, Yutaka; Murata, Yoshiharu; Hayashi, Yoshitaka

2012-01-01

Glucagon is believed to be one of the most important peptides for upregulating blood glucose levels. However, homozygous glucagon–green fluorescent protein (gfp) knock-in mice (Gcggfp/gfp: GCGKO) are normoglycemic despite the absence of proglucagon-derived peptides, including glucagon. To characterize metabolism in the GCGKO mice, we analyzed gene expression and metabolome in the liver. The expression of genes encoding rate-limiting enzymes for gluconeogenesis was only marginally altered. On the other hand, genes encoding enzymes involved in conversion of amino acids to metabolites available for the tricarboxylic acid cycle and/or gluconeogenesis showed lower expression in the GCGKO liver. The expression of genes involved in the metabolism of fatty acids and nicotinamide was also altered. Concentrations of the metabolites in the GCGKO liver were altered in manners concordant with alteration in the gene expression patterns, and the plasma concentrations of amino acids were elevated in the GCGKO mice. The insulin concentration in serum and phosphorylation of Akt protein kinase in liver were reduced in GCGKO mice. These results indicated that proglucagon-derived peptides should play important roles in regulating various metabolic pathways, especially that of amino acids. Serum insulin concentration is lowered to compensate the impacts of absent proglucagon-derived peptide on glucose metabolism. On the other hand, impacts on other metabolic pathways are only partially compensated by reduced insulin action. PMID:22187375
Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis.

PubMed

He, Congcong; Bassik, Michael C; Moresi, Viviana; Sun, Kai; Wei, Yongjie; Zou, Zhongju; An, Zhenyi; Loh, Joy; Fisher, Jill; Sun, Qihua; Korsmeyer, Stanley; Packer, Milton; May, Herman I; Hill, Joseph A; Virgin, Herbert W; Gilpin, Christopher; Xiao, Guanghua; Bassel-Duby, Rhonda; Scherer, Philipp E; Levine, Beth

2012-01-18

Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.
Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome–associated PTPN11 mutation

PubMed Central

Marin, Talita M.; Keith, Kimberly; Davies, Benjamin; Conner, David A.; Guha, Prajna; Kalaitzidis, Demetrios; Wu, Xue; Lauriol, Jessica; Wang, Bo; Bauer, Michael; Bronson, Roderick; Franchini, Kleber G.; Neel, Benjamin G.; Kontaridis, Maria I.

2011-01-01

LEOPARD syndrome (LS) is an autosomal dominant “RASopathy” that manifests with congenital heart disease. Nearly all cases of LS are caused by catalytically inactivating mutations in the protein tyrosine phosphatase (PTP), non-receptor type 11 (PTPN11) gene that encodes the SH2 domain-containing PTP-2 (SHP2). RASopathies typically affect components of the RAS/MAPK pathway, yet it remains unclear how PTPN11 mutations alter cellular signaling to produce LS phenotypes. We therefore generated knockin mice harboring the Ptpn11 mutation Y279C, one of the most common LS alleles. Ptpn11Y279C/+ (LS/+) mice recapitulated the human disorder, with short stature, craniofacial dysmorphia, and morphologic, histologic, echocardiographic, and molecular evidence of hypertrophic cardiomyopathy (HCM). Heart and/or cardiomyocyte lysates from LS/+ mice showed enhanced binding of Shp2 to Irs1, decreased Shp2 catalytic activity, and abrogated agonist-evoked Erk/Mapk signaling. LS/+ mice also exhibited increased basal and agonist-induced Akt and mTor activity. The cardiac defects in LS/+ mice were completely reversed by treatment with rapamycin, an inhibitor of mTOR. Our results demonstrate that LS mutations have dominant-negative effects in vivo, identify enhanced mTOR activity as critical for causing LS-associated HCM, and suggest that TOR inhibitors be considered for treatment of HCM in LS patients. PMID:21339643
Maternal transcription of non-protein coding RNAs from the PWS-critical region rescues growth retardation in mice.

PubMed

Rozhdestvensky, Timofey S; Robeck, Thomas; Galiveti, Chenna R; Raabe, Carsten A; Seeger, Birte; Wolters, Anna; Gubar, Leonid V; Brosius, Jürgen; Skryabin, Boris V

2016-02-05

Prader-Willi syndrome (PWS) is a neurogenetic disorder caused by loss of paternally expressed genes on chromosome 15q11-q13. The PWS-critical region (PWScr) contains an array of non-protein coding IPW-A exons hosting intronic SNORD116 snoRNA genes. Deletion of PWScr is associated with PWS in humans and growth retardation in mice exhibiting ~15% postnatal lethality in C57BL/6 background. Here we analysed a knock-in mouse containing a 5'HPRT-LoxP-Neo(R) cassette (5'LoxP) inserted upstream of the PWScr. When the insertion was inherited maternally in a paternal PWScr-deletion mouse model (PWScr(p-/m5'LoxP)), we observed compensation of growth retardation and postnatal lethality. Genomic methylation pattern and expression of protein-coding genes remained unaltered at the PWS-locus of PWScr(p-/m5'LoxP) mice. Interestingly, ubiquitous Snord116 and IPW-A exon transcription from the originally silent maternal chromosome was detected. In situ hybridization indicated that PWScr(p-/m5'LoxP) mice expressed Snord116 in brain areas similar to wild type animals. Our results suggest that the lack of PWScr RNA expression in certain brain areas could be a primary cause of the growth retardation phenotype in mice. We propose that activation of disease-associated genes on imprinted regions could lead to general therapeutic strategies in associated diseases.
Allele Compensation in Tip60+/− Mice Rescues White Adipose Tissue Function In Vivo

PubMed Central

Gao, Yuan; Hamers, Nicole; Rakhshandehroo, Maryam; Berger, Ruud; Lough, John; Kalkhoven, Eric

2014-01-01

Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis), a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/− mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/− mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/− displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/− mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice. PMID:24870614
New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.

PubMed

Srinivasan, Rahul; Lu, Tsai-Yi; Chai, Hua; Xu, Ji; Huang, Ben S; Golshani, Peyman; Coppola, Giovanni; Khakh, Baljit S

2016-12-21

Astrocytes exist throughout the nervous system and are proposed to affect neural circuits and behavior. However, studying astrocytes has proven difficult because of the lack of tools permitting astrocyte-selective genetic manipulations. Here, we report the generation of Aldh1l1-Cre/ERT2 transgenic mice to selectively target astrocytes in vivo. We characterized Aldh1l1-Cre/ERT2 mice using imaging, immunohistochemistry, AAV-FLEX-GFP microinjections, and crosses to RiboTag, Ai95, and new Cre-dependent membrane-tethered Lck-GCaMP6f knockin mice that we also generated. Two to three weeks after tamoxifen induction, Aldh1l1-Cre/ERT2 selectively targeted essentially all adult (P80) brain astrocytes with no detectable neuronal contamination, resulting in expression of cytosolic and Lck-GCaMP6f, and permitting subcellular astrocyte calcium imaging during startle responses in vivo. Crosses with RiboTag mice allowed sequencing of actively translated mRNAs and determination of the adult cortical astrocyte transcriptome. Thus, we provide well-characterized, easy-to-use resources with which to selectively study astrocytes in situ and in vivo in multiple experimental scenarios. Copyright © 2016 Elsevier Inc. All rights reserved.
Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene.

PubMed

Gerlach, Max; Kraft, Theresia; Brenner, Bernhard; Petersen, Björn; Niemann, Heiner; Montag, Judith

2018-06-13

During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7 -gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene ( GMNN ). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9- GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9- GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.
CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci.

PubMed

Tasan, Ipek; Sustackova, Gabriela; Zhang, Liguo; Kim, Jiah; Sivaguru, Mayandi; HamediRad, Mohammad; Wang, Yuchuan; Genova, Justin; Ma, Jian; Belmont, Andrew S; Zhao, Huimin

2018-06-15

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.
Ephrin-B3 coordinates timed axon targeting and amygdala spinogenesis for innate fear behaviour

PubMed Central

Zhu, Xiao-Na; Liu, Xian-Dong; Sun, Suya; Zhuang, Hanyi; Yang, Jing-Yu; Henkemeyer, Mark; Xu, Nan-Jie

2016-01-01

Innate emotion response to environmental stimuli is a fundamental brain function that is controlled by specific neural circuits. Dysfunction of early emotional circuits may lead to neurodevelopmental disorders such as autism and schizophrenia. However, how the functional circuits are formed to prime initial emotional behaviours remain elusive. We reveal here using gene-targeted mutations an essential role for ephrin-B3 ligand-like activity in the development of innate fear in the neonatal brain. We further demonstrate that ephrin-B3 controls axon targeting and coordinates spinogenesis and neuronal activity within the amygdala. The morphological and behavioural abnormalities in ephrin-B3 mutant mice are rescued by conditional knock-in of wild-type ephrin-B3 during the critical period when axon targeting and fear responses are initiated. Our results thus define a key axonal molecule that participates in the wiring of amygdala circuits and helps bring about fear emotion during the important adolescence period. PMID:27008987
Ephrin-B3 coordinates timed axon targeting and amygdala spinogenesis for innate fear behaviour.

PubMed

Zhu, Xiao-Na; Liu, Xian-Dong; Sun, Suya; Zhuang, Hanyi; Yang, Jing-Yu; Henkemeyer, Mark; Xu, Nan-Jie

2016-03-24

Innate emotion response to environmental stimuli is a fundamental brain function that is controlled by specific neural circuits. Dysfunction of early emotional circuits may lead to neurodevelopmental disorders such as autism and schizophrenia. However, how the functional circuits are formed to prime initial emotional behaviours remain elusive. We reveal here using gene-targeted mutations an essential role for ephrin-B3 ligand-like activity in the development of innate fear in the neonatal brain. We further demonstrate that ephrin-B3 controls axon targeting and coordinates spinogenesis and neuronal activity within the amygdala. The morphological and behavioural abnormalities in ephrin-B3 mutant mice are rescued by conditional knock-in of wild-type ephrin-B3 during the critical period when axon targeting and fear responses are initiated. Our results thus define a key axonal molecule that participates in the wiring of amygdala circuits and helps bring about fear emotion during the important adolescence period.
An essential cell-autonomous role for hepcidin in cardiac iron homeostasis

PubMed Central

Lakhal-Littleton, Samira; Wolna, Magda; Chung, Yu Jin; Christian, Helen C; Heather, Lisa C; Brescia, Marcella; Ball, Vicky; Diaz, Rebeca; Santos, Ana; Biggs, Daniel; Clarke, Kieran; Davies, Benjamin; Robbins, Peter A

2016-01-01

Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain. DOI: http://dx.doi.org/10.7554/eLife.19804.001 PMID:27897970
Intracellular APP Domain Regulates Serine-Palmitoyl-CoA Transferase Expression and Is Affected in Alzheimer's Disease

PubMed Central

Grimm, Marcus O. W.; Grösgen, Sven; Rothhaar, Tatjana L.; Burg, Verena K.; Hundsdörfer, Benjamin; Haupenthal, Viola J.; Friess, Petra; Müller, Ulrike; Fassbender, Klaus; Riemenschneider, Matthias; Grimm, Heike S.; Hartmann, Tobias

2011-01-01

Lipids play an important role as risk or protective factors in Alzheimer's disease (AD), a disease biochemically characterized by the accumulation of amyloid beta peptides (Aβ), released by proteolytic processing of the amyloid precursor protein (APP). Changes in sphingolipid metabolism have been associated to the development of AD. The key enzyme in sphingolipid de novo synthesis is serine-palmitoyl-CoA transferase (SPT). In the present study we identified a new physiological function of APP in sphingolipid synthesis. The APP intracellular domain (AICD) was found to decrease the expression of the SPT subunit SPTLC2, the catalytic subunit of the SPT heterodimer, resulting in that decreased SPT activity. AICD function was dependent on Fe65 and SPTLC2 levels are increased in APP knock-in mice missing a functional AICD domain. SPTLC2 levels are also increased in familial and sporadic AD postmortem brains, suggesting that SPT is involved in AD pathology. PMID:21660213
Murinization of Internalin Extends Its Receptor Repertoire, Altering Listeria monocytogenes Cell Tropism and Host Responses

PubMed Central

Tsai, Yu-Huan; Disson, Olivier; Bierne, Hélène; Lecuit, Marc

2013-01-01

Listeria monocytogenes (Lm) is an invasive foodborne pathogen that leads to severe central nervous system and maternal-fetal infections. Lm ability to actively cross the intestinal barrier is one of its key pathogenic properties. Lm crosses the intestinal epithelium upon the interaction of its surface protein internalin (InlA) with its host receptor E-cadherin (Ecad). InlA-Ecad interaction is species-specific, does not occur in wild-type mice, but does in transgenic mice expressing human Ecad and knock-in mice expressing humanized mouse Ecad. To study listeriosis in wild-type mice, InlA has been “murinized” to interact with mouse Ecad. Here, we demonstrate that, unexpectedly, murinized InlA (InlAm) mediates not only Ecad-dependent internalization, but also N-cadherin-dependent internalization. Consequently, InlAm-expressing Lm targets not only goblet cells expressing luminally-accessible Ecad, as does Lm in humanized mice, but also targets villous M cells, which express luminally-accessible N-cadherin. This aberrant Lm portal of entry results in enhanced innate immune responses and intestinal barrier damage, both of which are not observed in wild-type Lm-infected humanized mice. Murinization of InlA therefore not only extends the host range of Lm, but also broadens its receptor repertoire, providing Lm with artifactual pathogenic properties. These results challenge the relevance of using InlAm-expressing Lm to study human listeriosis and in vivo host responses to this human pathogen. PMID:23737746
Murinization of internalin extends its receptor repertoire, altering Listeria monocytogenes cell tropism and host responses.

PubMed

Tsai, Yu-Huan; Disson, Olivier; Bierne, Hélène; Lecuit, Marc

2013-01-01

Listeria monocytogenes (Lm) is an invasive foodborne pathogen that leads to severe central nervous system and maternal-fetal infections. Lm ability to actively cross the intestinal barrier is one of its key pathogenic properties. Lm crosses the intestinal epithelium upon the interaction of its surface protein internalin (InlA) with its host receptor E-cadherin (Ecad). InlA-Ecad interaction is species-specific, does not occur in wild-type mice, but does in transgenic mice expressing human Ecad and knock-in mice expressing humanized mouse Ecad. To study listeriosis in wild-type mice, InlA has been "murinized" to interact with mouse Ecad. Here, we demonstrate that, unexpectedly, murinized InlA (InlA(m)) mediates not only Ecad-dependent internalization, but also N-cadherin-dependent internalization. Consequently, InlA(m)-expressing Lm targets not only goblet cells expressing luminally-accessible Ecad, as does Lm in humanized mice, but also targets villous M cells, which express luminally-accessible N-cadherin. This aberrant Lm portal of entry results in enhanced innate immune responses and intestinal barrier damage, both of which are not observed in wild-type Lm-infected humanized mice. Murinization of InlA therefore not only extends the host range of Lm, but also broadens its receptor repertoire, providing Lm with artifactual pathogenic properties. These results challenge the relevance of using InlA(m)-expressing Lm to study human listeriosis and in vivo host responses to this human pathogen.

Ubiquitin-Positive Intranuclear Inclusions in Neuronal and Glial Cells in a Mouse Model of the Fragile-X Premutation

PubMed Central

Wenzel, H. Jürgen; Hunsaker, Michael R.; Greco, Claudia M.; Willemsen, Rob; Berman, Robert F.

2010-01-01

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder caused by CGG trinucleotide repeat expansions in the fragile X mental retardation 1 (FMR1) gene. The neuropathological hallmark of the disease is the presence of ubiquitin-positive intranuclear inclusions in neurons and in astrocytes. Ubiquitin-positive intranuclear inclusions have also been found in the neurons of transgenic mice model carrying an expanded CGG(98) trinucleotide repeat of human origin, but have not previously been described in glial cells. Therefore, we used immunocytochemical methods to determine the pathological features of nuclear and/or cytoplasmic inclusions in astrocytes, Bergmann glia and neurons, as well as relationships between inclusion patterns, age, and repeat length in CGG knock-in (KI) mice in comparison with wild type mice. In CGG KI mice, ubiquitin-positive intranuclear inclusions were found in neurons (e.g., pyramidal cells, GABAergic neurons) throughout the brain in cortical and subcortical brain regions; these inclusions increased in number and size with advanced age. Ubiquitin-positive intranuclear inclusions were also present in protoplasmic astrocytes, including Bergmann glia in the cerebellum. The morphology of intranuclear inclusions in CGG KI mice was compared to that of typical inclusions in human neurons and astrocytes in postmortem FXTAS brain tissue. This new finding of previously unreported pathology in astrocytes of CGG KI mice now provides an important mouse model to study astrocyte pathology in human FXTAS. PMID:20051238
Activation of Estrogen Response Element–Independent ERα Signaling Protects Female Mice From Diet-Induced Obesity

PubMed Central

Yasrebi, Ali; Rivera, Janelle A.; Krumm, Elizabeth A.; Yang, Jennifer A.

2017-01-01

17β-estradiol (E2) regulates central and peripheral mechanisms that control energy and glucose homeostasis predominantly through estrogen receptor α (ERα) acting via receptor binding to estrogen response elements (EREs). ERα signaling is also involved in mediating the effects of E2 on diet-induced obesity (DIO), although the roles of ERE-dependent and -independent ERα signaling in reducing the effects of DIO remain largely unknown. We hypothesize that ERE-dependent ERα signaling is necessary to ameliorate the effects of DIO. We addressed this question using ERα knockout (KO) and ERα knockin/knockout (KIKO) female mice, the latter expressing an ERα that lacks a functional ERE binding domain. Female mice were ovariectomized, fed a low-fat diet (LFD) or a high-fat diet (HFD), and orally dosed with vehicle or estradiol benzoate (EB) (300 μg/kg). After 9 weeks, body composition, glucose and insulin tolerance, peptide hormone and inflammatory cytokine levels, and hypothalamic arcuate nucleus and liver gene expression were assessed. EB reduced body weight and body fat in wild-type (WT) female mice, regardless of diet, and in HFD-fed KIKO female mice, in part by reducing energy intake and feeding efficiency. EB reduced fasting glucose levels in KIKO mice fed both diets but augmented glucose tolerance only in HFD-fed KIKO female mice. Plasma insulin and interleukin 6 were elevated in KIKO and KO female mice compared with LFD-fed WT female mice. Expression of arcuate neuropeptide and receptor genes and liver fatty acid biosynthesis genes was altered by HFD and by EB through ERE-dependent and -independent mechanisms. Therefore, ERE-independent signaling mechanisms in both the brain and peripheral organs mediate, in part, the effects of E2 during DIO. PMID:27901601
Patatin-like phospholipase domain–containing protein 3 promotes transfers of essential fatty acids from triglycerides to phospholipids in hepatic lipid droplets

PubMed Central

Mitsche, Matthew A.; Hobbs, Helen H.; Cohen, Jonathan C.

2018-01-01

Fatty liver disease (FLD) is a burgeoning health problem. A missense variant (I148M) in patatin-like phospholipase domain–containing protein 3 (PNPLA3) confers susceptibility to FLD, although the mechanism is not known. To glean first insights into the physiological function of PNPLA3, we performed detailed lipidomic profiling of liver lysates and lipid droplets (LDs) from WT and Pnpla3−/− (KO) mice and from knock-in (ki) mice expressing either the 148M variant (IM-ki mice) or a variant (S47A) that renders the protein catalytically inactive (SA-ki mice). The four strains differed in composition of very-long-chain polyunsaturated fatty acids (vLCPUFA) in hepatic LDs. In the LDs of IM-ki mice, vLCPUFAs were depleted from triglycerides and enriched in phospholipids. Conversely, vLCPUFAs were enriched in triglycerides and depleted from phospholipids in SA-ki and Pnpla3−/− mice. Release of vLCPUFAs from hepatic LDs incubated ex vivo was increased in droplets from IM-ki mice and decreased from droplets isolated from Pnpla3−/− and SA-ki mice relative to those of WT mice. Thus, the physiological role of PNPLA3 appears to be to remodel triglycerides and phospholipids in LDs, perhaps to accommodate changes in LD size in response to feeding. Because SA-ki and IM-ki both cause FLD and yet have opposite effects on the lipidomic profile of LDs, we conclude that the FLD associated with genetic variation in PNPLA3 is not related to the enzyme's role in remodeling LD lipids. PMID:29555681
Impairment of Vision in a Mouse Model of Usher Syndrome Type III.

PubMed

Tian, Guilian; Lee, Richard; Ropelewski, Philip; Imanishi, Yoshikazu

2016-03-01

The purpose of this study was to obtain an Usher syndrome type III mouse model with retinal phenotype. Speed congenic method was used to obtain Clrn1 exon 1 knockout (Clrn1-/-) and Clrn1N48K knockin (Clrn1N48K/N48K) mice under A/J background. To study the retinal functions of these mice, we measured scotopic and photopic ERG responses. To observe if there are any structural abnormalities, we conducted light and transmission electron microscopy of fixed retinal specimens. In 3-month-old Clrn1-/- mice, scotopic b-wave amplitude was reduced by more than 25% at the light intensities from -2.2 to 0.38 log cd·s/m2, but scotopic a-wave amplitudes were comparable to those of age-matched wild type mice at all the light intensities tested. In 9-month-old Clrn1-/- mice, scotopic b-wave amplitudes were further reduced by more than 35%, and scotopic a-wave amplitude also showed a small decline as compared with wild type mice. Photopic ERG responses were comparable between Clrn1-/- and wild type mice. Those electrophysiological defects were not associated with a loss of rods. In Clrn1N48K/N48K mice, both a- and b-wave amplitudes were not discernable from those of wild type mice aged up to 10 months. Mutations that are Clrn1-/- biallelic cause visual defects when placed under A/J background. The absence of apparent rod degeneration suggests that the observed phenotype is due to functional defects, and not due to loss of rods. Biallelic Clrn1N48K/N48K mutations did not cause discernible visual defects, suggesting that Clrn1- allele is more severely dysfunctional than ClrnN48K allele.
Generation and characterization of Dyt1 DeltaGAG knock-in mouse as a model for early-onset dystonia.

PubMed

Dang, Mai T; Yokoi, Fumiaki; McNaught, Kevin St P; Jengelley, Toni-Ann; Jackson, Tehone; Li, Jianyong; Li, Yuqing

2005-12-01

A trinucleotide deletion of GAG in the DYT1 gene that encodes torsinA protein is implicated in the neurological movement disorder of Oppenheim's early-onset dystonia. The mutation removes a glutamic acid in the carboxy region of torsinA, a member of the Clp protease/heat shock protein family. The function of torsinA and the role of the mutation in causing dystonia are largely unknown. To gain insight into these unknowns, we made a gene-targeted mouse model of Dyt1 DeltaGAG to mimic the mutation found in DYT1 dystonic patients. The mutated heterozygous mice had deficient performance on the beam-walking test, a measure of fine motor coordination and balance. In addition, they exhibited hyperactivity in the open-field test. Mutant mice also showed a gait abnormality of increased overlap. Mice at 3 months of age did not display deficits in beam-walking and gait, while 6-month mutant mice did, indicating an age factor in phenotypic expression as well. While striatal dopamine and 4-dihydroxyphenylacetic acid (DOPAC) levels in Dyt1 DeltaGAG mice were similar to that of wild-type mice, a 27% decrease in 4-hydroxy, 3-methoxyphenacetic acid (homovanillic acid) was detected in mutant mice. Dyt1 DeltaGAG tissues also have ubiquitin- and torsinA-containing aggregates in neurons of the pontine nuclei. A sex difference was noticed in the mutant mice with female mutant mice exhibiting fewer alterations in behavioral, neurochemical, and cellular changes. Our results show that knocking in a Dyt1 DeltaGAG allele in mouse alters their motor behavior and recapitulates the production of protein aggregates that are seen in dystonic patients. Our data further support alterations in the dopaminergic system as a part of dystonia's neuropathology.
Estrogen receptor 1 modulates circadian rhythms in adult female mice.

PubMed

Blattner, Margaret S; Mahoney, Megan M

2014-06-01

Estradiol influences the level and distribution of daily activity, the duration of the free-running period, and the behavioral phase response to light pulses. However, the mechanisms by which estradiol regulates daily and circadian rhythms are not fully understood. We tested the hypothesis that estrogens modulate daily activity patterns via both classical and "non-classical" actions at the estrogen receptor subtype 1 (ESR1). We used female transgenic mice with mutations in their estrogen response pathways; ESR1 knock-out (ERKO) mice and "non-classical" estrogen receptor knock-in (NERKI) mice. NERKI mice have an ESR1 receptor with a mutation in the estrogen-response-element binding domain, allowing only actions via "non-classical" genomic and second messenger pathways. Ovariectomized female NERKI, ERKO, and wildtype (WT) mice were given a subcutaneous capsule with low- or high-dose estradiol and compared with counterparts with no hormone replacement. We measured wheel-running activity in a light:dark cycle and constant darkness, and the behavioral phase response to light pulses given at different points during the subjective day and night. Estradiol increased average daily wheel-running, consolidated activity to the dark phase, and shortened the endogenous period in WT, but not NERKI and ERKO mice. The timing of activity onset during entrainment was advanced in all estradiol-treated animals regardless of genotype suggesting an ESR1-independent mechanism. We propose that estradiol modifies period, activity level, and distribution of activity via classical actions of ESR1 whereas an ESR1 independent mechanism regulates the phase of rhythms.
Pathogenesis of persistent hyperplastic primary vitreous in mice lacking the arf tumor suppressor gene.

PubMed

Martin, Amy C; Thornton, J Derek; Liu, Jiewiu; Wang, XiaoFei; Zuo, Jian; Jablonski, Monica M; Chaum, Edward; Zindy, Frederique; Skapek, Stephen X

2004-10-01

Persistent hyperplastic primary vitreous (PHPV) is an idiopathic developmental eye disease associated with failed involution of the hyaloid vasculature. The present work addressed the pathogenesis of PHPV in a mouse model that replicates many aspects of the human disease. Ophthalmoscopic and histologic analyses documented pathologic processes in eyes of mice lacking the Arf gene compared with Ink4a-deficient and wild-type control animals. Immunohistochemical staining, in situ hybridization, and RT-PCR demonstrated the expression of relevant gene products. Arf gene expression was determined by in situ hybridization using wholemounts of wild-type mouse eyes and by immunofluorescence staining for green fluorescent protein (GFP) in Arf(+/GFP) heterozygous knock-in mouse eyes. Abnormalities in Arf(-/-) mice mimicked those found in patients with severe PHPV. The mice had microphthalmia; fibrovascular, retrolental tissue containing retinal pigment epithelial cells and remnants of the hyaloid vascular system; posterior lens capsule destruction with lens degeneration and opacity; and severe retinal dysplasia and detachment. Eyes of mice lacking the overlapping Ink4a gene were normal. Arf was selectively expressed in perivascular cells within the vitreous of the postnatal eye. Cells composing the retrolental mass in Arf(-/-) mice expressed the Arf promoter. The remnant hyaloid vessels expressed Flk-1. Its ligand, vascular endothelial growth factor (Vegf), was expressed in the retrolental tissue and the adjacent dysplastic neuroretina. Arf(-/-) mice have features that accurately mimic severe PHPV. In the HVS, Arf expression in perivascular cells may block their accumulation or repress Vegf expression to promote HVS involution and prevent PHPV.
Pathogenesis of Persistent Hyperplastic Primary Vitreous in Mice Lacking the Arf Tumor Suppressor Gene

PubMed Central

Martin, Amy C.; Thornton, J. Derek; Liu, Jiewiu; Wang, XiaoFei; Zuo, Jian; Jablonski, Monica M.; Chaum, Edward; Zindy, Frederique; Skapek, Stephen X.

2006-01-01

Purpose Persistent hyperplastic primary vitreous (PHPV) is an idiopathic developmental eye disease associated with failed involution of the hyaloid vasculature. The present work addressed the pathogenesis of PHPV in a mouse model that replicates many aspects of the human disease. Methods Ophthalmoscopic and histologic analyses documented pathologic processes in eyes of mice lacking the Arf gene compared with Ink4a-deficient and wild-type control animals. Immunohistochemical staining, in situ hybridization, and RT-PCR demonstrated the expression of relevant gene products. Arf gene expression was determined by in situ hybridization using wholemounts of wild-type mouse eyes and by immunofluorescence staining for green fluores-cent protein (GFP) in Arf+/GFP heterozygous knock-in mouse eyes. Results Abnormalities in Arf−/− mice mimicked those found in patients with severe PHPV. The mice had microphthalmia; fibrovascular, retrolental tissue containing retinal pigment epithelial cells and remnants of the hyaloid vascular system; posterior lens capsule destruction with lens degeneration and opacity; and severe retinal dysplasia and detachment. Eyes of mice lacking the overlapping Ink4a gene were normal. Arf was selectively expressed in perivascular cells within the vitreous of the postnatal eye. Cells composing the retrolental mass in Arf−/− mice expressed the Arf promoter. The remnant hyaloid vessels expressed Flk-1. Its ligand, vascular endothelial growth factor (Vegf), was expressed in the retrolental tissue and the adjacent dysplastic neuroretina. Conclusions Arf−/− mice have features that accurately mimic severe PHPV. In the HVS, Arf expression in perivascular cells may block their accumulation or repress Vegf expression to promote HVS involution and prevent PHPV. PMID:15452040
Hematopoietic stem cells can differentiate into restricted myeloid progenitors before cell division in mice.

PubMed

Grinenko, Tatyana; Eugster, Anne; Thielecke, Lars; Ramasz, Beáta; Krüger, Anja; Dietz, Sevina; Glauche, Ingmar; Gerbaulet, Alexander; von Bonin, Malte; Basak, Onur; Clevers, Hans; Chavakis, Triantafyllos; Wielockx, Ben

2018-05-15

Hematopoietic stem cells (HSCs) continuously replenish all blood cell types through a series of differentiation steps and repeated cell divisions that involve the generation of lineage-committed progenitors. However, whether cell division in HSCs precedes differentiation is unclear. To this end, we used an HSC cell-tracing approach and Ki67 RFP knock-in mice, in a non-conditioned transplantation model, to assess divisional history, cell cycle progression, and differentiation of adult HSCs. Our results reveal that HSCs are able to differentiate into restricted progenitors, especially common myeloid, megakaryocyte-erythroid and pre-megakaryocyte progenitors, without undergoing cell division and even before entering the S phase of the cell cycle. Additionally, the phenotype of the undivided but differentiated progenitors correlated with the expression of lineage-specific genes and loss of multipotency. Thus HSC fate decisions can be uncoupled from physical cell division. These results facilitate a better understanding of the mechanisms that control fate decisions in hematopoietic cells.
Mechanism and treatment for the learning and memory deficits associated with mouse models of Noonan syndrome

PubMed Central

Lee, Yong-Seok; Ehninger, Dan; Zhou, Miou; Oh, Jun-Young; Kang, Minkyung; Kwak, Chuljung; Ryu, Hyun-Hee; Butz, Delana; Araki, Toshiyuki; Cai, Ying; Balaji, J.; Sano, Yoshitake; Nam, Christine I.; Kim, Hyong Kyu; Kaang, Bong-Kiun; Burger, Corinna; Neel, Benjamin G.; Silva, Alcino J.

2015-01-01

In Noonan Syndrome (NS) 30% to 50% of subjects show cognitive deficits of unknown etiology and with no known treatment. Here, we report that knock-in mice expressing either of two NS-associated Ptpn11 mutations show hippocampal-dependent spatial learning impairments and deficits in hippocampal long-term potentiation (LTP). In addition, viral overexpression of the PTPN11D61G in adult hippocampus results in increased baseline excitatory synaptic function, deficits in LTP and spatial learning, which can all be reversed by a MEK inhibitor. Furthermore, brief treatment with lovastatin reduces Ras-Erk activation in the brain, and normalizes the LTP and learning deficits in adult Ptpn11D61G/+ mice. Our results demonstrate that increased basal Erk activity and corresponding baseline increases in excitatory synaptic function are responsible for the LTP impairments and, consequently, the learning deficits in mouse models of NS. These data also suggest that lovastatin or MEK inhibitors may be useful for treating the cognitive deficits in NS. PMID:25383899
A failure in energy metabolism and antioxidant uptake precede symptoms of Huntington's disease in mice.

PubMed

Acuña, Aníbal I; Esparza, Magdalena; Kramm, Carlos; Beltrán, Felipe A; Parra, Alejandra V; Cepeda, Carlos; Toro, Carlos A; Vidal, René L; Hetz, Claudio; Concha, Ilona I; Brauchi, Sebastián; Levine, Michael S; Castro, Maite A

2013-01-01

Huntington's disease has been associated with a failure in energy metabolism and oxidative damage. Ascorbic acid is a powerful antioxidant highly concentrated in the brain where it acts as a messenger, modulating neuronal metabolism. Using an electrophysiological approach in R6/2 HD slices, we observe an abnormal ascorbic acid flux from astrocytes to neurons, which is responsible for alterations in neuronal metabolic substrate preferences. Here using striatal neurons derived from knock-in mice expressing mutant huntingtin (STHdhQ cells), we study ascorbic acid transport. When extracellular ascorbic acid concentration increases, as occurs during synaptic activity, ascorbic acid transporter 2 (SVCT2) translocates to the plasma membrane, ensuring optimal ascorbic acid uptake for neurons. In contrast, SVCT2 from cells that mimic HD symptoms (dubbed HD cells) fails to reach the plasma membrane under the same conditions. We reason that an early impairment of ascorbic acid uptake in HD neurons could lead to early metabolic failure promoting neuronal death.
A failure in energy metabolism and antioxidant uptake precede symptoms of Huntington’s disease in mice

PubMed Central

Acuña, Aníbal I.; Esparza, Magdalena; Kramm, Carlos; Beltrán, Felipe A.; Parra, Alejandra V.; Cepeda, Carlos; Toro, Carlos A.; Vidal, René L.; Hetz, Claudio; Concha, Ilona I.; Brauchi, Sebastián; Levine, Michael S.; Castro, Maite A.

2013-01-01

Huntington’s disease has been associated with a failure in energy metabolism and oxidative damage. Ascorbic acid is a powerful antioxidant highly concentrated in the brain where it acts as a messenger, modulating neuronal metabolism. Using an electrophysiological approach in R6/2 HD slices, we observe an abnormal ascorbic acid flux from astrocytes to neurons, which is responsible for alterations in neuronal metabolic substrate preferences. Here using striatal neurons derived from knock-in mice expressing mutant huntingtin (STHdhQ cells), we study ascorbic acid transport. When extracellular ascorbic acid concentration increases, as occurs during synaptic activity, ascorbic acid transporter 2 (SVCT2) translocates to the plasma membrane, ensuring optimal ascorbic acid uptake for neurons. In contrast, SVCT2 from cells that mimic HD symptoms (dubbed HD cells) fails to reach the plasma membrane under the same conditions. We reason that an early impairment of ascorbic acid uptake in HD neurons could lead to early metabolic failure promoting neuronal death. PMID:24336051
Anchored phosphatases modulate glucose homeostasis

PubMed Central

Hinke, Simon A; Navedo, Manuel F; Ulman, Allison; Whiting, Jennifer L; Nygren, Patrick J; Tian, Geng; Jimenez-Caliani, Antonio J; Langeberg, Lorene K; Cirulli, Vincenzo; Tengholm, Anders; Dell'Acqua, Mark L; Santana, L Fernando; Scott, John D

2012-01-01

Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic β-cells involves ion channels and mobilization of Ca2+ and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-β-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca2+ currents, and attenuates cytoplasmic accumulation of Ca2+ and cAMP in β-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity. PMID:22940692
A failure in energy metabolism and antioxidant uptake precede symptoms of Huntington’s disease in mice

NASA Astrophysics Data System (ADS)

Acuña, Aníbal I.; Esparza, Magdalena; Kramm, Carlos; Beltrán, Felipe A.; Parra, Alejandra V.; Cepeda, Carlos; Toro, Carlos A.; Vidal, René L.; Hetz, Claudio; Concha, Ilona I.; Brauchi, Sebastián; Levine, Michael S.; Castro, Maite A.

2013-12-01

Huntington’s disease has been associated with a failure in energy metabolism and oxidative damage. Ascorbic acid is a powerful antioxidant highly concentrated in the brain where it acts as a messenger, modulating neuronal metabolism. Using an electrophysiological approach in R6/2 HD slices, we observe an abnormal ascorbic acid flux from astrocytes to neurons, which is responsible for alterations in neuronal metabolic substrate preferences. Here using striatal neurons derived from knock-in mice expressing mutant huntingtin (STHdhQ cells), we study ascorbic acid transport. When extracellular ascorbic acid concentration increases, as occurs during synaptic activity, ascorbic acid transporter 2 (SVCT2) translocates to the plasma membrane, ensuring optimal ascorbic acid uptake for neurons. In contrast, SVCT2 from cells that mimic HD symptoms (dubbed HD cells) fails to reach the plasma membrane under the same conditions. We reason that an early impairment of ascorbic acid uptake in HD neurons could lead to early metabolic failure promoting neuronal death.
Loss of RUNX1/AML1 arginine-methylation impairs in peripheral T cell homeostasis

PubMed Central

Mizutani, Shinsuke; Yoshida, Tatsushi; Zhao, Xinyang; Nimer, Stephen D.; Taniwaki, Masafumi; Okuda, Tsukasa

2016-01-01

Summary RUNX1 (previously termed AML1) is a frequent target of human leukaemia-associated gene aberrations, and it encodes the DNA-binding subunit of the Core-Binding Factor transcription factor complex. RUNX1 expression is essential for the initiation of definitive haematopoiesis, for steady-state thrombopoiesis, and for normal lymphocytes development. Recent studies revealed that protein arginine methyltransferase 1 (PRMT1), which accounts for the majority of the type I PRMT activity in cells, methylates two arginine residues in RUNX1 (R206 and R210), and these modifications inhibit corepressor-binding to RUNX1 thereby enhancing its transcriptional activity. In order to elucidate the biological significance of these methylations, we established novel knock-in mouse lines with non-methylable, double arginine-to-lysine (RTAMR-to-KTAMK) mutations in RUNX1. Homozygous Runx1KTAMK/KTAMK mice are born alive and appear normal during adulthood. However, Runx1KTAMK/KTAMK mice showed a reduction in CD3+ T lymphoid cells and a decrease in CD4+ T cells in peripheral lymphoid organs, in comparison to their wild-type littermates, leading to a reduction in the CD4+ to CD8+ T-cell ratio. These findings suggest that arginine-methylation of RUNX1 in the RTAMR-motif is dispensable for the development of definitive haematopoiesis and for steady-state platelet production, however this modification affects the role of RUNX1 in the maintenance of the peripheral CD4+ T-cell population. PMID:26010396
Analysis of mouse models carrying the I26T and R160C substitutions in the transcriptional repressor HESX1 as models for septo-optic dysplasia and hypopituitarism

PubMed Central

Sajedi, Ezat; Gaston-Massuet, Carles; Signore, Massimo; Andoniadou, Cynthia L.; Kelberman, Daniel; Castro, Sandra; Etchevers, Heather C.; Gerrelli, Dianne; Dattani, Mehul T.; Martinez-Barbera, Juan Pedro

2008-01-01

SUMMARY A homozygous substitution of the highly conserved isoleucine at position 26 by threonine (I26T) in the transcriptional repressor HESX1 has been associated with anterior pituitary hypoplasia in a human patient, with no forebrain or eye defects. Two individuals carrying a homozygous substitution of the conserved arginine at position 160 by cysteine (R160C) manifest septo-optic dysplasia (SOD), a condition characterised by pituitary abnormalities associated with midline telencephalic structure defects and optic nerve hypoplasia. We have generated two knock-in mouse models containing either the I26T or R160C substitution in the genomic locus. Hesx1I26T/I26T embryos show pituitary defects comparable with Hesx1−/− mouse mutants, with frequent occurrence of ocular abnormalities, although the telencephalon develops normally. Hesx1R160C/R160C mutants display forebrain and pituitary defects that are identical to those observed in Hesx1−/− null mice. We also show that the expression pattern of HESX1 during early human development is very similar to that described in the mouse, suggesting that the function of HESX1 is conserved between the two species. Together, these results suggest that the I26T mutation yields a hypomorphic allele, whereas R160C produces a null allele and, consequently, a more severe phenotype in both mice and humans. PMID:19093031
A Natural Variant of the T Cell Receptor-Signaling Molecule Vav1 Reduces Both Effector T Cell Functions and Susceptibility to Neuroinflammation.

PubMed

Kassem, Sahar; Gaud, Guillaume; Bernard, Isabelle; Benamar, Mehdi; Dejean, Anne S; Liblau, Roland; Fournié, Gilbert J; Colacios, Céline; Malissen, Bernard; Saoudi, Abdelhadi

2016-07-01

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell antigen receptor signals and therefore plays an important role in T cell development and activation. Our previous genetic studies identified a locus on rat chromosome 9 that controls the susceptibility to neuroinflammation and contains a non-synonymous polymorphism in the major candidate gene Vav1. To formally demonstrate the causal implication of this polymorphism, we generated a knock-in mouse bearing this polymorphism (Vav1R63W). Using this model, we show that Vav1R63W mice display reduced susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide immunization. This is associated with a lower production of effector cytokines (IFN-γ, IL-17 and GM-CSF) by autoreactive CD4 T cells. Despite increased proportion of Foxp3+ regulatory T cells in Vav1R63W mice, we show that this lowered cytokine production is intrinsic to effector CD4 T cells and that Treg depletion has no impact on EAE development. Finally, we provide a mechanism for the above phenotype by showing that the Vav1R63W variant has normal enzymatic activity but reduced adaptor functions. Together, these data highlight the importance of Vav1 adaptor functions in the production of inflammatory cytokines by effector T cells and in the susceptibility to neuroinflammation.
A Natural Variant of the T Cell Receptor-Signaling Molecule Vav1 Reduces Both Effector T Cell Functions and Susceptibility to Neuroinflammation

PubMed Central

Kassem, Sahar; Bernard, Isabelle; Dejean, Anne S.; Liblau, Roland; Fournié, Gilbert J.; Colacios, Céline

2016-01-01

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell antigen receptor signals and therefore plays an important role in T cell development and activation. Our previous genetic studies identified a locus on rat chromosome 9 that controls the susceptibility to neuroinflammation and contains a non-synonymous polymorphism in the major candidate gene Vav1. To formally demonstrate the causal implication of this polymorphism, we generated a knock-in mouse bearing this polymorphism (Vav1R63W). Using this model, we show that Vav1R63W mice display reduced susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide immunization. This is associated with a lower production of effector cytokines (IFN-γ, IL-17 and GM-CSF) by autoreactive CD4 T cells. Despite increased proportion of Foxp3+ regulatory T cells in Vav1R63W mice, we show that this lowered cytokine production is intrinsic to effector CD4 T cells and that Treg depletion has no impact on EAE development. Finally, we provide a mechanism for the above phenotype by showing that the Vav1R63W variant has normal enzymatic activity but reduced adaptor functions. Together, these data highlight the importance of Vav1 adaptor functions in the production of inflammatory cytokines by effector T cells and in the susceptibility to neuroinflammation. PMID:27438086
Functional characterization of three MicroRNAs of the Asian Tiger Mosquito, Aedes albopictus

PubMed Central

2013-01-01

Background Temporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, which are temporally up-regulated in the various developmental stages of Ae. albopictus mosquitoes. Methods miRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, hatching rate and hatching time in embryos, eclosion rate in larvae, longevity and fecundity in the adult mosquitoes. Results The knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, both by in vitro expression in C6/36 mosquito cell lines after transfection as well as in in vivo expression in developmental stages of mosquitoes by microinjection. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in Ae. albopictus development, detected by a decrease in the hatching rate of embryos and eclosion rate in larvae and a marked reduction in longevity and fecundity in adults. Conclusion This study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection is a novel study to delineate the importance of the miRNA expression in regulating mosquito development. The knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development and maintenance of the vectorial population density and pathogen transmissibility of this mosquito vector. PMID:23924583
The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism.

PubMed

Kleinberger, Gernot; Brendel, Matthias; Mracsko, Eva; Wefers, Benedikt; Groeneweg, Linda; Xiang, Xianyuan; Focke, Carola; Deußing, Maximilian; Suárez-Calvet, Marc; Mazaheri, Fargol; Parhizkar, Samira; Pettkus, Nadine; Wurst, Wolfgang; Feederle, Regina; Bartenstein, Peter; Mueggler, Thomas; Arzberger, Thomas; Knuesel, Irene; Rominger, Axel; Haass, Christian

2017-07-03

Genetic variants in the triggering receptor expressed on myeloid cells 2 (TREM2) increase the risk for several neurodegenerative diseases including Alzheimer's disease and frontotemporal dementia (FTD). Homozygous TREM2 missense mutations, such as p.T66M, lead to the FTD-like syndrome, but how they cause pathology is unknown. Using CRISPR/Cas9 genome editing, we generated a knock-in mouse model for the disease-associated Trem2 p.T66M mutation. Consistent with a loss-of-function mutation, we observe an intracellular accumulation of immature mutant Trem2 and reduced generation of soluble Trem2 similar to patients with the homozygous p.T66M mutation. Trem2 p.T66M knock-in mice show delayed resolution of inflammation upon in vivo lipopolysaccharide stimulation and cultured macrophages display significantly reduced phagocytic activity. Immunohistochemistry together with in vivo TSPO small animal positron emission tomography (μPET) demonstrates an age-dependent reduction in microglial activity. Surprisingly, perfusion magnetic resonance imaging and FDG-μPET imaging reveal a significant reduction in cerebral blood flow and brain glucose metabolism. Thus, we demonstrate that a TREM2 loss-of-function mutation causes brain-wide metabolic alterations pointing toward a possible function of microglia in regulating brain glucose metabolism. © 2017 The Authors.

BC RNA Mislocalization in the Fragile X Premutation.

PubMed

Muslimov, Ilham A; Eom, Taesun; Iacoangeli, Anna; Chuang, Shih-Chieh; Hukema, Renate K; Willemsen, Rob; Stefanov, Dimitre G; Wong, Robert K S; Tiedge, Henri

2018-01-01

Fragile X premutation disorder is caused by CGG triplet repeat expansions in the 5' untranslated region of FMR1 mRNA. The question of how expanded CGG repeats cause disease is a subject of continuing debate. Our work indicates that CGG-repeat structures compete with regulatory BC1 RNA for access to RNA transport factor hnRNP A2. As a result, BC1 RNA is mislocalized in vivo, as its synapto-dendritic presence is severely diminished in brains of CGG-repeat knock-in animals (a premutation mouse model). Lack of BC1 RNA is known to cause seizure activity and cognitive dysfunction. Our working hypothesis thus predicted that absence, or significantly reduced presence, of BC1 RNA in synapto-dendritic domains of premutation animal neurons would engender cognate phenotypic alterations. Testing this prediction, we established epileptogenic susceptibility and cognitive impairments as major phenotypic abnormalities of CGG premutation mice. In CA3 hippocampal neurons of such animals, synaptic release of glutamate elicits neuronal hyperexcitability in the form of group I metabotropic glutamate receptor-dependent prolonged epileptiform discharges. CGG-repeat knock-in animals are susceptible to sound-induced seizures and are cognitively impaired as revealed in the Attentional Set Shift Task. These phenotypic disturbances occur in young-adult premutation animals, indicating that a neurodevelopmental deficit is an early-initial manifestation of the disorder. The data are consistent with the notion that RNA mislocalization can contribute to pathogenesis.
Appropriate suppression of Notch signaling by Mesp factors is essential for stripe pattern formation leading to segment boundary formation.

PubMed

Takahashi, Yu; Yasuhiko, Yukuto; Kitajima, Satoshi; Kanno, Jun; Saga, Yumiko

2007-04-15

Mesp1 and Mesp2 are homologous transcription factors that are co-expressed in the anterior presomitic mesoderm (PSM) during mouse somitogenesis. The loss of Mesp2 alone in our conventional Mesp2-null mice results in the complete disruption of somitogenesis, including segment border formation, rostro-caudal patterning and epithelialization of somitic mesoderm. This has led us to interpret that Mesp2 is solely responsible for somitogenesis. Our novel Mesp2 knock-in alleles, however, exhibit a remarkable upregulation of Mesp1. Removal of the pgk-neo cassette from the new allele leads to localization of Mesp1 and several gene expression, and somite formation in the tail region. Moreover, a reduction in the gene dosage of Mesp1 by one copy disrupts somite formation, confirming the involvement of Mesp1 in the rescue events. Furthermore, we find that activated Notch1 knock-in significantly upregulates Mesp1 expression, even in the absence of a Notch signal mediator, Psen1. This indicates that the Psen1-independent effects of activated Notch1 are mostly attributable to the induction of Mesp1. However, we have also confirmed that Mesp2 enhances the expression of the Notch1 receptor in the anterior PSM. The activation and subsequent suppression of Notch signaling might thus be a crucial event for both stripe pattern formation and boundary formation.
Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation

PubMed Central

Li, Defang; Liu, Jin; Guo, Baosheng; Liang, Chao; Dang, Lei; Lu, Cheng; He, Xiaojuan; Cheung, Hilda Yeuk-Siu; Xu, Liang; Lu, Changwei; He, Bing; Liu, Biao; Shaikh, Atik Badshah; Li, Fangfei; Wang, Luyao; Yang, Zhijun; Au, Doris Wai-Ting; Peng, Songlin; Zhang, Zongkang; Zhang, Bao-Ting; Pan, Xiaohua; Qian, Airong; Shang, Peng; Xiao, Lianbo; Jiang, Baohong; Wong, Chris Kong-Chu; Xu, Jiake; Bian, Zhaoxiang; Liang, Zicai; Guo, De-an; Zhu, Hailong; Tan, Weihong; Lu, Aiping; Zhang, Ge

2016-01-01

Emerging evidence indicates that osteoclasts direct osteoblastic bone formation. MicroRNAs (miRNAs) have a crucial role in regulating osteoclast and osteoblast function. However, whether miRNAs mediate osteoclast-directed osteoblastic bone formation is mostly unknown. Here, we show that increased osteoclastic miR-214-3p associates with both elevated serum exosomal miR-214-3p and reduced bone formation in elderly women with fractures and in ovariectomized (OVX) mice. Osteoclast-specific miR-214-3p knock-in mice have elevated serum exosomal miR-214-3p and reduced bone formation that is rescued by osteoclast-targeted antagomir-214-3p treatment. We further demonstrate that osteoclast-derived exosomal miR-214-3p is transferred to osteoblasts to inhibit osteoblast activity in vitro and reduce bone formation in vivo. Moreover, osteoclast-targeted miR-214-3p inhibition promotes bone formation in ageing OVX mice. Collectively, our results suggest that osteoclast-derived exosomal miR-214-3p transfers to osteoblasts to inhibit bone formation. Inhibition of miR-214-3p in osteoclasts may be a strategy for treating skeletal disorders involving a reduction in bone formation. PMID:26947250
Vav1 GEF activity is required for T cell mediated allograft rejection

PubMed Central

Haubert, Dirk; Li, Jianping; Saveliev, Alexander; Calzascia, Thomas; Sutter, Esther; Metzler, Barbara; Kaiser, Daniel; Tybulewicz, Victor L.J.; Weckbecker, Gisbert

2012-01-01

The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Vav1 has been shown to transduce signals both dependent and independent of its GEF function. The most promising approach to disrupt Vav1 activity by pharmacological inhibition would be to target its GEF function. However, the contribution of Vav1 GEF activity for allogeneic T cell activation has not been clarified yet. To address this question, we used knock-in mice bearing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, lack of Vav1 GEF activity strongly abrogated the in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings demonstrate a strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone is sufficient to induce significant immunosuppression. PMID:22456277
High-fidelity Glucagon-CreER mouse line generated by CRISPR-Cas9 assisted gene targeting.

PubMed

Ackermann, Amanda M; Zhang, Jia; Heller, Aryel; Briker, Anna; Kaestner, Klaus H

2017-03-01

α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for β-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER "knock-in" mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreER T2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing. We utilized CRISPR-Cas9 technology to insert an IRES-CreER T2 sequence into the 3' UTR of the Glucagon ( Gcg ) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreER T2 mice. Recombination efficiency in GCG + pancreatic α-cells and glucagon-like peptide 1 positive (GLP1 + ) enteroendocrine L-cells was measured in Gcg-CreER T2 ; Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood. Tamoxifen injection of Gcg-CreER T2 ; Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively), as well as in first-wave fetal α-cells (36%) and adult enteroendocrine L-cells (33%). Mice homozygous for the Gcg-CreER T2 allele were phenotypically normal. We successfully derived a Gcg-CreER T2 mouse line that expresses CreER T2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types.
Hypertension from chronic central sodium chloride in mice is mediated by the ouabain-binding site on the Na,K-ATPase α2-isoform

PubMed Central

Dostanic, Iva; Lingrel, Jerry B.; Hou, Xiaohong; Wu, Hengwei

2011-01-01

A chronic increase in the concentration of sodium chloride in the cerebrospinal fluid (CSF) (↑CSF [NaCl]) appears to be critically important for the development of salt-dependent hypertension. In agreement with this concept, increasing CSF [NaCl] chronically by intracerebroventricular (icv) infusion of NaCl-rich artificial CSF (aCSF-HiNaCl) in rats produces hypertension by the same mechanisms (i.e., aldosterone-ouabain pathway in the brain) as that produced by dietary sodium in salt-sensitive strains. We first demonstrate here that icv aCSF-HiNaCl for 10 days also causes hypertension in wild-type (WT) mice. We then used both WT and gene-targeted mice to explore the mechanisms. In WT mice with a ouabain-sensitive Na,K-ATPase α2-isoform (α2S/S), mean arterial pressure rose by ∼25 mmHg within 2 days of starting aCSF-HiNaCl (0.6 nmol Na/min) and remained elevated throughout the study. Ouabain (171 pmol/day icv) increased blood pressure to a similar extent. aCSF-HiNaCl or ouabain given at the same rates subcutaneously instead of intracerebroventricularly had no effect on blood pressure. The pressor response to icv aCSF-HiNaCl was abolished by an anti-ouabain antibody given intracerebroventricularly but not subcutaneously, indicating that it is mediated by an endogenous ouabain-like substance in the brain. We compared the effects of icv aCSF-HiNaCl or icv ouabain on blood pressure in α2S/S versus knockout/knockin mice with a ouabain-resistant endogenous α2-subunit (α2R/R). In α2R/R, there was no pressor response to icv aCSF-HiNaCl in contrast to WT mice. The α2R/R genotype also lacked a pressor response to icv ouabain. These data demonstrate that chronic ↑CSF [NaCl] causes hypertension in mice and that the blood pressure response is mediated by the ouabain-like substance in the brain, specifically by its binding to the α2-isoform of the Na,K-ATPase. PMID:21856907
Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes.

PubMed

Koehler, Sybille; Brähler, Sebastian; Braun, Fabian; Hagmann, Henning; Rinschen, Markus M; Späth, Martin R; Höhne, Martin; Wunderlich, F Thomas; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T

2017-06-01

Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type -mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2 fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
A Balanced Diet Is Necessary for Proper Entrainment Signals of the Mouse Liver Clock

PubMed Central

Hirao, Akiko; Tahara, Yu; Kimura, Ichiro; Shibata, Shigenobu

2009-01-01

Background The peripheral circadian clock in mice is entrained not only by light-dark cycles but also by daily restricted feeding schedules. Behavioral and cell culture experiments suggest an increase in glucose level as a factor in such feeding-induced entrainment. For application of feeding-induced entrainment in humans, nutrient content and dietary variations should be considered. Principal Finding To elucidate the food composition necessary for dietary entrainment, we examined whether complete or partial substitution of dietary nutrients affected phase shifts in liver clocks of mice. Compared with fasting mice or ad libitum fed mice, the liver bioluminescence rhythm advanced by 3–4 h on the middle day in Per2::luciferase knock-in mice that were administered a standard mouse diet, i.e. AIN-93M formula [0.6–0.85 g/10 g mouse BW] (composition: 14% casein, 47% cornstarch, 15% gelatinized cornstarch, 10% sugar, 4% soybean oil, and 10% other [fiber, vitamins, minerals, etc.]), for 2 days. When each nutrient was tested alone (100% nutrient), an insignificant weak phase advance was found to be induced by cornstarch and soybean oil, but almost no phase advance was induced by gelatinized cornstarch, high-amylose cornstarch, glucose, sucrose, or casein. A combination of glucose and casein without oil, vitamin, or fiber caused a significant phase advance. When cornstarch in AIN-93M was substituted with glucose, sucrose, fructose, polydextrose, high-amylose cornstarch, or gelatinized cornstarch, the amplitude of phase advance paralleled the increase in blood glucose concentration. Conclusions Our results strongly suggest the following: (1) balanced diets containing carbohydrates/sugars and proteins are good for restricted feeding-induced entrainment of the peripheral circadian clock and (2) a balanced diet that increases blood glucose, but not by sugar alone, is suitable for entrainment. These findings may assist in the development of dietary recommendations for on-board meals served to air travelers and shift workers to reduce jet lag-like symptoms. PMID:19738906
Serine 302 Phosphorylation of Mouse Insulin Receptor Substrate 1 (IRS1) Is Dispensable for Normal Insulin Signaling and Feedback Regulation by Hepatic S6 Kinase*

PubMed Central

Copps, Kyle D.; Hançer, Nancy J.; Qiu, Wei; White, Morris F.

2016-01-01

Constitutive activation of the mammalian target of rapamycin complex 1 and S6 kinase (mTORC1→ S6K) attenuates insulin-stimulated Akt activity in certain tumors in part through “feedback” phosphorylation of the upstream insulin receptor substrate 1 (IRS1). However, the significance of this mechanism for regulating insulin sensitivity in normal tissue remains unclear. We investigated the function of Ser-302 in mouse IRS1, the major site of its phosphorylation by S6K in vitro, through genetic knock-in of a serine-to-alanine mutation (A302). Although insulin rapidly stimulated feedback phosphorylation of Ser-302 in mouse liver and muscle, homozygous A302 mice (A/A) and their knock-in controls (S/S) exhibited similar glucose homeostasis and muscle insulin signaling. Furthermore, both A302 and control primary hepatocytes from which Irs2 was deleted showed marked inhibition of insulin-stimulated IRS1 tyrosine phosphorylation and PI3K binding after emetine treatment to raise intracellular amino acids and activate mTORC1 → S6K signaling. To specifically activate mTORC1 in mouse tissue, we deleted hepatic Tsc1 using Cre adenovirus. Although it moderately decreased IRS1/PI3K association and Akt phosphorylation in liver, Tsc1 deletion failed to cause glucose intolerance or promote hyperinsulinemia in mixed background A/A or S/S mice. Moreover, Tsc1 deletion failed to stimulate phospho-Ser-302 or other putative S6K sites within IRS1, whereas ribosomal S6 protein was constitutively phosphorylated. Following acute Tsc1 deletion from hepatocytes, Akt phosphorylation, but not IRS1/PI3K association, was rapidly restored by treatment with the mTORC1 inhibitor rapamycin. Thus, within the hepatic compartment, mTORC1 → S6K signaling regulates Akt largely through IRS-independent means with little effect upon physiologic insulin sensitivity. PMID:26846849
Measuring discrimination- and reversal learning in mouse models within 4 days and without prior food deprivation

PubMed Central

Smit, August B.; Verhage, Matthijs

2016-01-01

Many neurological and psychiatric disorders are characterized by deficits in cognitive flexibility. Modeling cognitive flexibility in mice enables the investigation of mechanisms underlying these deficits. The majority of currently available behavioral tests targeting this cognitive domain are reversal learning tasks that require scheduled food restriction, extended training periods and labor-intensive, and stress-inducing animal handling. Here, we describe a novel 4-day (4-d) continuously running task measuring discrimination- and reversal learning in an automated home cage (CognitionWall DL/RL task) that largely eliminates these limitations. In this task, mice can earn unlimited number of food rewards by passing through the correct hole of the three-holed CognitionWall. To assess the validity and sensitivity of this novel task, the performance of C57BL/6J mice, amyloid precursor protein/presenilin1 transgenic (APP/PS1) mice, α-calmodulin kinase-II (αCaMKII) T305D knock-in mice, and mice with an orbitofrontal cortex lesion were examined. We found that C57BL/6J mice reach stable performance levels within the 4 d of the task, while experiencing only slight reductions in weight and no major effects on circadian rhythm. The task detected learning deficits in APP/PS1 transgenic and αCaMKII T305D mutant mice. Additionally, we established that the orbitofrontal cortex underlies reversal learning performance in our task. Because of its short duration and the absence of food deprivation and concurrent weight loss, this novel automated home-cage task substantially improves comprehensive preclinical assessment of cognitive functions in mouse models of psychiatric and neurological disorders and also enables analysis during specific developmental stages. PMID:27918287
Mast cells mediate the immune suppression induced by dermal exposure to JP-8 jet fuel.

PubMed

Limón-Flores, Alberto Y; Chacón-Salinas, Rommel; Ramos, Gerardo; Ullrich, Stephen E

2009-11-01

Applying jet propulsion-8 (JP-8) jet fuel to the skin of mice induces immune suppression. Applying JP-8 to the skin of mice suppresses T-cell-mediated immune reactions including, contact hypersensitivity (CHS) delayed-type hypersensitivity and T-cell proliferation. Because dermal mast cells play an important immune regulatory role in vivo, we tested the hypothesis that mast cells mediate jet fuel-induced immune suppression. When we applied JP-8 to the skin of mast cell deficient mice CHS was not suppressed. Reconstituting mast cell deficient mice with wild-type bone marrow derived mast cells (mast cell "knock-in mice") restored JP-8-induced immune suppression. When, however, mast cells from prostaglandin E(2) (PGE(2))-deficient mice were used, the ability of JP-8 to suppress CHS was not restored, indicating that mast cell-derived PGE(2) was activating immune suppression. Examining the density of mast cells in the skin and lymph nodes of JP-8-treated mice indicated that jet fuel treatment caused an initial increase in mast cell density in the skin, followed by increased numbers of mast cells in the subcutaneous space and then in draining lymph nodes. Applying JP-8 to the skin increased mast cell expression of CXCR4, and increased the expression of CXCL12 by draining lymph node cells. Because CXCL12 is a chemoattractant for CXCR4+ mast cells, we treated JP-8-treated mice with AMD3100, a CXCR4 antagonist. AMD3100 blocked the mobilization of mast cells to the draining lymph node and inhibited JP-8-induced immune suppression. Our findings demonstrate the importance of mast cells in mediating jet fuel-induced immune suppression.
Measuring discrimination- and reversal learning in mouse models within 4 days and without prior food deprivation.

PubMed

Remmelink, Esther; Smit, August B; Verhage, Matthijs; Loos, Maarten

2016-11-01

Many neurological and psychiatric disorders are characterized by deficits in cognitive flexibility. Modeling cognitive flexibility in mice enables the investigation of mechanisms underlying these deficits. The majority of currently available behavioral tests targeting this cognitive domain are reversal learning tasks that require scheduled food restriction, extended training periods and labor-intensive, and stress-inducing animal handling. Here, we describe a novel 4-day (4-d) continuously running task measuring discrimination- and reversal learning in an automated home cage (CognitionWall DL/RL task) that largely eliminates these limitations. In this task, mice can earn unlimited number of food rewards by passing through the correct hole of the three-holed CognitionWall. To assess the validity and sensitivity of this novel task, the performance of C57BL/6J mice, amyloid precursor protein/presenilin1 transgenic (APP/PS1) mice, α-calmodulin kinase-II (αCaMKII) T305D knock-in mice, and mice with an orbitofrontal cortex lesion were examined. We found that C57BL/6J mice reach stable performance levels within the 4 d of the task, while experiencing only slight reductions in weight and no major effects on circadian rhythm. The task detected learning deficits in APP/PS1 transgenic and αCaMKII T305D mutant mice. Additionally, we established that the orbitofrontal cortex underlies reversal learning performance in our task. Because of its short duration and the absence of food deprivation and concurrent weight loss, this novel automated home-cage task substantially improves comprehensive preclinical assessment of cognitive functions in mouse models of psychiatric and neurological disorders and also enables analysis during specific developmental stages. © 2016 Remmelink et al.; Published by Cold Spring Harbor Laboratory Press.
Gelsolin: a novel thyroid hormone receptor-beta interacting protein that modulates tumor progression in a mouse model of follicular thyroid cancer.

PubMed

Kim, Caroline S; Furuya, Fumihiko; Ying, Hao; Kato, Yasuhito; Hanover, John A; Cheng, Sheue-yann

2007-03-01

Follicular thyroid cancer (FTC) is known to metastasize to distant sites via hematogenous spread; however, the underlying pathways that contribute to metastasis remain unknown. Recent creation of a knockin mutant mouse that expresses a mutant thyroid hormone receptor-beta (TRbeta(PV/PV) mouse) that spontaneously develops thyroid cancer with metastasis similar to humans has provided new opportunities to study contributors to FTC metastasis. This study evaluates the role of gelsolin, an actin-regulatory protein, in modulating the metastatic potential of FTC. Gelsolin was previously found by cDNA microarray analysis to be down-regulated in TRbeta(PV/PV) mice as compared with wild-type mice. This study found an age-dependent reduction of gelsolin protein abundance in TRbeta(PV/PV) mice as tumorigenesis progressed. Knockdown of gelsolin by small interfering RNA resulted in increased tumor cell motility and increased gelsolin expression by histone deacetylase inhibitor (trichostatin A) led to decreased cell motility. Additional biochemical analyses demonstrated that gelsolin physically interacted with TRbeta1 or PV in vivo and in vitro. The interaction regions were mapped to the C terminus of gelsolin and the DNA binding domain of TR. The physical interaction of gelsolin with PV reduced its binding to actin, leading to disarrayed cytoskeletal architectures. These results suggest that PV-induced alteration of the actin/gelsolin cytoskeleton contributes to increased cell motility. Thus, the present study uncovered a novel PV-mediated oncogenic pathway that could contribute to the local tumor progression and metastatic potential of thyroid carcinogenesis.
Model of pediatric pituitary hormone deficiency separates the endocrine and neural functions of the LHX3 transcription factor in vivo.

PubMed

Colvin, Stephanie C; Malik, Raleigh E; Showalter, Aaron D; Sloop, Kyle W; Rhodes, Simon J

2011-01-04

The etiology of most pediatric hormone deficiency diseases is poorly understood. Children with combined pituitary hormone deficiency (CPHD) have insufficient levels of multiple anterior pituitary hormones causing short stature, metabolic disease, pubertal failure, and often have associated nervous system symptoms. Mutations in developmental regulatory genes required for the specification of the hormone-secreting cell types of the pituitary gland underlie severe forms of CPHD. To better understand these diseases, we have created a unique mouse model of CPHD with a targeted knockin mutation (Lhx3 W227ter), which is a model for the human LHX3 W224ter disease. The LHX3 gene encodes a LIM-homeodomain transcription factor, which has essential roles in pituitary and nervous system development in mammals. The introduced premature termination codon results in deletion of the carboxyl terminal region of the LHX3 protein, which is critical for pituitary gene activation. Mice that lack all LHX3 function do not survive beyond birth. By contrast, the homozygous Lhx3 W227ter mice survive, but display marked dwarfism, thyroid disease, and female infertility. Importantly, the Lhx3 W227ter mice have no apparent nervous system deficits. The Lhx3 W227ter mouse model provides a unique array of hormone deficits and facilitates experimental approaches that are not feasible with human patients. These experiments demonstrate that the carboxyl terminus of the LHX3 transcription factor is not required for viability. More broadly, this study reveals that the in vivo actions of a transcription factor in different tissues are molecularly separable.
Disruption of Protein Processing in the Endoplasmic Reticulum of DYT1 Knock-in Mice Implicates Novel Pathways in Dystonia Pathogenesis.

PubMed

Beauvais, Genevieve; Bode, Nicole M; Watson, Jaime L; Wen, Hsiang; Glenn, Kevin A; Kawano, Hiroyuki; Harata, N Charles; Ehrlich, Michelle E; Gonzalez-Alegre, Pedro

2016-10-05

Dystonia type 1 (DYT1) is a dominantly inherited neurological disease caused by mutations in TOR1A, the gene encoding the endoplasmic reticulum (ER)-resident protein torsinA. Previous work mostly completed in cell-based systems suggests that mutant torsinA alters protein processing in the secretory pathway. We hypothesized that inducing ER stress in the mammalian brain in vivo would trigger or exacerbate mutant torsinA-induced dysfunction. To test this hypothesis, we crossed DYT1 knock-in with p58(IPK)-null mice. The ER co-chaperone p58(IPK) interacts with BiP and assists in protein maturation by helping to fold ER cargo. Its deletion increases the cellular sensitivity to ER stress. We found a lower generation of DYT1 knock-in/p58 knock-out mice than expected from this cross, suggesting a developmental interaction that influences viability. However, surviving animals did not exhibit abnormal motor function. Analysis of brain tissue uncovered dysregulation of eiF2α and Akt/mTOR translational control pathways in the DYT1 brain, a finding confirmed in a second rodent model and in human brain. Finally, an unbiased proteomic analysis identified relevant changes in the neuronal protein landscape suggesting abnormal ER protein metabolism and calcium dysregulation. Functional studies confirmed the interaction between the DYT1 genotype and neuronal calcium dynamics. Overall, these findings advance our knowledge on dystonia, linking translational control pathways and calcium physiology to dystonia pathogenesis and identifying potential new pharmacological targets. Dystonia type 1 (DYT1) is one of the different forms of inherited dystonia, a neurological disorder characterized by involuntary, disabling movements. DYT1 is caused by mutations in the gene that encodes the endoplasmic reticulum (ER)-resident protein torsinA. How mutant torsinA causes neuronal dysfunction remains unknown. Here, we show the behavioral and molecular consequences of stressing the ER in DYT1 mice by increasing the amount of misfolded proteins. This resulted in the generation of a reduced number of animals, evidence of abnormal ER protein processing and dysregulation of translational control pathways. The work described here proposes a shared mechanism for different forms of dystonia, links for the first time known biological pathways to dystonia pathogenesis, and uncovers potential pharmacological targets for its treatment. Copyright © 2016 the authors 0270-6474/16/3610245-12$15.00/0.
Mesodermal expression of integrin α5β1 regulates neural crest development and cardiovascular morphogenesis

PubMed Central

Liang, Dong; Wang, Xia; Mittal, Ashok; Dhiman, Sonam; Hou, Shuan-Yu; Degenhardt, Karl; Astrof, Sophie

2014-01-01

Integrin α5-null embryos die in mid-gestation from severe defects in cardiovascular morphogenesis, which stem from defective development of the neural crest, heart and vasculature. To investigate the role of integrin α5β1 in cardiovascular development, we used the Mesp1Cre knock-in strain of mice to ablate integrin α5 in the anterior mesoderm, which gives rise to all of the cardiac and many of the vascular and muscle lineages in the anterior portion of the embryo. Surprisingly, we found that mutant embryos displayed numerous defects related to the abnormal development of the neural crest such as cleft palate, ventricular septal defect, abnormal development of hypoglossal nerves, and defective remodeling of the aortic arch arteries. We found that defects in arch artery remodeling stem from the role of mesodermal integrin α5β1 in neural crest proliferation and differentiation into vascular smooth muscle cells, while proliferation of pharyngeal mesoderm and differentiation of mesodermal derivatives into vascular smooth muscle cells was not defective. Taken together our studies demonstrate a requisite role for mesodermal integrin α5β1 in signaling between the mesoderm and the neural crest, thereby regulating neural crest-dependent morphogenesis of essential embryonic structures. PMID:25242040
Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV.

PubMed

Xiao, Qing; Min, Taishan; Ma, Shuangping; Hu, Lingna; Chen, Hongyan; Lu, Daru

2018-04-18

Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis. Combing Cas9 plasmids targeting genome and ITR with AAV donor delivery, the precise knock-in of gene cassette was achieved, with 13-14% of the donor insertion events being mediated by MMEJ in HEK 293T cells. This study describes a novel method to integrate large single-strand transgene cassettes into the genomes, increasing knock-in efficiency by 13.6-19.5-fold relative to conventional AAV-mediated method. It also provides a comprehensive solution to the challenges of complicated production and difficult delivery with large exogenous fragments.
Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts.

PubMed

Xie, Zicong; Pang, Daxin; Wang, Kankan; Li, Mengjing; Guo, Nannan; Yuan, Hongming; Li, Jianing; Zou, Xiaodong; Jiao, Huping; Ouyang, Hongsheng; Li, Zhanjun; Tang, Xiaochun

2017-06-08

Genetically modified pigs have important roles in agriculture and biomedicine. However, genome-specific knock-in techniques in pigs are still in their infancy and optimal strategies have not been extensively investigated. In this study, we performed electroporation to introduce a targeting donor vector (a non-linearized vector that did not contain a promoter or selectable marker) into Porcine Foetal Fibroblasts (PFFs) along with a CRISPR/Cas9 vector. After optimization, the efficiency of the EGFP site-specific knock-in could reach up to 29.6% at the pRosa26 locus in PFFs. Next, we used the EGFP reporter PFFs to address two key conditions in the process of achieving transgenic pigs, the limiting dilution method and the strategy to evaluate the safety and feasibility of the knock-in locus. This study demonstrates that we establish an efficient procedures for the exogenous gene knock-in technique and creates a platform to efficiently generate promoter-less and selectable marker-free transgenic PFFs through the CRISPR/Cas9 system. This study should contribute to the generation of promoter-less and selectable marker-free transgenic pigs and it may provide insights into sophisticated site-specific genome engineering techniques for additional species.
Development of a one-step gene knock-out and knock-in method for metabolic engineering of Aureobasidium pullulans.

PubMed

Guo, Jian; Wang, Yuanhua; Li, Baozhong; Huang, Siyao; Chen, Yefu; Guo, Xuewu; Xiao, Dongguang

2017-06-10

Aureobasidium pullulans is an increasingly attractive host for bio-production of pullulan, heavy oil, polymalic acid, and a large spectrum of extracellular enzymes. To date, genetic manipulation of A. pullulans mainly relies on time-consuming conventional restriction enzyme digestion and ligation methods. In this study, we present a one-step homologous recombination-based method for rapid genetic manipulation in A. pullulans. Overlaps measuring >40bp length and 10μg DNA segments for homologous recombination provided maximum benefits to transformation of A. pullulans. This optimized method was successfully applied to PKSIII gene (encodes polyketide synthase) knock-out and gltP gene (encodes glycolipid transfer protein) knock-in. After disruption of PKSIII gene, secretion of melanin decreased slightly. The melanin purified from disruptant showed lower reducing capacity compared with that of the parent strain, leading to a decrease in exopolysaccharide production. Knock-in of gltP gene resulted in at least 4.68-fold increase in heavy oil production depending on the carbon source used, indicating that gltP can regulate heavy oil synthesis in A. pullulans. Copyright © 2017 Elsevier B.V. All rights reserved.
A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation.

PubMed

Adam, Frédéric; Casari, Caterina; Prévost, Nicolas; Kauskot, Alexandre; Loubière, Cécile; Legendre, Paulette; Repérant, Christelle; Baruch, Dominique; Rosa, Jean-Philippe; Bryckaert, Marijke; de Groot, Philip G; Christophe, Olivier D; Lenting, Peter J; Denis, Cécile V

2016-05-23

von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.

The Matricellular Protein CCN1 Promotes Mucosal Healing in Murine Colitis through IL-6

PubMed Central

Choi, Jacob S.; Kim, Ki-Hyun; Lau, Lester F.

2015-01-01

The matricellular protein CCN1 (CYR61) is known to function in wound healing and is upregulated in colons of patients with Crohn’s disease and ulcerative colitis, yet its specific role in colitis is unknown. Here we have used Ccn1dm/dm knockin mice expressing a CCN1 mutant unable to bind integrins α6β1 and αMβ2 as a model to probe CCN1 function in dextran sodium sulfate (DSS)-induced colitis. Ccn1dm/dm mice exhibited high mortality, impaired mucosal healing, and diminished IL-6 expression during the repair phase of DSS-induced colitis compared to wild type mice, despite having comparable severity of initial inflammation and tissue injury. CCN1 induced IL-6 expression in macrophages through integrin αMβ2 and in fibroblasts through α6β1, and IL-6 promoted intestinal epithelial cell (IEC) proliferation. Administration of purified CCN1 protein fully rescued Ccn1dm/dm mice from DSS-induced mortality, restored IEC proliferation and enhanced mucosal healing, whereas delivery of IL-6 partially rectified these defects. CCN1 therapy accelerated mucosal healing and recovery from DSS-induced colitis even in wild type mice. These findings reveal a critical role for CCN1 in restoring mucosal homeostasis after intestinal injury in part through integrin-mediated induction of IL-6 expression, and suggest a therapeutic potential for activating the CCN1/IL-6 axis for treating inflammatory bowel disease. PMID:25807183
Voluntary scheduled exercise alters diurnal rhythms of behaviour, physiology and gene expression in wild-type and vasoactive intestinal peptide-deficient mice

PubMed Central

Schroeder, Analyne M; Truong, Danny; Loh, Dawn H; Jordan, Maria C; Roos, Kenneth P; Colwell, Christopher S

2012-01-01

The circadian system co-ordinates the temporal patterning of behaviour and many underlying biological processes. In some cases, the regulated outputs of the circadian system, such as activity, may be able to feed back to alter core clock processes. In our studies, we used four wheel-access conditions (no access; free access; early night; and late night) to manipulate the duration and timing of activity while under the influence of a light–dark cycle. In wild-type mice, scheduled wheel access was able to increase ambulatory activity, inducing a level of exercise driven at various phases of the light–dark cycle. Scheduled exercise also manipulated the magnitude and phasing of the circadian-regulated outputs of heart rate and body temperature. At a molecular level, the phasing and amplitude of PER2::LUCIFERASE (PER2::LUC) expression rhythms in the SCN and peripheral tissues of Per2::Luc knockin mice were altered by scheduled exercise. We then tested whether scheduled wheel access could improve deficits observed in vasointestinal polypeptide-deficient mice under the influence of a light–dark cycle. We found that scheduled wheel access during the late night improved many of the behavioural, physiological and molecular deficits previously described in vasointestinal polypeptide-deficient mice. Our results raise the possibility that scheduled exercise could be used as a tool to modulate daily rhythms and, when applied, may counteract some of the negative impacts of ageing and disease on the circadian system. PMID:22988135
Maternal transcription of non-protein coding RNAs from the PWS-critical region rescues growth retardation in mice

PubMed Central

Rozhdestvensky, Timofey S.; Robeck, Thomas; Galiveti, Chenna R.; Raabe, Carsten A.; Seeger, Birte; Wolters, Anna; Gubar, Leonid V.; Brosius, Jürgen; Skryabin, Boris V.

2016-01-01

Prader-Willi syndrome (PWS) is a neurogenetic disorder caused by loss of paternally expressed genes on chromosome 15q11-q13. The PWS-critical region (PWScr) contains an array of non-protein coding IPW-A exons hosting intronic SNORD116 snoRNA genes. Deletion of PWScr is associated with PWS in humans and growth retardation in mice exhibiting ~15% postnatal lethality in C57BL/6 background. Here we analysed a knock-in mouse containing a 5′HPRT-LoxP-NeoR cassette (5′LoxP) inserted upstream of the PWScr. When the insertion was inherited maternally in a paternal PWScr-deletion mouse model (PWScrp−/m5′LoxP), we observed compensation of growth retardation and postnatal lethality. Genomic methylation pattern and expression of protein-coding genes remained unaltered at the PWS-locus of PWScrp−/m5′LoxP mice. Interestingly, ubiquitous Snord116 and IPW-A exon transcription from the originally silent maternal chromosome was detected. In situ hybridization indicated that PWScrp−/m5′LoxP mice expressed Snord116 in brain areas similar to wild type animals. Our results suggest that the lack of PWScr RNA expression in certain brain areas could be a primary cause of the growth retardation phenotype in mice. We propose that activation of disease-associated genes on imprinted regions could lead to general therapeutic strategies in associated diseases. PMID:26848093
The unique and cooperative roles of the Grainy head-like transcription factors in epidermal development reflect unexpected target gene specificity.

PubMed

Boglev, Yeliz; Wilanowski, Tomasz; Caddy, Jacinta; Parekh, Vishwas; Auden, Alana; Darido, Charbel; Hislop, Nikki R; Cangkrama, Michael; Ting, Stephen B; Jane, Stephen M

2011-01-15

The Grainy head-like 3 (Grhl3) gene encodes a transcription factor that plays essential roles in epidermal morphogenesis during embryonic development, with deficient mice exhibiting failed skin barrier formation, defective wound repair, and loss of eyelid fusion. Despite sharing significant sequence homology, overlapping expression patterns, and an identical core consensus DNA binding site, the other members of the Grhl family (Grhl1 and -2) fail to compensate for the loss of Grhl3 in these processes. Here, we have employed diverse genetic models, coupled with biochemical studies, to define the inter-relationships of the Grhl factors in epidermal development. We show that Grhl1 and Grhl3 have evolved complete functional independence, as evidenced by a lack of genetic interactions in embryos carrying combinations of targeted alleles of these genes. In contrast, compound heterozygous Grhl2/Grhl3 embryos displayed failed wound repair, and loss of a single Grhl2 allele in Grhl3-null embryos results in fully penetrant eyes open at birth. Expression of Grhl2 from the Grhl3 locus in homozygous knock-in mice corrects the wound repair defect, but these embryos still display a complete failure of skin barrier formation. This functional dissociation is due to unexpected differences in target gene specificity, as both GRHL2 and GRHL3 bind to and regulate expression of the wound repair gene Rho GEF 19, but regulation of the barrier forming gene, Transglutaminase 1 (TGase1), is unique to GRHL3. Our findings define the mechanisms underpinning the unique and cooperative roles of the Grhl genes in epidermal development. Copyright © 2010 Elsevier Inc. All rights reserved.
Androgen receptor polyglutamine expansion drives age-dependent quality control defects and muscle dysfunction.

PubMed

Nath, Samir R; Yu, Zhigang; Gipson, Theresa A; Marsh, Gregory B; Yoshidome, Eriko; Robins, Diane M; Todi, Sokol V; Housman, David E; Lieberman, Andrew P

2018-05-29

Skeletal muscle has emerged as a critical, disease-relevant target tissue in spinal and bulbar muscular atrophy, a degenerative disorder of the neuromuscular system caused by a CAG/polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. Here, we used RNA-Seq to identify pathways that are disrupted in diseased muscle using AR113Q knock-in mice. This analysis unexpectedly identified significantly diminished expression of numerous ubiquitin-proteasome pathway genes in AR113Q muscle, encoding approximately 30% of proteasome subunits and 20% of E2 ubiquitin conjugases. These changes were age-, hormone- and glutamine length-dependent and arose due to a toxic gain-of-function conferred by the mutation. Moreover, altered gene expression was associated with decreased level of the proteasome transcription factor NRF1 and its activator DDI2 and resulted in diminished proteasome activity. Ubiquitinated ADRM1 was detected in AR113Q muscle, indicating the occurrence of stalled proteasomes in mutant mice. Finally, diminished expression of Drosophila orthologues of NRF1 or ADRM1 promoted the accumulation of polyQ AR protein and increased toxicity. Collectively, these data indicate that AR113Q muscle develops progressive proteasome dysfunction that leads to the impairment of quality control and the accumulation of polyQ AR protein, key features that contribute to the age-dependent onset and progression of this disorder.
A mouse model for a partially inactive obesity-associated human MC3R variant

PubMed Central

Lee, Bonggi; Koo, Jashin; Yun Jun, Joo; Gavrilova, Oksana; Lee, Yongjun; Seo, Arnold Y.; Taylor-Douglas, Dezmond C.; Adler-Wailes, Diane C.; Chen, Faye; Gardner, Ryan; Koutzoumis, Dimitri; Sherafat Kazemzadeh, Roya; Roberson, Robin B.; Yanovski, Jack A.

2016-01-01

We previously reported children homozygous for two MC3R sequence variants (C17A+G241A) have greater fat mass than controls. Here we show, using homozygous knock-in mouse models in which we replace murine Mc3r with wild-type human (MC3RhWT/hWT) and double-mutant (C17A+G241A) human (MC3RhDM/hDM) MC3R, that MC3RhDM/hDM have greater weight and fat mass, increased energy intake and feeding efficiency, but reduced length and fat-free mass compared with MC3RhWT/hWT. MC3RhDM/hDM mice do not have increased adipose tissue inflammatory cell infiltration or greater expression of inflammatory markers despite their greater fat mass. Serum adiponectin levels are increased in MC3RhDM/hDM mice and MC3RhDM/hDM human subjects. MC3RhDM/hDM bone- and adipose tissue-derived mesenchymal stem cells (MSCs) differentiate into adipocytes that accumulate more triglyceride than MC3RhWT/hWT MSCs. MC3RhDM/hDM impacts nutrient partitioning to generate increased adipose tissue that appears metabolically healthy. These data confirm the importance of MC3R signalling in human metabolism and suggest a previously-unrecognized role for the MC3R in adipose tissue development. PMID:26818770
Inactivation of the F4/80 glycoprotein in the mouse germ line.

PubMed

Schaller, Evelyne; Macfarlane, Alison J; Rupec, Rudolf A; Gordon, Siamon; McKnight, Andrew J; Pfeffer, Klaus

2002-11-01

Macrophages play a crucial role in the defense against pathogens. Distinct macrophage populations can be defined by the expression of restricted cell surface proteins. Resident tissue macrophages, encompassing Kupffer cells of the liver and red pulp macrophages of the spleen, characteristically express the F4/80 molecule, a cell surface glycoprotein related to the seven transmembrane-spanning family of hormone receptors. In this study, gene targeting was used to simultaneously inactivate the F4/80 molecule in the germ line of the mouse and to produce a mouse line that expresses the Cre recombinase under the direct control of the F4/80 promoter (F4/80-Cre knock-in). F4/80-deficient mice are healthy and fertile. Macrophage populations in tissues can develop in the absence of F4/80 expression. Functional analysis revealed that the generation of T-cell-independent B-cell responses and macrophage antimicrobial defense after infection with Listeria monocytogenes are not impaired in the absence of F4/80. Interestingly, tissues of F4/80-deficient mice could not be labeled with anti-BM8, another macrophage subset-specific marker with hitherto undefined molecular antigenic structure. Recombinant expression of a F4/80 cDNA in heterologous cells confirmed this observation, indicating that the targets recognized by the F4/80 and BM8 monoclonal antibodies are identical.
Characterization of Oxygen-Induced Retinopathy in Mice Carrying an Inactivating Point Mutation in the Catalytic Site of ADAM15

PubMed Central

Maretzky, Thorsten; Blobel, Carl P.; Guaiquil, Victor

2014-01-01

Purpose. Retinal neovascularization is found in diseases such as macular degeneration, diabetic retinopathy, or retinopathy of prematurity and is usually caused by alterations in oxygen supply. We have previously described that mice lacking the membrane-anchored metalloproteinase ADAM15 (a Disintegrin and Metalloprotease 15) have decreased pathological neovascularization of the retina in the oxygen-induced retinopathy (OIR) model. The main purpose of the present study was to determine the contribution of the catalytic activity of ADAM15 to OIR. Methods. To address this question, we generated knock-in mice carrying an inactivating Glutamate to Alanine (E>A) point mutation in the catalytic site of ADAM15 (Adam15E>A mice) and subjected these animals to the OIR model and a heterotopic tumor model. Moreover, we used cell-based assays to determine whether ADAM15 can process cell surface receptors involved in angiogenesis. Results. We found that pathological neovascularization in the OIR model in Adam15E>A mice was comparable to that observed in wild type mice, but tumor implantation by heterotopically injected melanoma cells was reduced. In cell-based assays, overexpressed ADAM15 could process the FGFR2iiib, but was unable to process several receptors with roles in angiogenesis. Conclusions. Collectively, these results suggest that the catalytic activity of ADAM15 is not crucial for its function in promoting pathological neovascularization in the mouse OIR model, most likely because of the very limited substrate repertoire of ADAM15. Instead, other noncatalytic functions of ADAM15 must be important for its role in the OIR model. PMID:25249606
Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.

PubMed

Xue, Zhaoyu; Ren, Mengda; Wu, Menghua; Dai, Junbiao; Rong, Yikang S; Gao, Guanjun

2014-03-21

Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor. Copyright © 2014 Xue et al.
Cloning-free CRISPR

PubMed Central

Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I.

2015-01-01

Summary We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis. PMID:26527385
A low-cost, high-quality new drug discovery process using patient-derived induced pluripotent stem cells.

PubMed

Giri, Shibashish; Bader, Augustinus

2015-01-01

Knockout, knock-in and conditional mutant gene-targeted mice are routinely used for disease modeling in the drug discovery process, but the human response is often difficult to predict from these models. It is believed that patient-derived induced pluripotent stem cells (iPSCs) could replace millions of animals currently sacrificed in preclinical testing and provide a route to new safer pharmaceutical products. In this review, we discuss the use of IPSCs in the drug discovery process. We highlight how they can be used to assess the toxicity and clinical efficacy of drug candidates before the latter are moved into costly and lengthy preclinical and clinical trials. Copyright © 2014 Elsevier Ltd. All rights reserved.
Abnormal neurofilament inclusions and segregations in dorsal root ganglia of a Charcot-Marie-Tooth type 2E mouse model.

PubMed

Zhao, Jian; Brown, Kristy; Liem, Ronald K H

2017-01-01

Charcot-Marie-Tooth (CMT) disease or hereditary motor and sensory neuropathy is the most prevalent inherited peripheral neuropathy and is associated with over 90 causative genes. Mutations in neurofilament light polypeptide gene, NEFL cause CMT2E, an axonal form of CMT that results in abnormal structures and/or functions of peripheral axons in spinal cord motor neurons and dorsal root ganglion neurons. We have previously generated and characterized a knock-in mouse model of CMT2E with the N98S mutation in Nefl that presented with multiple inclusions in spinal cord neurons. In this report, we conduct immunofluorescence studies of cultured dorsal root ganglia (DRG) from NeflN98S/+ mice, and show that inclusions found in DRG neurites can occur in embryonic stages. Ultrastructural analyses reveal that the inclusions are disordered neurofilaments packed in high density, segregated from other organelles. Immunochemical studies show decreased NFL protein levels in DRG, cerebellum and spinal cord in NeflN98S/+ mice, and total NFL protein pool is shifted toward the triton-insoluble fraction. Our findings reveal the nature of the inclusions in NeflN98S/+ mice, provide useful information to understand mechanisms of CMT2E disease, and identify DRG from NeflN98S/+ mice as a useful cell line model for therapeutic discoveries.
[RS-1 enhanced the efficiency of CRISPR-Cas9 mediated knock-in of human lactoferrin].

PubMed

Zhou, Wenjun; Guo, Rihong; Deng, Mingtian; Wang, Feng; Zhang, Yanli

2017-08-25

This study aims to knock out the goat β-lactoglobulin (BLG) gene using CRISPR-Cas9 system and knock in human lactoferrin (hLF) at the BLG locus, and further study the effect of RAD51 stimulatory compound (RS-1) on homologous recombination efficiency. First, we designed an sgRNA targeting the first exon of goat BLG gene and constructed a co-expression vector pCas9-sgBLG. This sgRNA vector was then transfected into goat ear fibroblasts (GEFs), and the target region was examined by T7EN1 assay and sequencing. Second, we constructed a targeting vector pBHA-hLF-NIE including NEO and EGFP genes based on BLG gene locus. This targeting vector together with pCas9-sgBLG expression vector was co-transfected into GEFs. Transfected cells were then treated with 0, 5, 10 and 20 μmol/L RS-1 for 72 h to analyse the EGFP expression efficiency. Next, we used 800 μg/mL G418 to screen G418-resistent cell clones, and studied hLF site-specific knock-in cell clones by PCR and sequencing. The editing efficiency of sgBLG was between 25% and 31%. The EGFP expression efficiency indicated that the gene knock-in efficiency was improved by RS-1 in a dose-dependent manner, which could reach 3.5-fold compared to the control group. The percentage of positive cells with hLF knock-in was increased to 32.61% when 10 μmol/L RS-1 was used. However, when the concentration of RS-1 increased to 20 μmol/L, the percentage of positive cells decreased to 22.22% and resulted in an increase of senescent cell clone number. These results suggested that hLF knock-in and BLG knock-out in GEFs were achieved by using CRISPR/Cas9 system, and optimum concentration of RS-1 could improve knock-in efficiency, which provides a reference for efficiently obtaining gene knock-in cells using CRISPR/Cas9 in the future.
Phosphorylation of Smad2/3 at specific linker threonine indicates slow-cycling intestinal stem-like cells before reentry to cell cycle.

PubMed

Kishimoto, Masanobu; Fukui, Toshiro; Suzuki, Ryo; Takahashi, Yu; Sumimoto, Kimi; Okazaki, Takashi; Sakao, Masayuki; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2015-02-01

Quiescent (slow-cycling) and active (rapid-cycling) stem cells are demonstrated in small intestines. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in murine stomach, and suggested these cells are epithelial stem cells. Here, we explore whether pSmad2/3L-Thr could serve as a biomarker for small intestine and colon stem cells. We examined small intestines and colons from C57BL/6 mice and colons with dextran sulfate sodium (DSS)-induced colitis. We performed double-immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, chromogranin A, CDK4, DCAMKL1, and Musashi-1. Small intestines and colons from Lgr5-EGFP knock-in mice were examined by pSmad2/3L-Thr immunofluorescent staining. To examine BrdU label retention of pSmad2/3L-Thr immunostaining-positive cells, we collected specimens after BrdU administration and observed double-immunofluorescent staining of pSmad2/3L-Thr with BrdU. In small intestines and colons, pSmad2/3L-Thr immunostaining-strongly positive cells were detected around crypt bases. Immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was not observed. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with cytokeratin 8, CDK4, and Musashi-1 and different localization from chromogranin A and DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-strongly positive cells were morphologically undifferentiated. In Lgr5-EGFP knock-in mice, some but not all pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with Lgr5. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with BrdU at 5, 10, and 15 days after administration. In DSS-induced colitis, pSmad2/3L-Thr and Ki67 immunostaining-positive cells increased in the regeneration phase and decreased in the injury phase. In murine small intestines and colons, we suggest pSmad2/3L-Thr immunostaining-strongly positive cells are epithelial stem-like cells just before reentry to the cell cycle.
Altered fast- and slow-twitch muscle fibre characteristics in female mice with a (S248F) knock-in mutation of the brain neuronal nicotinic acetylcholine receptor.

PubMed

Cannata, David J; Finkelstein, David I; Gantois, Ilse; Teper, Yaroslav; Drago, John; West, Jan M

2009-01-01

We generated a mouse line with a missense mutation (S248F) in the gene (CHRNA4) encoding the alpha4 subunit of neuronal nicotinic acetylcholine receptor (nAChR). Mutant mice demonstrate brief nicotine induced dystonia that resembles the clinical events seen in patients with the same mutation. Drug-induced dystonia is more pronounced in female mice, thus our aim was to determine if the S248F mutation changed the properties of fast- and slow-twitch muscle fibres from female mutant mice. Reverse transcriptase-PCR confirmed CHRNA4 gene expression in the brain but not skeletal muscles in normal and mutant mice. Ca(2+) and Sr(2+) force activation curves were obtained using skinned muscle fibres prepared from slow-twitch (soleus) and fast-twitch (EDL) muscles. Two significant results were found: (1) the (pCa(50) - pSr(50)) value from EDL fibres was smaller in mutant mice than in wild type (1.01 vs. 1.30), (2) the percentage force produced at pSr 5.5 was larger in mutants than in wild type (5.76 vs. 0.24%). Both results indicate a shift to slow-twitch characteristics in the mutant. This conclusion is supported by the identification of the myosin heavy chain (MHC) isoforms. Mutant EDL fibres expressed MHC I (usually only found in slow-twitch fibres) as well as MHC IIa. Despite the lack of spontaneous dystonic events, our findings suggest that mutant mice may be having subclinical events or the mutation results in a chronic alteration to muscle neural input.
Differential molecular and behavioural alterations in mouse models of GABRG2 haploinsufficiency versus dominant negative mutations associated with human epilepsy

PubMed Central

Warner, Timothy A.; Shen, Wangzhen; Huang, Xuan; Liu, Zhong; Macdonald, Robert L.; Kang, Jing-Qiong

2016-01-01

Genetic epilepsy is a common disorder with phenotypic variation, but the basis for the variation is unknown. Comparing the molecular pathophysiology of mutations in the same epilepsy gene may provide mechanistic insights into the phenotypic heterogeneity. GABRG2 is an established epilepsy gene, and mutations in it produce epilepsy syndromes with varying severities. The disease phenotype in some cases may be caused by simple loss of subunit function (functional haploinsufficiency), while others may be caused by loss-of-function plus dominant negative suppression and other cellular toxicity. Detailed molecular defects and the corresponding seizures and related comorbidities resulting from haploinsufficiency and dominant negative mutations, however, have not been compared. Here we compared two mouse models of GABRG2 loss-of-function mutations associated with epilepsy with different severities, Gabrg2+/Q390X knockin (KI) and Gabrg2+/- knockout (KO) mice. Heterozygous Gabrg2+/Q390XKI mice are associated with a severe epileptic encephalopathy due to a dominant negative effect of the mutation, while heterozygous Gabrg2+/- KO mice are associated with mild absence epilepsy due to simple haploinsufficiency. Unchanged at the transcriptional level, KI mice with severe epilepsy had neuronal accumulation of mutant γ2 subunits, reduced remaining functional wild-type subunits in dendrites and synapses, while KO mice with mild epilepsy had no intracellular accumulation of the mutant subunits and unaffected biogenesis of the remaining wild-type subunits. Consequently, KI mice with dominant negative mutations had much less wild-type receptor expression, more severe seizures and behavioural comorbidities than KO mice. This work provides insights into the pathophysiology of epilepsy syndrome heterogeneity and designing mechanism-based therapies. PMID:27340224
Effects of the BDNF Val66Met Polymorphism on Anxiety-Like Behavior Following Nicotine Withdrawal in Mice

PubMed Central

Lee, Bridgin G.; Anastasia, Agustin; Hempstead, Barbara L.; Lee, Francis S.

2015-01-01

Introduction: Nicotine withdrawal is characterized by both affective and cognitive symptoms. Identifying genetic polymorphisms that could affect the symptoms associated with nicotine withdrawal are important in predicting withdrawal sensitivity and identifying personalized cessation therapies. In the current study we used a mouse model of a non-synonymous single nucleotide polymorphism in the translated region of the brain-derived neurotrophic factor (BDNF) gene that substitutes a valine (Val) for a methionine (Met) amino acid (Val66Met) to examine the relationship between the Val66Met single nucleotide polymorphism and nicotine dependence. Methods: This study measured proBDNF and the BDNF prodomain levels following nicotine and nicotine withdrawal and examined a mouse model of a common polymorphism in this protein (BDNFMet/Met) in three behavioral paradigms: novelty-induced hypophagia, marble burying, and the open-field test. Results: Using the BDNF knock-in mouse containing the BDNF Val66Met polymorphism we found: (1) blunted anxiety-like behavior in BDNFMet/Met mice following withdrawal in three behavioral paradigms: novelty-induced hypophagia, marble burying, and the open-field test; (2) the anxiolytic effects of chronic nicotine are absent in BDNFMet/Met mice; and (3) an increase in BDNF prodomain in BDNFMet/Met mice following nicotine withdrawal. Conclusions: Our study is the first to examine the effect of the BDNF Val66Met polymorphism on the affective symptoms of withdrawal from nicotine in mice. In these mice, a single-nucleotide polymorphism in the translated region of the BDNF gene can result in a blunted withdrawal, as measured by decreased anxiety-like behavior. The significant increase in the BDNF prodomain in BDNFMet/Met mice following nicotine cessation suggests a possible role of this ligand in the circuitry remodeling after withdrawal. PMID:25744957
The importance of basonuclin 2 in adult mice and its relation to basonuclin 1.

PubMed

Vanhoutteghem, Amandine; Delhomme, Brigitte; Hervé, Françoise; Nondier, Isabelle; Petit, Jean-Maurice; Araki, Masatake; Araki, Kimi; Djian, Philippe

2016-05-01

BNC2 is an extremely conserved zinc finger protein with important functions in the development of craniofacial bones and male germ cells. Because disruption of the Bnc2 gene in mice causes neonatal lethality, the function of the protein in adult animals has not been studied. Until now BNC2 was considered to have a wider tissue distribution than its paralog, BNC1, but the precise cell types expressing Bnc2 are largely unknown. We identify here the cell types containing BNC2 in the mouse and we show the unexpected presence of BNC1 in many BNC2-containing cells. BNC1 and BNC2 are colocalized in male and female germ cells, ovarian epithelial cells, sensory neurons, hair follicle keratinocytes and connective cells of organ capsules. In many cell lineages, the two basonuclins appear and disappear synchronously. Within the male germ cell lineage, BNC1 and BNC2 are found in prospermatogonia and undifferentiated spermatogonia, and disappear abruptly from differentiating spermatogonia. During oogenesis, the two basonuclins accumulate specifically in maturing oocytes. During the development of hair follicles, BNC1 and BNC2 concentrate in the primary hair germs. As follicle morphogenesis proceeds, cells possessing BNC1 and BNC2 invade the dermis and surround the papilla. During anagen, BNC1 and BNC2 are largely restricted to the basal layer of the outer root sheath and the matrix. During catagen, the compartment of cells possessing BNC1 and BNC2 regresses, and in telogen, the two basonuclins are confined to the secondary hair germ. During the next anagen, the BNC1/BNC2-containing cell population regenerates the hair follicle. By examining Bnc2(-/-) mice that have escaped the neonatal lethality usually associated with lack of BNC2, we demonstrate that BNC2 possesses important functions in many of the cell types where it resides. Hair follicles of postnatal Bnc2(-/-) mice do not fully develop during the first cycle and thereafter remain blocked in telogen. It is concluded that the presence of BNC2 in the secondary hair germ is required to regenerate the transient segment of the follicle. Postnatal Bnc2(-/-) mice also show severe dwarfism, defects in oogenesis and alterations of palatal rugae. Although the two basonuclins possess very similar zinc fingers and are largely coexpressed, BNC1 cannot substitute for BNC2. This is shown incontrovertibly in knockin mice expressing Bnc1 instead of Bnc2 as these mice invariably die at birth with craniofacial abnormalities undistinguishable from those of Bnc2(-/-) mice. The function of the basonuclins in the secondary hair germ is of particular interest. Copyright © 2016 Elsevier B.V. All rights reserved.
Infantile Pain Episodes Associated with Novel Nav1.9 Mutations in Familial Episodic Pain Syndrome in Japanese Families.

PubMed

Okuda, Hiroko; Noguchi, Atsuko; Kobayashi, Hatasu; Kondo, Daiki; Harada, Kouji H; Youssefian, Shohab; Shioi, Hirotomo; Kabata, Risako; Domon, Yuki; Kubota, Kazufumi; Kitano, Yutaka; Takayama, Yasunori; Hitomi, Toshiaki; Ohno, Kousaku; Saito, Yoshiaki; Asano, Takeshi; Tominaga, Makoto; Takahashi, Tsutomu; Koizumi, Akio

2016-01-01

Painful peripheral neuropathy has been correlated with various voltage-gated sodium channel mutations in sensory neurons. Recently Nav1.9, a voltage-gated sodium channel subtype, has been established as a genetic influence for certain peripheral pain syndromes. In this study, we performed a genetic study in six unrelated multigenerational Japanese families with episodic pain syndrome. Affected participants (n = 23) were characterized by infantile recurrent pain episodes with spontaneous mitigation around adolescence. This unique phenotype was inherited in an autosomal-dominant mode. Linkage analysis was performed for two families with 12 affected and nine unaffected members, and a single locus was identified on 3p22 (LOD score 4.32). Exome analysis (n = 14) was performed for affected and unaffected members in these two families and an additional family. Two missense variants were identified: R222H and R222S in SCN11A. Next, we generated a knock-in mouse model harboring one of the mutations (R222S). Behavioral tests (Hargreaves test and cold plate test) using R222S and wild-type C57BL/6 (WT) mice, young (8-9 weeks old; n = 10-12 for each group) and mature (36-38 weeks old; n = 5-6 for each group), showed that R222S mice were significantly (p < 0.05) more hypersensitive to hot and cold stimuli than WT mice. Electrophysiological studies using dorsal root ganglion neurons from 8-9-week-old mice showed no significant difference in resting membrane potential, but input impedance and firing frequency of evoked action potentials were significantly increased in R222S mice compared with WT mice. However, there was no significant difference among Nav1.9 (WT, R222S, and R222H)-overexpressing ND7/23 cell lines. These results suggest that our novel mutation is a gain-of-function mutation that causes infantile familial episodic pain. The mouse model developed here will be useful for drug screening for familial episodic pain syndrome associated with SCN11A mutations.
Skeletal, cardiac, and respiratory muscle function and histopathology in the P448Lneo- mouse model of FKRP-deficient muscular dystrophy.

PubMed

Yu, Qing; Morales, Melissa; Li, Ning; Fritz, Alexander G; Ruobing, Ren; Blaeser, Anthony; Francois, Ershia; Lu, Qi-Long; Nagaraju, Kanneboyina; Spurney, Christopher F

2018-04-06

Fukutin-related protein (FKRP) mutations are the most common cause of dystroglycanopathies known to cause both limb girdle and congenital muscular dystrophy. The P448Lneo- mouse model has a knock-in mutation in the FKRP gene and develops skeletal, respiratory, and cardiac muscle disease. We studied the natural history of the P448Lneo- mouse model over 9 months and the effects of twice weekly treadmill running. Forelimb and hindlimb grip strength (Columbus Instruments) and overall activity (Omnitech Electronics) assessed skeletal muscle function. Echocardiography was performed using VisualSonics Vevo 770 (FujiFilm VisualSonics). Plethysmography was performed using whole body system (ADInstruments). Histological evaluations included quantification of inflammation, fibrosis, central nucleation, and fiber size variation. P448Lneo- mice had significantly increased normalized tissue weights compared to controls at 9 months of age for the heart, gastrocnemius, soleus, tibialis anterior, quadriceps, and triceps. There were no significant differences seen in forelimb or hindlimb grip strength or activity monitoring in P448Lneo- mice with or without exercise compared to controls. Skeletal muscles demonstrated increased inflammation, fibrosis, central nucleation, and variation in fiber size compared to controls (p < 0.05) and worsened with exercise. Plethysmography showed significant differences in respiratory rates and decreased tidal and minute volumes in P448Lneo- mice (p < 0.01). There was increased fibrosis in the diaphragm compared to controls (p < 0.01). Echocardiography demonstrated decreased systolic function in 9-month-old mutant mice (p < 0.01). There was increased myocardial wall thickness and mass (p < 0.001) with increased fibrosis in 9-month-old P448Lneo- mice compared to controls (p < 0.05). mRNA expression for natriuretic peptide type A (Nppa) was significantly increased in P448Lneo- mice compared to controls at 6 months (p < 0.05) and for natriuretic peptide type B (Nppb) at 6 and 9 months of age (p < 0.05). FKRP-deficient P448Lneo- mice demonstrate significant deficits in cardiac and respiratory functions compared to control mice, and this is associated with increased inflammation and fibrosis. This study provides new functional outcome measures for preclinical trials of FKRP-related muscular dystrophies.

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