Working-for-Food Behaviors: A Preclinical Study in Prader-Willi Mutant Mice.

PubMed

Lassi, Glenda; Maggi, Silvia; Balzani, Edoardo; Cosentini, Ilaria; Garcia-Garcia, Celina; Tucci, Valter

2016-11-01

Abnormal feeding behavior is one of the main symptoms of Prader-Willi syndrome (PWS). By studying a PWS mouse mutant line, which carries a paternally inherited deletion of the small nucleolar RNA 116 (Snord116), we observed significant changes in working-for-food behavioral responses at various timescales. In particular, we report that PWS mutant mice show a significant delay compared to wild-type littermate controls in responding to both hour-scale and seconds-to-minutes-scale time intervals. This timing shift in mutant mice is associated with better performance in the working-for-food task, and results in better decision making in these mutant mice. The results of our study reveal a novel aspect of the organization of feeding behavior, and advance the understanding of the interplay between the metabolic functions and cognitive mechanisms of PWS. Copyright © 2016 by the Genetics Society of America.

Initial Experience with Balloon-Occluded Trans-catheter Arterial Chemoembolization (B-TACE) for Hepatocellular Carcinoma

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

Maruyama, Mitsunari, E-mail: mitunari@med-shimane.u.ac.jp; Yoshizako, Takeshi, E-mail: yosizako@med.shimane-u.ac.jp; Nakamura, Tomonori, E-mail: t-naka@med.shimane-u.ac.jp

2016-03-15

PurposeThis study was performed to evaluate the accumulation of lipiodol emulsion (LE) and adverse events during our initial experience of balloon-occluded trans-catheter arterial chemoembolization (B-TACE) for hepatocellular carcinoma (HCC) compared with conventional TACE (C-TACE).MethodsB-TACE group (50 cases) was compared with C-TACE group (50 cases). The ratio of the LE concentration in the tumor to that in the surrounding embolized liver parenchyma (LE ratio) was calculated after each treatment. Adverse events were evaluated according to the Common Terminology Criteria for Adverse Effects (CTCAE) version 4.0.ResultsThe LE ratio at the level of subsegmental showed a statistically significant difference between the groups (tmore » test: P < 0.05). Only elevation of alanine aminotransferase was more frequent in the B-TACE group, showing a statistically significant difference (Mann–Whitney test: P < 0.05). While B-TACE caused severe adverse events (liver abscess and infarction) in patients with bile duct dilatation, there was no statistically significant difference in incidence between the groups. Multivariate logistic regression analysis suggested that the significant risk factor for liver abscess/infarction was bile duct dilatation (P < 0.05).ConclusionThe LE ratio at the level of subsegmental showed a statistically significant difference between the groups (t test: P < 0.05). B-TACE caused severe adverse events (liver abscess and infarction) in patients with bile duct dilatation.« less

Differentiation-induced skin cancer suppression by FOS, p53, and TACE/ADAM17

PubMed Central

Guinea-Viniegra, Juan; Zenz, Rainer; Scheuch, Harald; Jiménez, María; Bakiri, Latifa; Petzelbauer, Peter; Wagner, Erwin F.

2012-01-01

Squamous cell carcinomas (SCCs) are heterogeneous and aggressive skin tumors for which innovative, targeted therapies are needed. Here, we identify a p53/TACE pathway that is negatively regulated by FOS and show that the FOS/p53/TACE axis suppresses SCC by inducing differentiation. We found that epidermal Fos deletion in mouse tumor models or pharmacological FOS/AP-1 inhibition in human SCC cell lines induced p53 expression. Epidermal cell differentiation and skin tumor suppression were caused by a p53-dependent transcriptional activation of the metalloprotease TACE/ADAM17 (TNF-α–converting enzyme), a previously unknown p53 target gene that was required for NOTCH1 activation. Although half of cutaneous human SCCs display p53-inactivating mutations, restoring p53/TACE activity in mouse and human skin SCCs induced tumor cell differentiation independently of the p53 status. We propose FOS/AP-1 inhibition or p53/TACE reactivating strategies as differentiation-inducing therapies for SCCs. PMID:22772468

Harnessing the natural inhibitory domain to control TNFα Converting Enzyme (TACE) activity in vivo.

PubMed

Wong, Eitan; Cohen, Tal; Romi, Erez; Levin, Maxim; Peleg, Yoav; Arad, Uri; Yaron, Avraham; Milla, Marcos E; Sagi, Irit

2016-12-16

Dysregulated activity of A Disintegrin And Metalloproteinase 17 (ADAM17)/TNFα Converting Enzyme (TACE) is associated with inflammatory disorders and cancer progression by releasing regulatory membrane-tethered proteins like TNFα, IL6R and EGFR ligands. Although specific inhibition of TACE is thought to be a viable strategy for inflammatory disorders and for malignancies treatment, the generation of effective inhibitors in vivo has been proven to be challenging. Here we report on the development of a protein inhibitor that leverages the endogenous modulator of TACE. We have generated a stable form of the auto-inhibitory TACE prodomain (TPD), which specifically inhibits in vitro and cell-surface TACE, but not the related ADAM10, and effectively modulated TNFα secretion in cells. TPD significantly attenuated TACE-mediated disease models of sepsis, rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and reduced TNFα in synovial fluids from RA patients. Our results demonstrate that intervening with endogenous ADAM sheddase modulatory mechanisms holds potential as a general strategy for the design of ADAM inhibitors.

Genes and signaling pathways involved in memory enhancement in mutant mice

PubMed Central

2014-01-01

Mutant mice have been used successfully as a tool for investigating the mechanisms of memory at multiple levels, from genes to behavior. In most cases, manipulating a gene expressed in the brain impairs cognitive functions such as memory and their underlying cellular mechanisms, including synaptic plasticity. However, a remarkable number of mutations have been shown to enhance memory in mice. Understanding how to improve a system provides valuable insights into how the system works under normal conditions, because this involves understanding what the crucial components are. Therefore, more can be learned about the basic mechanisms of memory by studying mutant mice with enhanced memory. This review will summarize the genes and signaling pathways that are altered in the mutants with enhanced memory, as well as their roles in synaptic plasticity. Finally, I will discuss how knowledge of memory-enhancing mechanisms could be used to develop treatments for cognitive disorders associated with impaired plasticity. PMID:24894914

The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice.

PubMed

Field, H J; Wildy, P

1978-10-01

The pathogenicity for mice of two mutants of herpes simplex virus (type 1 and type 2), which fail to induce thymidine kinase, were compared with their respective parent strains. The mutants were much less virulent than the parents following either intracerebral or peripheral inoculation. The replication of the virus at the site of inoculation and its progression into the nervous system were studied. Following a very large inoculum in the ear, the type 1 mutant was found to establish a latent infection in the cervical dorsal root ganglia. Mice inoculated intracerebrally with small doses of the mutant viruses were solidly immune to challenge with lethal doses of the parent strain.

Correlation of Multislice CT and Histomorphology in HCC Following TACE: Predictors of Outcome

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

Herber, S., E-mail: herber@radiologie.klinik.uni-mainz.d; Biesterfeld, S.; Franz, U.

2008-07-15

The purpose of this study was to correlate histopathological with CT findings in patients suffering from hepatocellular carcinoma (HCC) eligible for orthotopic liver transplantation (OLT), with a special focus on the antitumoral effect of transarterial chemoembolization (TACE) therapy. A total of 42 consecutive patients suffering from HCC had been treated prior to OLT by means of TACE. TACE was carried out with a mixture of Lipiodol (10-20 ml) and mitomycin C (max. dosage, 10 mg). TACE was performed at 6- to 8-week intervals. Follow-up investigation included contrast-enhanced multislice CT controls and laboratory control. Liver explants were evaluated macroscopically and microscopicallymore » to determine the number and size of the tumor lesions as well as the degree of tumor necrosis. Necrosis was investigated in H and E-stained sections. The degree of necrosis was classified as follows: 0-25%, 26-50%, 51-75%, 75-99%, and complete necrosis. Two hundred thirty-one TACE procedures (5.5 {+-} 2.9; range, 1-14) were performed. Mean tumor size in CT before and after TACE was 4.1 {+-} 2.4 (range, 1.0-12.0 cm) and 2.7 {+-} 1.2 (range, 1.0-6.0 cm; p < 0.001). Mean tumor number before and after TACE in CT was 2.5 {+-} 1.5 (n = 105; range, 1-8) and 2.4 {+-} 2.0 (n = 103; range, 1-6; p = 0.99). In the surgical specimen tumor size and tumor number were 2.8 {+-} 1.6 (range, 1.0-7.0 cm; p = 0.78) and 1.9 {+-} 1.2 (range, 1-7; p = 0.003). Mean tumor necrosis was 67.8% {+-} 28.1%. Tumor necrosis was subtotal or complete in 17 of 42 (40.5%) patients. Tumor necrosis correlated significantly with the degree of arterial devascularization in CT (p = 0.001), the amount of Lipiodol washout (p = 0.002), and the number of tumor lesions (i.e., unifocal vs. multifocal). Furthermore, elevated serum levels of bilirubin (p = 0.005) and decreased albumin (p = 0.004) affected the local antitumoral effect. A poor necrosis rate (< 25%) significantly correlated with the number of TACE procedures

The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice.

PubMed Central

Field, H. J.; Wildy, P.

1978-01-01

The pathogenicity for mice of two mutants of herpes simplex virus (type 1 and type 2), which fail to induce thymidine kinase, were compared with their respective parent strains. The mutants were much less virulent than the parents following either intracerebral or peripheral inoculation. The replication of the virus at the site of inoculation and its progression into the nervous system were studied. Following a very large inoculum in the ear, the type 1 mutant was found to establish a latent infection in the cervical dorsal root ganglia. Mice inoculated intracerebrally with small doses of the mutant viruses were solidly immune to challenge with lethal doses of the parent strain. PMID:212476

Evaluation of nigrostriatal dopaminergic function in adult +/+ and +/- BDNF mutant mice.

PubMed

Dluzen, D E; Gao, X; Story, G M; Anderson, L I; Kucera, J; Walro, J M

2001-07-01

Deletion of a single copy of the BDNF gene has been shown to affect the nigrostriatal dopaminergic system of young adult BDNF mice. In the present report we evaluated various indices of nigrostriatal dopaminergic function between 9-month-old wild-type (+/+) and heterozygous (+/-) BDNF mutant mice. Performance in a sensorimotor beam walking task was significantly decreased in +/- mice as indicated by increased times required to traverse both a wide (21 mm) and narrow (6 mm) beam. No differences in spontaneous locomotor behavior were observed between the +/+ and +/- mice. Amphetamine-stimulated (5 mg/kg) locomotor behavior was increased to a greater degree in the +/- mice, with the number of movements performed by these mice being significantly greater than their +/+ controls. Corpus striatal dopamine concentrations were significantly greater in the +/- BDNF mice. The absence of any significant differences for dopamine concentrations within the hypothalamus and olfactory bulb of these mice, as well as an absence of any difference in striatal norepinephrine concentrations, suggested a relative specificity of these effects to the corpus striatum. Both the +/- and +/+ mice showed similar reductions in striatal dopamine concentrations in response to a neurotoxic regimen of methamphetamine (20 mg/kg). Collectively these data show increased levels of striatal dopamine concentrations associated with altered behavioral responses involving the nigrostriatal dopaminergic system within the heterozygous BDNF mutant mice. Copyright 2001 Academic Press.

Anti-H-Y responses of H-2b mutant mice.

PubMed

Simpson, E; Gordon, R D; Chandler, P R; Bailey, D

1978-10-01

Two strains of H-2b mutant mice, H-2ba and H-2bf, in which the mutational event took place at H-2K, make anti-H-Y cytotoxic T cell responses which are H-2-restricted, Db-associated and indistinguishable in target cell specificity from those of H-2b mice. Thus, alteration of the H-2K molecule affects neither the Ir gene controlling the response, nor the associative antigen. On the other hand, one H-2Db mutant strain, H-2bo, although it makes a good anti-H-Y cytotoxic response, shows target cell specificity restricted to its own Dbo antigen(s), and neither H-2b, H-2ba or H-2bf anti-H-Y cytotoxic cells kill H-2bo male target cells. Thus, the alteration of the H-2Db molecule does not affect the Ir gene of H-2b mice, but it does alter the H-2Db-associative antigen.

Phenotype detection in morphological mutant mice using deformation features.

PubMed

Roy, Sharmili; Liang, Xi; Kitamoto, Asanobu; Tamura, Masaru; Shiroishi, Toshihiko; Brown, Michael S

2013-01-01

Large-scale global efforts are underway to knockout each of the approximately 25,000 mouse genes and interpret their roles in shaping the mammalian embryo. Given the tremendous amount of data generated by imaging mutated prenatal mice, high-throughput image analysis systems are inevitable to characterize mammalian development and diseases. Current state-of-the-art computational systems offer only differential volumetric analysis of pre-defined anatomical structures between various gene-knockout mice strains. For subtle anatomical phenotypes, embryo phenotyping still relies on the laborious histological techniques that are clearly unsuitable in such big data environment. This paper presents a system that automatically detects known phenotypes and assists in discovering novel phenotypes in muCT images of mutant mice. Deformation features obtained from non-linear registration of mutant embryo to a normal consensus average image are extracted and analyzed to compute phenotypic and candidate phenotypic areas. The presented system is evaluated using C57BL/10 embryo images. All cases of ventricular septum defect and polydactyly, well-known to be present in this strain, are successfully detected. The system predicts potential phenotypic areas in the liver that are under active histological evaluation for possible phenotype of this mouse line.

GH and IGF1: roles in energy metabolism of long-living GH mutant mice.

PubMed

Brown-Borg, Holly M; Bartke, Andrzej

2012-06-01

Of the multiple theories to explain exceptional longevity, the most robust of these has centered on the reduction of three anabolic protein hormones, growth hormone (GH), insulin-like growth factor, and insulin. GH mutant mice live 50% longer and exhibit significant differences in several aspects of energy metabolism as compared with wild-type mice. Mitochondrial metabolism is upregulated in the absence of GH, whereas in GH transgenic mice and dwarf mice treated with GH, multiple aspects of these pathways are suppressed. Core body temperature is markedly lower in dwarf mice, yet whole-body metabolism, as measured by indirect calorimetry, is surprisingly higher in Ames dwarf and Ghr-/- mice compared with normal controls. Elevated adiponectin, a key antiinflammatory cytokine, is also very likely to contribute to longevity in these mice. Thus, several important components related to energy metabolism are altered in GH mutant mice, and these differences are likely critical in aging processes and life-span extension.

Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis.

PubMed

Marty, Caroline; Pecquet, Christian; Nivarthi, Harini; El-Khoury, Mira; Chachoua, Ilyas; Tulliez, Micheline; Villeval, Jean-Luc; Raslova, Hana; Kralovics, Robert; Constantinescu, Stefan N; Plo, Isabelle; Vainchenker, William

2016-03-10

Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms, we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After 6 months, CALRdel52-, in contrast to rare CALRins5-, transduced mice developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover, a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease, suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell-line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of Janus kinase 2 and signal transducer and activator of transcription 5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation. © 2016 by The American Society of Hematology.

mPeriod2 Brdm1 and other single Period mutant mice have normal food anticipatory activity.

PubMed

Pendergast, Julie S; Wendroth, Robert H; Stenner, Rio C; Keil, Charles D; Yamazaki, Shin

2017-11-14

Animals anticipate the timing of food availability via the food-entrainable oscillator (FEO). The anatomical location and timekeeping mechanism of the FEO are unknown. Several studies showed the circadian gene, Period 2, is critical for FEO timekeeping. However, other studies concluded that canonical circadian genes are not essential for FEO timekeeping. In this study, we re-examined the effects of the Per2 Brdm1 mutation on food entrainment using methods that have revealed robust food anticipatory activity in other mutant lines. We examined food anticipatory activity, which is the output of the FEO, in single Period mutant mice. Single Per1, Per2, and Per3 mutant mice had robust food anticipatory activity during restricted feeding. In addition, we found that two different lines of Per2 mutant mice (ldc and Brdm1) anticipated restricted food availability. To determine if FEO timekeeping persisted in the absence of the food cue, we assessed activity during fasting. Food anticipatory (wheel-running) activity in all Period mutant mice was also robust during food deprivation. Together, our studies demonstrate that the Period genes are not necessary for the expression of food anticipatory activity.

Comparison of efficacy between TACE combined with apatinib and TACE alone in the treatment of intermediate and advanced hepatocellular carcinoma: A single-center randomized controlled trial.

PubMed

Lu, Wei; Jin, Xin-Li; Yang, Chao; Du, Peng; Jiang, Fu-Qiang; Ma, Jun-Peng; Yang, Jian; Xie, Peng; Zhang, Zhe

2017-06-03

This study was designed to compare the clinical efficacy and safety of transcatheter arterial chemoembolization (TACE) combined with apatinib and TACE alone in the treatment of intermediate and advanced hepatocellular carcinoma (HCC). From March 2015 to August 2015, a total of 44 patients with moderate and advanced HCC, who were admitted in the Navy General Hospital of China, were included into this study. These patients were randomly divided into 2 groups: group A and group B. Patients in group A underwent TACE alone, while patients in group B underwent the combined treatment of TACE with apatinib. Differences in preoperative general data between these 2 groups were not statistically significant (P > 0.05). All patients were followed up for 12-18 months. Changes in α-fetal protein (AFP) at 3 months after treatment and the objective response rate (ORR) at 3, 6, 9 and 12 months after treatment were compared between these 2 groups. Furthermore, progression-free survival (PFS) and the incidence of adverse reactions were also compared between these 2 groups. AFP levels in groups A and B significantly decreased after 3 months of treatment, compared with the levels before treatment, and the differences were statistically significant (P < 0.05). However, at 3 months after treatment, the difference between these 2 groups was not statistically significant (P > 0.05). ORR at 3, 6, 9 and 12 months after treatment was 36.36%, 27.27%, 13.64% and 9.09%, respectively, in group A; and 60%, 50%, 45% and 35%, respectively, in group B. At 3 and 6 months after treatment, the differences between these 2 groups were not statistically significant (P > 0.05); while at 9 and 12 months after treatment, the differences between these 2 groups were statistically significant (P < 0.05). The median PFS was 6.0 months in group A and 12.5 months in group B, and the difference was statistically significant (P < 0.05). The incidences of complications were related to oral apatinib, such as

Fear-related behaviors in Lurcher mutant mice exposed to a predator.

PubMed

Lorivel, T; Roy, V; Hilber, P

2014-11-01

The Lurcher mutant mice are characterized by massive cerebellar cortex degeneration. Besides their motor and cognitive disturbances, they exhibit both exaggerated blood corticosterone (CORT) level surge and behavioral disinhibition when confronted to anxiogenic conditions (i.e. to a potential threat). In this study, we assessed if such physiological and behavioral hyperactivity was also detectable in a fear-eliciting situation (actual threat). For this purpose, the behaviors and CORT level elevations in Lurcher mice were compared with those of littermate controls in the predator exposure test: mice were exposed either to a rat (exposure) or to a brief wave of the experimenter's hand (sham exposure). While the basal CORT concentrations (24 h before testing) were not significantly different between mice of both genotypes, the post-exposure ones were higher in Lurcher than in control mice whatever the condition of the experimental design (exposure or sham exposure). Predator exposure did not provoke significant increase of CORT levels whatever the genotype. On the contrary, our data clearly showed that fear-related behaviors of cerebellar mutants facing a real threat were exacerbated in comparison to those of control mice. These results suggest that the cerebellar cortex not only participates to fear conditioning and anxiety but also actively contributes to the modulation of the innate fear-related behaviors. © 2014 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Parametric response mapping cut-off values that predict survival of hepatocellular carcinoma patients after TACE.

PubMed

Nörthen, Aventinus; Asendorf, Thomas; Shin, Hoen-Oh; Hinrichs, Jan B; Werncke, Thomas; Vogel, Arndt; Kirstein, Martha M; Wacker, Frank K; Rodt, Thomas

2018-04-21

Parametric response mapping (PRM) is a novel image-analysis technique applicable to assess tumor viability and predict intrahepatic recurrence of hepatocellular carcinoma (HCC) patients treated with transarterial chemoembolization (TACE). However, to date, the prognostic value of PRM for prediction of overall survival in HCC patients undergoing TACE is unclear. The objective of this explorative, single-center study was to identify cut-off values for voxel-specific PRM parameters that predict the post TACE overall survival in HCC patients. PRM was applied to biphasic CT data obtained at baseline and following 3 TACE treatments of 20 patients with HCC tumors ≥ 2 cm. The individual portal venous phases were registered to the arterial phases followed by segmentation of the largest lesion, i.e., the region of interest (ROI). Segmented voxels with their respective arterial and portal venous phase density values were displayed as a scatter plot. Voxel-specific PRM parameters were calculated and compared to patients' survival at 1, 2, and 3 years post treatment to identify the maximal predictive parameters. The hypervascularized tissue portion of the ROI was found to represent an independent predictor of the post TACE overall survival. For this parameter, cut-off values of 3650, 2057, and 2057 voxels, respectively, were determined to be optimal to predict overall survival at 1, 2, and 3 years after TACE. Using these cut points, patients were correctly classified as having died with a sensitivity of 80, 92, and 86% and as still being alive with a specificity of 60, 75, and 83%, respectively. The prognostic accuracy measured by area under the curve (AUC) values ranged from 0.73 to 0.87. PRM may have prognostic value to predict post TACE overall survival in HCC patients.

Overexpression of mutant HSP27 causes axonal neuropathy in mice.

PubMed

Lee, Jinho; Jung, Sung-Chul; Joo, Jaesoon; Choi, Yu-Ri; Moon, Hyo Won; Kwak, Geon; Yeo, Ha Kyung; Lee, Ji-Su; Ahn, Hye-Jee; Jung, Namhee; Hwang, Sunhee; Rheey, Jingeun; Woo, So-Youn; Kim, Ji Yon; Hong, Young Bin; Choi, Byung-Ok

2015-06-19

Mutations in heat shock 27 kDa protein 1 (HSP27 or HSPB1) cause distal hereditary motor neuropathy (dHMN) or Charcot-Marie-Tooth disease type 2 F (CMT2F) according to unknown factors. Mutant HSP27 proteins affect axonal transport by reducing acetylated tubulin. We generated a transgenic mouse model overexpressing HSP27-S135F mutant protein driven by Cytomegalovirus (CMV) immediate early promoter. The mouse phenotype was similar to dHMN patients in that they exhibit motor neuropathy. To determine the phenotypic aberration of transgenic mice, behavior test, magnetic resonance imaging (MRI), electrophysiological study, and pathology were performed. Rotarod test showed that founder mice exhibited lowered motor performance. MRI also revealed marked fatty infiltration in the anterior and posterior compartments at calf level. Electrophysiologically, compound muscle action potential (CMAP) but not motor nerve conduction velocity (MNCV) was reduced in the transgenic mice. Toluidine staining with semi-thin section of sciatic nerve showed the ratio of large myelinated axon fiber was reduced, which might cause reduced locomotion in the transgenic mice. Electron microscopy also revealed abundant aberrant myelination. Immunohistochemically, neuronal dysfunctions included elevated level of phosphorylated neurofilament and reduced level of acetylated tubulin in the sural nerve of transgenic mice. There was no additional phenotype besides motor neuronal defects. Overexpression of HSP27-S135F protein causes peripheral neuropathy. The mouse model can be applied to future development of therapeutic strategies for dHMN or CMT2F.

Sucrose intake and fasting glucose levels in 5-HT(1A) and 5-HT(1B) receptor mutant mice.

PubMed

Bechtholt, Anita J; Smith, Karen; Gaughan, Stephanie; Lucki, Irwin

2008-03-18

Serotonin (5-HT)(1A) and 5-HT(1B) receptors have been implicated in the incidence and treatment of depression in part through the examination of animals lacking these receptors. Although these receptors have been repeatedly implicated in ingestive behavior there is little information about how 5-HT(1A) and 5-HT(1B) receptor mutant mice react to solutions of varying palatability. In the present experiment male and female 5-HT(1A) and 5-HT(1B) mutant and wild-type mice were presented with increasing concentrations of sucrose using a two-bottle choice procedure. In addition fasting blood glucose levels were assessed. Both male and female 5-HT(1B) mutant mice drank more sucrose than WT mice but also consumed more water. Female, but not male, 5-HT(1A) mutant mice similarly showed increased sucrose consumption, but did not demonstrate increased consumption of water. In addition, the pattern of increased sucrose consumption over genotype and sex was related to fasting blood glucose concentrations such that levels in male 5-HT(1B) mutant mice were reduced relative to wild-type and 5-HT(1A) mutant males, but similar to those of females. The findings in 5-HT(1B) mutant mice emphasize the role of the 5-HT(1B) receptor in regulating ingestive behavior, whereas female sex hormones and 5-HT(1A) receptors may interact to alter sucrose consumption in 5-HT(1A) mutant mice. In addition, these findings may have implications for the role of these receptors in the incidence and treatment of depression since the intake of sucrose has been used as an index of anhedonia in animal models of depression and antidepressant efficacy.

Comparison of efficacy between TACE combined with apatinib and TACE alone in the treatment of intermediate and advanced hepatocellular carcinoma: A single-center randomized controlled trial

PubMed Central

Lu, Wei; Jin, Xin-Li; Yang, Chao; Du, Peng; Jiang, Fu-Qiang; Ma, Jun-Peng; Yang, Jian; Xie, Peng; Zhang, Zhe

2017-01-01

ABSTRACT Objective: This study was designed to compare the clinical efficacy and safety of transcatheter arterial chemoembolization (TACE) combined with apatinib and TACE alone in the treatment of intermediate and advanced hepatocellular carcinoma (HCC). Methods: From March 2015 to August 2015, a total of 44 patients with moderate and advanced HCC, who were admitted in the Navy General Hospital of China, were included into this study. These patients were randomly divided into 2 groups: group A and group B. Patients in group A underwent TACE alone, while patients in group B underwent the combined treatment of TACE with apatinib. Differences in preoperative general data between these 2 groups were not statistically significant (P > 0.05). All patients were followed up for 12–18 months. Changes in α-fetal protein (AFP) at 3 months after treatment and the objective response rate (ORR) at 3, 6, 9 and 12 months after treatment were compared between these 2 groups. Furthermore, progression-free survival (PFS) and the incidence of adverse reactions were also compared between these 2 groups. Results: AFP levels in groups A and B significantly decreased after 3 months of treatment, compared with the levels before treatment, and the differences were statistically significant (P < 0.05). However, at 3 months after treatment, the difference between these 2 groups was not statistically significant (P > 0.05). ORR at 3, 6, 9 and 12 months after treatment was 36.36%, 27.27%, 13.64% and 9.09%, respectively, in group A; and 60%, 50%, 45% and 35%, respectively, in group B. At 3 and 6 months after treatment, the differences between these 2 groups were not statistically significant (P > 0.05); while at 9 and 12 months after treatment, the differences between these 2 groups were statistically significant (P < 0.05). The median PFS was 6.0 months in group A and 12.5 months in group B, and the difference was statistically significant (P < 0.05). The incidences of complications

[Comparative analysis of TACE alone or plus RFA in the treatment of 167 cases of intermediate and advanced staged primary hepatocellular carcinoma].

PubMed

Zhao, Ming; Wang, Jian-peng; Wu, Pei-hong; Zhang, Fu-jun; Huang, Zi-lin; Li, Wang; Zhang, Liang; Pan, Chang-chuan; Li, Chuan-xing; Jiang, Yong

2010-11-09

To evaluate the clinical efficacy and survival rate of transarterial chemoembolization (TACE) alone or plus radiofrequency ablation (RFA) in patients with intermediate or advanced stage primary hepatocellular carcinoma (HCC). In this retrospective study, 467 cases received RFA or TACE plus RFA. Among them, 167 cases with strict clinical procedure (TACE alone or plus RFA) and complete follow-up data were included. Eighty-seven cases received TACE and 80 cases had TACE plus RFA between January 2000 and December 2006. Hierarchical analyses were performed using log-rank tests and survival curve was estimated by Kaplan-Meier method. A total of 167 patients received TACE alone or plus RFA for a follow-up period of 1 to 89 months. In the TACE alone group, the time-to-progression (TTP) was an average of 3.6 months. The median survival was 13 months, one-year survival rate 52.9%, three-year survival rate 11.5% and five-year survival rate 4.6%. In the TACE plus RFA group, the TTP time was an average of 10.8 months. The median survival time was 30 months, one-year survival rate 85.0%, three-year survival rate 45.0% and five-year survival rate 11.3%. In the TACE alone group, the median survival of intermediate stage HCC was 14 months, one-year survival rate 62.2%, three-year survival rate 13.3% and five-year survival rate 4.4%; In the TACE plus RFA group, the median survival of intermediate stage HCC was 14 months, one-year survival rate 90.1%, three-year survival rate 52.9% and five-year survival rate 13.7%. All differences of two groups has statistical significance (P < 0.05). In intermediate stage HCC, the median survival of TACE alone group was 14 months, one-year survival rate 62.2%, three-year survival rate 13.3%, five-year survival rate 4.4% versus 32 months, 90.1%, 52.9%, 13.7% in the TACE plus RFA group respectively. For the advanced stage HCC, the median survival time was 12 months, one-year survival rate 35%, three-year survival rate 7.1% and five-year survival rate

Cp/Heph mutant mice have iron-induced neurodegeneration diminished by deferiprone

PubMed Central

Zhao, Liangliang; Hadziahmetovic, Majda; Wang, Chenguang; Xu, Xueying; Song, Ying; Jinnah, H.A.; Wodzinska, Jolanta; Iacovelli, Jared; Wolkow, Natalie; Krajacic, Predrag; Weissberger, Alyssa Cwanger; Connelly, John; Spino, Michael; Lee, Michael K.; Connor, James; Giasson, Benoit; Harris, Z. Leah; Dunaief, Joshua L.

2016-01-01

Brain iron accumulates in several neurodegenerative diseases and can cause oxidative damage, but mechanisms of brain iron homeostasis are incompletely understood. Patients with mutations in the cellular iron-exporting ferroxidase ceruloplasmin (Cp) have brain iron accumulation causing neurodegeneration. Here, we assessed the brains of mice with combined mutation of Cp and its homolog hephaestin. Compared to single mutants, brain iron accumulation was accelerated in double mutants in the cerebellum, substantia nigra, and hippocampus. Iron accumulated within glia, while neurons were iron deficient. There was loss of both neurons and glia. Mice developed ataxia and tremor, and most died by 9 months. Treatment with the oral iron chelator deferiprone diminished brain iron levels, protected against neuron loss, and extended lifespan. Ferroxidases play important, partially overlapping roles in brain iron homeostasis by facilitating iron export from glia, making iron available to neurons. PMID:26303407

An improved Red/ET recombineering system and mouse ES cells culture conditions for the generation of targeted mutant mice.

PubMed

Kumagai, Katsuyoshi; Takanashi, Masakatsu; Ohno, Shin-Ichiro; Kuroda, Masahiko; Sudo, Katsuko

2017-05-03

Targeted mutant mice generated on a C57BL/6 background are powerful tools for analysis of the biological functions of genes, and gene targeting technologies using mouse embryonic stem (ES) cells have been used to generate such mice. Recently, a bacterial artificial chromosome (BAC) recombineering system was established for the construction of targeting vectors. However, gene retrieval from BACs for the generation of gene targeting vectors using this system remains difficult. Even when construction of a gene targeting vector is successful, the efficiency of production of targeted mutant mice from ES cells derived from C57BL/6 mice are poor. Therefore, in this study, we first improved the strategy for the retrieval of genes from BACs and their transfer into a DT-A plasmid, for the generation of gene targeting vectors using the BAC recombineering system. Then, we attempted to generate targeted mutant mice from ES cell lines derived from C57BL/6 mice, by culturing in serum-free medium. In conclusion, we established an improved strategy for the efficient generation of targeted mutant mice on a C57BL/6 background, which are useful for the in vivo analysis of gene functions and regulation.

Targeted inactivation of the mouse locus encoding coagulation factor XIII-A: hemostatic abnormalities in mutant mice and characterization of the coagulation deficit.

PubMed

Lauer, Peter; Metzner, Hubert J; Zettlmeissl, Gerd; Li, Meng; Smith, Austin G; Lathe, Richard; Dickneite, Gerhard

2002-12-01

Blood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin gamma-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.

Distinct Neurobehavioural Effects of Cannabidiol in Transmembrane Domain Neuregulin 1 Mutant Mice

PubMed Central

Long, Leonora E.; Chesworth, Rose; Huang, Xu-Feng; Wong, Alexander; Spiro, Adena; McGregor, Iain S.; Arnold, Jonathon C.; Karl, Tim

2012-01-01

The cannabis constituent cannabidiol (CBD) possesses anxiolytic and antipsychotic properties. We have previously shown that transmembrane domain neuregulin 1 mutant (Nrg1 TM HET) mice display altered neurobehavioural responses to the main psychoactive constituent of cannabis, Δ9-tetrahydrocannabinol. Here we investigated whether Nrg1 TM HET mice respond differently to CBD and whether CBD reverses schizophrenia-related phenotypes expressed by these mice. Adult male Nrg1 TM HET and wild type-like littermates (WT) received vehicle or CBD (1, 50 or 100 mg/kg i.p.) for 21 days. During treatment and 48 h after withdrawal we measured behaviour, whole blood CBD concentrations and autoradiographic receptor binding. Nrg1 HET mice displayed locomotor hyperactivity, PPI deficits and reduced 5-HT2A receptor binding density in the substantia nigra, but these phenotypes were not reversed by CBD. However, long-term CBD (50 and 100 mg/kg) selectively enhanced social interaction in Nrg1 TM HET mice. Furthermore, acute CBD (100 mg/kg) selectively increased PPI in Nrg1 TM HET mice, although tolerance to this effect was manifest upon repeated CBD administration. Long-term CBD (50 mg/kg) also selectively increased GABAA receptor binding in the granular retrosplenial cortex in Nrg1 TM HET mice and reduced 5-HT2A binding in the substantia nigra in WT mice. Nrg1 appears necessary for CBD-induced anxiolysis since only WT mice developed decreased anxiety-related behaviour with repeated CBD treatment. Altered pharmacokinetics in mutant mice could not explain our findings since no genotype differences existed in CBD blood concentrations. Here we demonstrate that Nrg1 modulates acute and long-term neurobehavioural effects of CBD, which does not reverse the schizophrenia-relevant phenotypes. PMID:22509273

Distinct neurobehavioural effects of cannabidiol in transmembrane domain neuregulin 1 mutant mice.

PubMed

Long, Leonora E; Chesworth, Rose; Huang, Xu-Feng; Wong, Alexander; Spiro, Adena; McGregor, Iain S; Arnold, Jonathon C; Karl, Tim

2012-01-01

The cannabis constituent cannabidiol (CBD) possesses anxiolytic and antipsychotic properties. We have previously shown that transmembrane domain neuregulin 1 mutant (Nrg1 TM HET) mice display altered neurobehavioural responses to the main psychoactive constituent of cannabis, Δ(9)-tetrahydrocannabinol. Here we investigated whether Nrg1 TM HET mice respond differently to CBD and whether CBD reverses schizophrenia-related phenotypes expressed by these mice. Adult male Nrg1 TM HET and wild type-like littermates (WT) received vehicle or CBD (1, 50 or 100 mg/kg i.p.) for 21 days. During treatment and 48 h after withdrawal we measured behaviour, whole blood CBD concentrations and autoradiographic receptor binding. Nrg1 HET mice displayed locomotor hyperactivity, PPI deficits and reduced 5-HT(2A) receptor binding density in the substantia nigra, but these phenotypes were not reversed by CBD. However, long-term CBD (50 and 100 mg/kg) selectively enhanced social interaction in Nrg1 TM HET mice. Furthermore, acute CBD (100 mg/kg) selectively increased PPI in Nrg1 TM HET mice, although tolerance to this effect was manifest upon repeated CBD administration. Long-term CBD (50 mg/kg) also selectively increased GABA(A) receptor binding in the granular retrosplenial cortex in Nrg1 TM HET mice and reduced 5-HT(2A) binding in the substantia nigra in WT mice. Nrg1 appears necessary for CBD-induced anxiolysis since only WT mice developed decreased anxiety-related behaviour with repeated CBD treatment. Altered pharmacokinetics in mutant mice could not explain our findings since no genotype differences existed in CBD blood concentrations. Here we demonstrate that Nrg1 modulates acute and long-term neurobehavioural effects of CBD, which does not reverse the schizophrenia-relevant phenotypes.

Partial Müllerian Duct Retention in Smad4 Conditional Mutant Male Mice.

PubMed

Petit, Fabrice G; Deng, Chuxia; Jamin, Soazik P

2016-01-01

Müllerian duct regression is a complex process which involves the AMH signalling pathway. We have previously demonstrated that besides AMH and its specific type II receptor (AMHRII), BMPR-IA and Smad5 are two essential factors implicated in this mechanism. Mothers against decapentaplegic homolog 4 (Smad4) is a transcription factor and the common Smad (co-Smad) involved in transforming growth factor beta (TGF-β) signalling pathway superfamily. Since Smad4 null mutants die early during gastrulation, we have inactivated Smad4 in the Müllerian duct mesenchyme. Specific inactivation of Smad4 in the urogenital ridge leads to the partial persistence of the Müllerian duct in adult male mice. Careful examination of the urogenital tract reveals that the Müllerian duct retention is randomly distributed either on one side or both sides. Histological analysis shows a uterus-like structure, which is confirmed by the expression of estrogen receptor α. As previously described in a β-catenin conditional mutant mouse model, β-catenin contributes to Müllerian duct regression. In our mutant male embryos, it appears that β-catenin expression is locally reduced along the urogenital ridge as compared to control mice. Moreover, the expression pattern is similar to those observed in control female mice. This study shows that reduced Smad4 expression disrupts the Wnt/β-catenin signalling leading to the partial persistence of Müllerian duct.

Developmental alterations in anxiety and cognitive behavior in serotonin transporter mutant mice.

PubMed

Sakakibara, Yasufumi; Kasahara, Yoshiyuki; Hall, F Scott; Lesch, Klaus-Peter; Murphy, Dennis L; Uhl, George R; Sora, Ichiro

2014-10-01

A promoter variant of the serotonin transporter (SERT) gene is known to affect emotional and cognitive regulation. In particular, the "short" allelic variant is implicated in the etiology of multiple neuropsychiatric disorders. Heterozygous (SERT(+/-)) and homozygous (SERT(-/-)) SERT mutant mice are valuable tools for understanding the mechanisms of altered SERT levels. Although these genetic effects are well investigated in adulthood, the developmental trajectory of altered SERT levels for behavior has not been investigated. We assessed anxiety-like and cognitive behaviors in SERT mutant mice in early adolescence and adulthood to examine the developmental consequences of reduced SERT levels. Spine density of pyramidal neurons was also measured in corticolimbic brain regions. Adult SERT(-/-) mice exhibited increased anxiety-like behavior, but these differences were not observed in early adolescent SERT(-/-) mice. Conversely, SERT(+/-) and SERT(-/-) mice did display higher spontaneous alternation during early adolescence and adulthood. SERT(+/-) and SERT(-/-) also exhibited greater neuronal spine densities in the orbitofrontal but not the medial prefrontal cortices. Adult SERT(-/-) mice also showed an increased spine density in the basolateral amygdala. Developmental alterations of the serotonergic system caused by genetic inactivation of SERT can have different influences on anxiety-like and cognitive behaviors through early adolescence into adulthood, which may be associated with changes of spine density in the prefrontal cortex and amygdala. The altered maturation of serotonergic systems may lead to specific age-related vulnerabilities to psychopathologies that develop during adolescence.

Recovery from impaired muscle growth arises from prolonged postnatal accretion of myonuclei in Atrx mutant mice

PubMed Central

Yan, Keqin; Price-O’Dea, Tina

2017-01-01

Reduced muscle mass due to pathological development can occur through several mechanisms, including the loss or reduced proliferation of muscle stem cells. Muscle-specific ablation of the α-thalassemia mental retardation syndrome mutant protein, Atrx, in transgenic mice results in animals with a severely reduced muscle mass at three weeks of age; yet this muscle mass reduction resolves by adult age. Here, we explore the cellular mechanism underlying this effect. Analysis of Atrx mutant mice included testing for grip strength and rotorod performance. Muscle fiber length, fiber volume and numbers of myofiber-associated nuclei were determined from individual EDL or soleus myofibers isolated at three, five, or eight weeks. Myofibers from three week old Atrx mutant mice are smaller with fewer myofiber-associated nuclei and reduced volume compared to control animals, despite similar fiber numbers. Nonetheless, the grip strength of Atrx mutant mice was comparable to control mice when adjusted for body weight. Myofiber volume remained smaller at five weeks, becoming comparable to controls by 8 weeks of age. Concomitantly, increased numbers of myofiber-associated nuclei and Ki67+ myoblasts indicated that the recovery of muscle mass likely arises from the prolonged accretion of new myonuclei. This suggests that under disease conditions the muscle satellite stem cell niche can remain in a prolonged active state, allowing for the addition of a minimum number of myonuclei required to achieve a normal muscle size. PMID:29095838

Hypervitaminosis D and premature aging: lessons learned from Fgf23 and Klotho mutant mice.

PubMed

Razzaque, Mohammed S; Lanske, Beate

2006-07-01

The essential role of low levels of vitamin D during aging is well documented. However, possible effects of high levels of vitamin D on the aging process are not yet clear. Recent in vivo genetic-manipulation studies have shown increased serum level of vitamin D and altered mineral-ion homeostasis in mice that lack either fibroblast growth factor 23 (Fgf23) or klotho (Kl) genes. These mice develop identical phenotypes consistent with premature aging. Elimination or reduction of vitamin-D activity from Fgf23 and Kl mutant mice, either by dietary restriction or genetic manipulation could rescue premature aging-like features and ectopic calcifications, resulting in prolonged survival of both mutants. Such in vivo experimental studies indicated that excessive vitamin-D activity and altered mineral-ion homeostasis could accelerate the aging process.

Increased Tau Phosphorylation and Tau Truncation, and Decreased Synaptophysin Levels in Mutant BRI2/Tau Transgenic Mice

PubMed Central

Garringer, Holly J.; Murrell, Jill; Sammeta, Neeraja; Gnezda, Anita; Ghetti, Bernardino; Vidal, Ruben

2013-01-01

Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease caused by a 10-nucleotide duplication-insertion in the BRI2 gene. FDD is clinically characterized by loss of vision, hearing impairment, cerebellar ataxia and dementia. The main neuropathologic findings in FDD are the deposition of Danish amyloid (ADan) and the presence of neurofibrillary tangles (NFTs). Here we investigated tau accumulation and truncation in double transgenic (Tg-FDD-Tau) mice generated by crossing transgenic mice expressing human Danish mutant BRI2 (Tg-FDD) with mice expressing human 4-repeat mutant Tau-P301S (Tg-Tau). Compared to Tg-Tau mice, we observed a significant enhancement of tau deposition in Tg-FDD-Tau mice. In addition, a significant increase in tau cleaved at aspartic acid (Asp) 421 was observed in Tg-FDD-Tau mice. Tg-FDD-Tau mice also showed a significant decrease in synaptophysin levels, occurring before widespread deposition of fibrillar ADan and tau can be observed. Thus, the presence of soluble ADan/mutant BRI2 can lead to significant changes in tau metabolism and synaptic dysfunction. Our data provide new in vivo insights into the pathogenesis of FDD and the pathogenic pathway(s) by which amyloidogenic peptides, regardless of their primary amino acid sequence, can cause neurodegeneration. PMID:23418567

Increased tau phosphorylation and tau truncation, and decreased synaptophysin levels in mutant BRI2/tau transgenic mice.

PubMed

Garringer, Holly J; Murrell, Jill; Sammeta, Neeraja; Gnezda, Anita; Ghetti, Bernardino; Vidal, Ruben

2013-01-01

Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease caused by a 10-nucleotide duplication-insertion in the BRI(2) gene. FDD is clinically characterized by loss of vision, hearing impairment, cerebellar ataxia and dementia. The main neuropathologic findings in FDD are the deposition of Danish amyloid (ADan) and the presence of neurofibrillary tangles (NFTs). Here we investigated tau accumulation and truncation in double transgenic (Tg-FDD-Tau) mice generated by crossing transgenic mice expressing human Danish mutant BRI(2) (Tg-FDD) with mice expressing human 4-repeat mutant Tau-P301S (Tg-Tau). Compared to Tg-Tau mice, we observed a significant enhancement of tau deposition in Tg-FDD-Tau mice. In addition, a significant increase in tau cleaved at aspartic acid (Asp) 421 was observed in Tg-FDD-Tau mice. Tg-FDD-Tau mice also showed a significant decrease in synaptophysin levels, occurring before widespread deposition of fibrillar ADan and tau can be observed. Thus, the presence of soluble ADan/mutant BRI(2) can lead to significant changes in tau metabolism and synaptic dysfunction. Our data provide new in vivo insights into the pathogenesis of FDD and the pathogenic pathway(s) by which amyloidogenic peptides, regardless of their primary amino acid sequence, can cause neurodegeneration.

[Accumulation of the bvg- Bordetella pertussis a virulent mutants in the process of experimental whooping cough in mice].

PubMed

Medkova, A Iu; Siniashina, L N; Rumiantseva, Iu P; Voronina, O L; Kunda, M S; Karataev, G I

2013-01-01

The duration of the persistence and dynamics of accumulation of insertion bvg- Bordetella pertussis mutants were studied in lungs of laboratory mice after intranasal and intravenous challenge by virulent bacteria of the causative agent of whooping cough. The capability of the virulent B. pertussis bacteria to long-term persistence in the body of mice was tested. Using the real-time PCR approximately hundred genome equivalents of the B. pertussis DNA were detected in lungs of mice in two months after infection regardless of the way of challenge. Using the bacterial test bacteria were identified during only four weeks after challenge. Bvg- B. pertussis avirulent mutants were accumulated for the infection time. The percentage of the avirulent bacteria in the B. pertussis population reached 50% in 7-9 weeks after challenge. The obtained results show that the laboratory mice can be used for study of the B. pertussis insertion mutant formation dynamics in vivo and confirm the hypothesis about insertional bvg- B. pertussis virulent mutants accumulation during development of pertussis infection in human.

Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.

PubMed

Kwok, Hang Fai; Botkjaer, Kenneth A; Tape, Christopher J; Huang, Yanchao; McCafferty, John; Murphy, Gillian

2014-06-01

We previously showed that a human anti-TACE antibody, D1(A12), is a potent inhibitor of TNF-α converting enzyme (TACE) ectodomain proteolysis and has pharmacokinetic properties suitable for studies of the inhibition of TACE-dependent growth factor shedding in relation to possible therapeutic applications. However, the lack of murine TACE immunoreactivity limits pre-clinical in vivo studies to human xenograft models which are poor analogies to in situ pathology and are not considered clinically predictive. Here, to overcome these limitations, we set out to develop a 'mouse and human cross-reactive' specific anti-TACE antibody. We first re-investigated the originally selected anti-TACE ectodomain phage-display clones, and isolated a lead 'mouse-human cross-reactive' anti-TACE scFv, clone A9. We reformatted scFv-A9 into an IgG2 framework for comprehensive biochemical and cellular characterization and further demonstrated that A9 is an exosite TACE inhibitor. However, surface plasmon resonance analysis and quenched-fluorescent (QF) peptide assay indicated that IgG reformatting of A9 caused low binding affinity and an 80-fold reduction in TACE ectodomain inhibition, severely limiting its efficacy. To address this, we constructed second generation phage-display randomization libraries focused on the complementarity-determining region 3, and carried out affinity selections shuffling between human and mouse TACE ectodomain as antigen in addition to an off-rate selection to increase the chance of affinity improvement. The bespoke 'three-step' selections enabled a 100-fold affinity enhancement of A9 IgG, and also improved its IC50 in a QF peptide assay to 0.2 nM. In human and mouse cancer cell assays, matured A9 IgG showed significant cell-surface TACE inhibition as a monotherapy or combination therapy with chemotherapeutic agent. Collectively, these data suggest that we successfully developed an exosite inhibitor of TACE with sub-nanomolar affinity, which possesses both

eIF4E/Fmr1 double mutant mice display cognitive impairment in addition to ASD-like behaviors.

PubMed

Huynh, Thu N; Shah, Manan; Koo, So Yeon; Faraud, Kirsten S; Santini, Emanuela; Klann, Eric

2015-11-01

Autism spectrum disorder (ASD) is a group of heritable disorders with complex and unclear etiology. Classic ASD symptoms include social interaction and communication deficits as well as restricted, repetitive behaviors. In addition, ASD is often comorbid with intellectual disability. Fragile X syndrome (FXS) is the leading genetic cause of ASD, and is the most commonly inherited form of intellectual disability. Several mouse models of ASD and FXS exist, however the intellectual disability observed in ASD patients is not well modeled in mice. Using the Fmr1 knockout mouse and the eIF4E transgenic mouse, two previously characterized mouse models of fragile X syndrome and ASD, respectively, we generated the eIF4E/Fmr1 double mutant mouse. Our study shows that the eIF4E/Fmr1 double mutant mice display classic ASD behaviors, as well as cognitive dysfunction. Importantly, the learning impairments displayed by the double mutant mice spanned multiple cognitive tasks. Moreover, the eIF4E/Fmr1 double mutant mice display increased levels of basal protein synthesis. The results of our study suggest that the eIF4E/Fmr1 double mutant mouse may be a reliable model to study cognitive dysfunction in the context of ASD. Copyright © 2015 Elsevier Inc. All rights reserved.

Prospective Evaluation of Transcatheter Arterial Chemoembolization (TACE) with Multiple Anti-Cancer Drugs (Epirubicin, Cisplatin, Mitomycin C, 5-Fluorouracil) Compared with TACE with Epirubicin for Treatment of Hepatocellular Carcinoma

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

Sahara, Shinya; Kawai, Nobuyuki; Sato, Morio, E-mail: morisato@mail.wakayama-med.ac.jp

Purpose: To compare the efficacy of transcatheter arterial chemoembolization (TACE) using multiple anticancer drugs (epirubicin, cisplatin, mitomycin C, and 5-furuorouracil: Multi group) with TACE using epirubicin (EP group) for hepatocellular carcinoma (HCC). Materials and Methods: The study design was a single-center, prospective, randomized controlled trial. Patients with unrespectable HCC confined to the liver, unsuitable for radiofrequency ablation, were assigned to the Multi group or the EP group. We assessed radiographic response as the primary endpoint; secondary endpoints were progression-free survival (PFS), safety, and hepatic branch artery abnormality (Grade I, no damage or mild vessel wall irregularity; Grade II, overt stenosis;more » Grade III, occlusion; Grades II and III indicated significant hepatic artery damage). A total of 51 patients were enrolled: 24 in the Multi group vs. 27 in the EP group. Results: No significant difference in HCC patient background was found between the groups. Radiographic response, PFS, and 1- and 2-year overall survival of the Multi vs. EP group were 54% vs. 48%, 6.1 months vs. 8.7 months, and 95% and 65% vs. 85% and 76%, respectively, with no significant difference. Significantly greater Grade 3 transaminase elevation was found in the Multi group (p = 0.023). Hepatic artery abnormality was observed in 34% of the Multi group and in 17.1% of the EP group (p = 0.019). Conclusion: TACE with multiple anti-cancer drugs was tolerable but appeared not to contribute to an increase in radiographic response or PFS, and caused significantly more hepatic arterial abnormalities compared with TACE with epirubicin alone.« less

Cortico-striatal synaptic defects and OCD-like behaviors in SAPAP3 mutant mice

PubMed Central

Welch, Jeffrey M.; Lu, Jing; Rodriguiz, Ramona M.; Trotta, Nicholas C.; Peca, Joao; Ding, Jin-Dong; Feliciano, Catia; Chen, Meng; Adams, J. Paige; Luo, Jianhong; Dudek, Serena M.; Weinberg, Richard J.; Calakos, Nicole; Wetsel, William C.; Feng, Guoping

2008-01-01

Obsessive-compulsive disorder (OCD) is an anxiety-spectrum disorder characterized by persistent intrusive thoughts (obsessions) and repetitive actions (compulsions). Dysfunction of cortico-striato-thalamo-cortical circuitry is implicated in OCD, though the underlying pathogenic mechanisms are unknown. SAP90/PSD95-associated protein 3 (SAPAP3) is a postsynaptic scaffolding protein at excitatory synapses that is highly expressed in the striatum. Here we show that mice with genetic deletion of SAPAP3 exhibit increased anxiety and compulsive grooming behavior leading to facial hair loss and skin lesions; both behaviors are alleviated by a selective serotonin reuptake inhibitor. Electrophysiological, structural, and biochemical studies of SAPAP3 mutant mice reveal defects in cortico-striatal synapses. Furthermore, lentiviral-mediated selective expression of SAPAP3 in the striatum rescues the synaptic and behavioral defects of SAPAP3 mutant mice. These findings demonstrate a critical role for SAPAP3 at cortico-striatal synapses and emphasize the importance of cortico-striatal circuitry in OCD-like behaviors. PMID:17713528

Alcohol reversibly disrupts TNF-α/TACE interactions in the cell membrane

PubMed Central

Song, Kejing; Zhao, Xue-Jun; Marrero, Luis; Oliver, Peter; Nelson, Steve; Kolls, Jay K

2005-01-01

Background Alcohol abuse has long been known to adversely affect innate and adaptive immune responses and pre-dispose to infections. One cellular mechanism responsible for this effect is alcohol-induced suppression of TNF-α (TNF) by mononuclear phagocytes. We have previously shown that alcohol in part inhibits TNF-α processing by TNF converting enzyme (TACE) in human monocytes. We hypothesized that the chain length of the alcohol is critical for post-transcriptional suppression of TNF secretion. Methods Due to the complex transcriptional and post-transcriptional regulation of TNF in macrophages, to specifically study TNF processing at the cell membrane we performed transient transfections of A549 cells with the TNF cDNA driven by the heterologous CMV promoter. TNF/TACE interactions at the cell surface were assessed using fluorescent resonance energy transfer (FRET) microscopy. Results The single carbon alcohol, methanol suppressed neither TNF secretion nor FRET efficiency between TNF and TACE. However, 2, 3, and 4 carbon alcohols were potent suppressors of TNF processing and FRET efficiency. The effect of ethanol, a 2-carbon alcohol was reversible. Conclusion These data show that inhibition of TNF-α processing by acute ethanol is a direct affect of ethanol on the cell membrane and is reversible upon cessation or metabolism. PMID:16246259

Studies on novel 2-imidazolidinones and tetrahydropyrimidin-2(1H)-ones as potential TACE inhibitors: design, synthesis, molecular modeling, and preliminary biological evaluation.

PubMed

DasGupta, Shirshendu; Murumkar, Prashant R; Giridhar, Rajani; Yadav, Mange Ram

2009-05-15

Compounds belonging to the class of 2-imidazolidinones and tetrahydropyrimidin-2(1H)-ones were synthesized and evaluated for their TACE inhibitory activity. Most of the compounds showed very good TACE inhibitory activity. Docking study clearly indicates importance of the P1' group of the inhibitor for the TACE inhibitory activity. This work proves that these two classes of molecules could be used as potential leads for the development of TACE inhibitors.

Abnormalities in brain structure and behavior in GSK-3alpha mutant mice

PubMed Central

2009-01-01

Background Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by two genes that generate two related proteins: GSK-3α and GSK-3β. Mice lacking a functional GSK-3α gene were engineered in our laboratory; they are viable and display insulin sensitivity. In this study, we have characterized brain functions of GSK-3α KO mice by using a well-established battery of behavioral tests together with neurochemical and neuroanatomical analysis. Results Similar to the previously described behaviours of GSK-3β+/-mice, GSK-3α mutants display decreased exploratory activity, decreased immobility time and reduced aggressive behavior. However, genetic inactivation of the GSK-3α gene was associated with: decreased locomotion and impaired motor coordination, increased grooming activity, loss of social motivation and novelty; enhanced sensorimotor gating and impaired associated memory and coordination. GSK-3α KO mice exhibited a deficit in fear conditioning, however memory formation as assessed by a passive avoidance test was normal, suggesting that the animals are sensitized for active avoidance of a highly aversive stimulus in the fear-conditioning paradigm. Changes in cerebellar structure and function were observed in mutant mice along with a significant decrease of the number and size of Purkinje cells. Conclusion Taken together, these data support a role for the GSK-3α gene in CNS functioning and possible involvement in the development of psychiatric disorders. PMID:19925672

Impaired Spermatogenesis, Muscle, and Erythrocyte Function in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

PubMed

Horiuchi, Keiko; Perez-Cerezales, Serafín; Papasaikas, Panagiotis; Ramos-Ibeas, Priscila; López-Cardona, Angela Patricia; Laguna-Barraza, Ricardo; Fonseca Balvís, Noelia; Pericuesta, Eva; Fernández-González, Raul; Planells, Benjamín; Viera, Alberto; Suja, Jose Angel; Ross, Pablo Juan; Alén, Francisco; Orio, Laura; Rodriguez de Fonseca, Fernando; Pintado, Belén; Valcárcel, Juan; Gutiérrez-Adán, Alfonso

2018-04-03

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Further studies on cortical tangential migration in wild type and Pax-6 mutant mice.

PubMed

Jiménez, D; López-Mascaraque, L; de Carlos, J A; Valverde, F

2002-01-01

In this study we present new data concerning the tangential migration from the medial and lateral ganglionic eminences (MGE and LGE) to the cerebral cortex during development. We have used Calbindin as a useful marker to follow the itinerary of tangential migratory cells during early developmental stages in wild-type and Pax-6 homozygous mutant mice. In the wild-type mice, at early developmental stages, migrating cells advance through the intermediate zone (IZ) and preplate (PP). At more advanced stages, migrating cells were present in the subplate (SP) and cortical plate (CP) to reach the entire developing cerebral cortex. We found that, in the homozygous mutant mice (Pax-6(Sey-Neu)/Pax-6(Sey-Neu)), this tangential migration is severely affected at early developmental stages: migrating cells were absent in the IZ, which were only found some days later, suggesting that in the mutant mice, there is a temporal delay in tangential migration. We have also defined some possible mechanisms to explain certain migratory routes from the basal telencephalon to the cerebral cortex. We describe the existence of two factors, which we consider to be essential for the normal migration; the first one is the cell adhesion molecule PSA-NCAM, whose role in other migratory systems is well known. The second factor is Robo-2, whose expression delimits a channel for the passage of migratory cells from the basal telencephalon to the cerebral cortex.

Ectopic Mineralization and Conductive Hearing Loss in Enpp1asj Mutant Mice, a New Model for Otitis Media and Tympanosclerosis.

PubMed

Tian, Cong; Harris, Belinda S; Johnson, Kenneth R

2016-01-01

Otitis media (OM), inflammation of the middle ear, is a common cause of hearing loss in children and in patients with many different syndromic diseases. Studies of the human population and mouse models have revealed that OM is a multifactorial disease with many environmental and genetic contributing factors. Here, we report on otitis media-related hearing loss in asj (ages with stiffened joints) mutant mice, which bear a point mutation in the Enpp1 gene. Auditory-evoked brainstem response (ABR) measurements revealed that around 90% of the mutant mice (Enpp1asj/asj) tested had moderate to severe hearing impairment in at least one ear. The ABR thresholds were variable and generally elevated with age. We found otitis media with effusion (OME) in all of the hearing-impaired Enpp1asj/asj mice by anatomic and histological examinations. The volume and inflammatory cell content of the effusion varied among the asj mutant mice, but all mutants exhibited a thickened middle ear epithelium with fibrous polyps and more mucin-secreting goblet cells than controls. Other abnormalities observed in the Enpp1 mutant mice include over-ossification at the round window ridge, thickened and over-calcified stapedial artery, fusion of malleus and incus, and white patches on the inside of tympanic membrane, some of which are typical symptoms of tympanosclerosis. An excessive yellow discharge was detected in the outer ear canal of older asj mutant mice, with 100% penetrance by 5 months of age, and contributes to the progressive nature of the hearing loss. This is the first report of hearing loss and ear pathology associated with an Enpp1 mutation in mice. The Enpp1asj mutant mouse provides a new animal model for studying tympanosclerotic otitis and otitis media with effusion, and also provides a specific model for the hearing loss recently reported to be associated with human ENPP1 mutations causing generalized arterial calcification of infancy and hypophosphatemic rickets.

Ectopic Mineralization and Conductive Hearing Loss in Enpp1asj Mutant Mice, a New Model for Otitis Media and Tympanosclerosis

PubMed Central

Tian, Cong; Harris, Belinda S.; Johnson, Kenneth R.

2016-01-01

Otitis media (OM), inflammation of the middle ear, is a common cause of hearing loss in children and in patients with many different syndromic diseases. Studies of the human population and mouse models have revealed that OM is a multifactorial disease with many environmental and genetic contributing factors. Here, we report on otitis media-related hearing loss in asj (ages with stiffened joints) mutant mice, which bear a point mutation in the Enpp1 gene. Auditory-evoked brainstem response (ABR) measurements revealed that around 90% of the mutant mice (Enpp1asj/asj) tested had moderate to severe hearing impairment in at least one ear. The ABR thresholds were variable and generally elevated with age. We found otitis media with effusion (OME) in all of the hearing-impaired Enpp1asj/asj mice by anatomic and histological examinations. The volume and inflammatory cell content of the effusion varied among the asj mutant mice, but all mutants exhibited a thickened middle ear epithelium with fibrous polyps and more mucin-secreting goblet cells than controls. Other abnormalities observed in the Enpp1 mutant mice include over-ossification at the round window ridge, thickened and over-calcified stapedial artery, fusion of malleus and incus, and white patches on the inside of tympanic membrane, some of which are typical symptoms of tympanosclerosis. An excessive yellow discharge was detected in the outer ear canal of older asj mutant mice, with 100% penetrance by 5 months of age, and contributes to the progressive nature of the hearing loss. This is the first report of hearing loss and ear pathology associated with an Enpp1 mutation in mice. The Enpp1asj mutant mouse provides a new animal model for studying tympanosclerotic otitis and otitis media with effusion, and also provides a specific model for the hearing loss recently reported to be associated with human ENPP1 mutations causing generalized arterial calcification of infancy and hypophosphatemic rickets. PMID:27959908

Schwann cell hyperplasia and tumors in transgenic mice expressing a naturally occurring mutant NF2 protein

PubMed Central

Giovannini, Marco; Robanus-Maandag, Els; Niwa-Kawakita, Michiko; van der Valk, Martin; Woodruff, James M.; Goutebroze, Laurence; Mérel, Philippe; Berns, Anton; Thomas, Gilles

1999-01-01

Specific mutations in some tumor suppressor genes such as p53 can act in a dominant fashion. We tested whether this mechanism may also apply for the neurofibromatosis type-2 gene (NF2) which, when mutated, leads to schwannoma development. Transgenic mice were generated that express, in Schwann cells, mutant NF2 proteins prototypic of natural mutants observed in humans. Mice expressing a NF2 protein with an interstitial deletion in the amino-terminal domain showed high prevalence of Schwann cell-derived tumors and Schwann cell hyperplasia, whereas those expressing a carboxy-terminally truncated protein were normal. Our results indicate that a subset of mutant NF2 alleles observed in patients may encode products with dominant properties when overexpressed in specific cell lineages. PMID:10215625

Motor hypertonia and lack of locomotor coordination in mutant mice lacking DSCAM.

PubMed

Lemieux, Maxime; Laflamme, Olivier D; Thiry, Louise; Boulanger-Piette, Antoine; Frenette, Jérôme; Bretzner, Frédéric

2016-03-01

Down syndrome cell adherence molecule (DSCAM) contributes to the normal establishment and maintenance of neural circuits. Whereas there is abundant literature regarding the role of DSCAM in the neural patterning of the mammalian retina, less is known about motor circuits. Recently, DSCAM mutation has been shown to impair bilateral motor coordination during respiration, thus causing death at birth. DSCAM mutants that survive through adulthood display a lack of locomotor endurance and coordination in the rotarod test, thus suggesting that the DSCAM mutation impairs motor control. We investigated the motor and locomotor functions of DSCAM(2J) mutant mice through a combination of anatomical, kinematic, force, and electromyographic recordings. With respect to wild-type mice, DSCAM(2J) mice displayed a longer swing phase with a limb hyperflexion at the expense of a shorter stance phase during locomotion. Furthermore, electromyographic activity in the flexor and extensor muscles was increased and coactivated over 20% of the step cycle over a wide range of walking speeds. In contrast to wild-type mice, which used lateral walk and trot at walking speed, DSCAM(2J) mice used preferentially less coordinated gaits, such as out-of-phase walk and pace. The neuromuscular junction and the contractile properties of muscles, as well as their muscle spindles, were normal, and no signs of motor rigidity or spasticity were observed during passive limb movements. Our study demonstrates that the DSCAM mutation induces dystonic hypertonia and a disruption of locomotor gaits. Copyright © 2016 the American Physiological Society.

Role of tumour necrosis factor alpha converting enzyme (TACE/ADAM17) and associated proteins in coronary artery disease and cardiac events.

PubMed

Chemaly, Melody; McGilligan, Victoria; Gibson, Mark; Clauss, Matthias; Watterson, Steven; Alexander, H Denis; Bjourson, Anthony John; Peace, Aaron

2017-12-01

Tumour necrosis factor alpha converting enzyme (TACE/ADAM17) is a member of the A disintegrin and metalloproteinase (ADAM) family of ectodomain shedding proteinases. It regulates many inflammatory processes by cleaving several transmembrane proteins, including tumour necrosis factor alpha (TNFα) and its receptors tumour necrosis factor alpha receptor 1 and tumour necrosis factor alpha receptor 2. There is evidence that TACE is involved in several inflammatory diseases, such as ischaemia, heart failure, arthritis, atherosclerosis, diabetes and cancer as well as neurological and immune diseases. This review summarizes the latest discoveries regarding the mechanism of action and regulation of TACE. It also focuses on the role of TACE in atherosclerosis and coronary artery disease (CAD), highlighting clinical studies that have investigated its expression and protein activity. The multitude of substrates cleaved by TACE make this enzyme an attractive target for therapy and a candidate for biomarker research and development in CAD. Crown Copyright © 2017. Published by Elsevier Masson SAS. All rights reserved.

Comparing the effectiveness of TWEAK and T-ACE in determining problem drinkers in pregnancy.

PubMed

Sarkar, M; Einarson, T; Koren, G

2010-01-01

The TWEAK and T-ACE screening tools are validated methods of identifying problem drinking in a pregnant population. The objective of this study was to compare the effectiveness of the TWEAK and T-ACE screening tools in identifying problem drinking using traditional cut-points (CP). Study participants consisted of women calling the Motherisk Alcohol Helpline for information regarding their alcohol use in pregnancy. In this cohort, concerns surrounding underreporting are not likely as women self-report their alcohol consumption. Participant's self-identification, confirmed by her amount of alcohol use, determined whether she was a problem drinker or not. The TWEAK and T-ACE tools were administered on both groups and subsequent analysis was done to determine if one tool was more effective in predicting problem drinking. The study consisted of 75 problem and 100 non-problem drinkers. Using traditional CP, the TWEAK and T-ACE tools both performed similarly at identifying potential at-risk women (positive predictive value = 0.54), with very high sensitivity rates (100-99% and 100-93%, respectively) but poor specificity rates (36-43% and 19-34%, respectively). Upon comparison, there was no statistical difference in the effectiveness for one test performing better than next using either CP of 2 (P = 0.66) or CP of 3 (P = 0.38). Despite the lack of difference in performance, improved specificity associated with TWEAK suggests that it may be better suited to screen at-risk populations seeking advice from a helpline.

Tissue-specific and time-dependent clonal expansion of ENU-induced mutant cells in gpt delta mice.

PubMed

Nakayama, Takafumi; Sawai, Tomoko; Masuda, Ikuko; Kaneko, Shinya; Yamauchi, Kazumi; Blyth, Benjamin J; Shimada, Yoshiya; Tachibana, Akira; Kakinuma, Shizuko

2017-10-01

DNA mutations play a crucial role in the origins of cancer, and the clonal expansion of mutant cells is one of the fundamental steps in multistage carcinogenesis. In this study, we correlated tumor incidence in B6C3F1 mice during the period after exposure to N-ethyl-N-nitrosourea (ENU) with the persistence of ENU-induced mutant clones in transgenic gpt delta B6C3F1 mice. The induced gpt mutations afforded no selective advantage in the mouse cells and could be distinguished by a mutational spectrum that is characteristic of ENU treatment. The gpt mutations were passengers of the mutant cell of origin and its daughter cells and thus could be used as neutral markers of clones that arose and persisted in the tissues. Female B6C3F1 mice exposed for 1 month to 200 ppm ENU in the drinking water developed early thymic lymphomas and late liver and lung tumors. To assay gpt mutations, we sampled the thymus, liver, lung, and small intestine of female gpt delta mice at 3 days, 4 weeks, and 8 weeks after the end of ENU exposure. Our results reveal that, in all four tissues, the ENU-induced gpt mutations persisted for weeks after the end of mutagen exposure. Clonal expansion of mutant cells was observed in the thymus and small intestine, with the thymus showing larger clone sizes. These results indicate that the clearance of mutant cells and the potential for clonal expansion during normal tissue growth depends on tissue type and that these factors may affect the sensitivity of different tissues to carcinogenesis. Environ. Mol. Mutagen. 58:592-606, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Seizure susceptibility of neuropeptide-Y null mutant mice in amygdala kindling and chemical-induced seizure models.

PubMed

Shannon, Harlan E; Yang, Lijuan

2004-01-01

Neuropeptide Y (NPY) administered exogenously is anticonvulsant, and, NPY null mutant mice are more susceptible to kainate-induced seizures. In order to better understand the potential role of NPY in epileptogenesis, the present studies investigated the development of amygdala kindling, post-kindling seizure thresholds, and anticonvulsant effects of carbamazepine and levetiracetam in 129S6/SvEv NPY(+/+) and NPY(-/-) mice. In addition, susceptibility to pilocarpine- and kainate-induced seizures was compared in NPY(+/+) and (-/-) mice. The rate of amygdala kindling development did not differ in the NPY(-/-) and NPY(+/+) mice either when kindling stimuli were presented once daily for at least 20 days, or, 12 times daily for 2 days. However, during kindling development, the NPY(-/-) mice had higher seizure severity scores and longer afterdischarge durations than the NPY(+/+) mice. Post-kindling, the NPY(-/-) mice had markedly lower afterdischarge thresholds and longer afterdischarge durations than NPY (+/+) mice. Carbamazepine and levetiracetam increased the seizure thresholds of both NPY (-/-) and (+/+) mice. In addition, NPY (-/-) mice had lower thresholds for both kainate- and pilocarpine-induced seizures. The present results in amygdala kindling and chemical seizure models suggest that NPY may play a more prominent role in determining seizure thresholds and severity of seizures than in events leading to epileptogenesis. In addition, a lack of NPY does not appear to confer drug-resistance in that carbamazepine and levetiracetam were anticonvulsant in both wild type (WT) and NPY null mutant mice.

Modeled changes of cerebellar activity in mutant mice are predictive of their learning impairments

NASA Astrophysics Data System (ADS)

Badura, Aleksandra; Clopath, Claudia; Schonewille, Martijn; de Zeeuw, Chris I.

2016-11-01

Translating neuronal activity to measurable behavioral changes has been a long-standing goal of systems neuroscience. Recently, we have developed a model of phase-reversal learning of the vestibulo-ocular reflex, a well-established, cerebellar-dependent task. The model, comprising both the cerebellar cortex and vestibular nuclei, reproduces behavioral data and accounts for the changes in neural activity during learning in wild type mice. Here, we used our model to predict Purkinje cell spiking as well as behavior before and after learning of five different lines of mutant mice with distinct cell-specific alterations of the cerebellar cortical circuitry. We tested these predictions by obtaining electrophysiological data depicting changes in neuronal spiking. We show that our data is largely consistent with the model predictions for simple spike modulation of Purkinje cells and concomitant behavioral learning in four of the mutants. In addition, our model accurately predicts a shift in simple spike activity in a mutant mouse with a brainstem specific mutation. This combination of electrophysiological and computational techniques opens a possibility of predicting behavioral impairments from neural activity.

Modeled changes of cerebellar activity in mutant mice are predictive of their learning impairments

PubMed Central

Badura, Aleksandra; Clopath, Claudia; Schonewille, Martijn; De Zeeuw, Chris I.

2016-01-01

Translating neuronal activity to measurable behavioral changes has been a long-standing goal of systems neuroscience. Recently, we have developed a model of phase-reversal learning of the vestibulo-ocular reflex, a well-established, cerebellar-dependent task. The model, comprising both the cerebellar cortex and vestibular nuclei, reproduces behavioral data and accounts for the changes in neural activity during learning in wild type mice. Here, we used our model to predict Purkinje cell spiking as well as behavior before and after learning of five different lines of mutant mice with distinct cell-specific alterations of the cerebellar cortical circuitry. We tested these predictions by obtaining electrophysiological data depicting changes in neuronal spiking. We show that our data is largely consistent with the model predictions for simple spike modulation of Purkinje cells and concomitant behavioral learning in four of the mutants. In addition, our model accurately predicts a shift in simple spike activity in a mutant mouse with a brainstem specific mutation. This combination of electrophysiological and computational techniques opens a possibility of predicting behavioral impairments from neural activity. PMID:27805050

Deficient plasticity in the primary visual cortex of alpha-calcium/calmodulin-dependent protein kinase II mutant mice.

PubMed

Gordon, J A; Cioffi, D; Silva, A J; Stryker, M P

1996-09-01

The recent characterization of plasticity in the mouse visual cortex permits the use of mutant mice to investigate the cellular mechanisms underlying activity-dependent development. As calcium-dependent signaling pathways have been implicated in neuronal plasticity, we examined visual cortical plasticity in mice lacking the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha CaMKII). In wild-type mice, brief occlusion of vision in one eye during a critical period reduces responses in the visual cortex. In half of the alpha CaMKII-deficient mice, visual cortical responses developed normally, but visual cortical plasticity was greatly diminished. After intensive training, spatial learning in the Morris water maze was severely impaired in a similar fraction of mutant animals. These data indicate that loss of alpha CaMKII results in a severe but variable defect in neuronal plasticity.

Tissue-specific oxidative stress and loss of mitochondria in CoQ-deficient Pdss2 mutant mice.

PubMed

Quinzii, Catarina M; Garone, Caterina; Emmanuele, Valentina; Tadesse, Saba; Krishna, Sindu; Dorado, Beatriz; Hirano, Michio

2013-02-01

Primary human CoQ(10) deficiencies are clinically heterogeneous diseases caused by mutations in PDSS2 and other genes required for CoQ(10) biosynthesis. Our in vitro studies of PDSS2 mutant fibroblasts, with <20% CoQ(10) of control cells, revealed reduced activity of CoQ(10)-dependent complex II+III and ATP synthesis, without amplification of reactive oxygen species (ROS), markers of oxidative damage, or antioxidant defenses. In contrast, COQ2 and ADCK3 mutant fibroblasts, with 30-50% CoQ(10) of controls, showed milder bioenergetic defects but significantly increased ROS and oxidation of lipids and proteins. We hypothesized that absence of oxidative stress markers and cell death in PDSS2 mutant fibroblasts were due to the extreme severity of CoQ(10) deficiency. Here, we have investigated in vivo effects of Pdss2 deficiency in affected and unaffected organs of CBA/Pdss2(kd/kd) mice at presymptomatic, phenotypic-onset, and end-stages of the disease. Although Pdss2 mutant mice manifest widespread CoQ(9) deficiency and mitochondrial respiratory chain abnormalities, only affected organs show increased ROS production, oxidative stress, mitochondrial DNA depletion, and reduced citrate synthase activity, an index of mitochondrial mass. Our data indicate that kidney-specific loss of mitochondria triggered by oxidative stress may be the cause of renal failure in Pdss2(kd/kd) mice.

Improvement in motor performance of Weaver mutant mice following lesions of the cerebellum.

PubMed

Grüsser, C; Grüsser-Cornehls, U

1998-12-01

In Weaver mutants (B6CBA wv/wv) cerebellar granule cells degenerate almost completely postnatally. A partial loss of Purkinje cells (PC) and a degeneration of dopaminergic cells in the substantia nigra have also been found. Weaver mice suffer from striking motor symptoms, including difficulty in walking without toppling over. In an attempt to influence the poor motor performance, the cerebellum in young animals was removed, thus eliminating the faulty output of surviving PCs, demonstrated electrophysiologically. Unoperated Weaver, lesioned wildtypes and one sham mouse were used as controls. Before and after operation, a battery of behavioural tests was performed. In Weaver mice, tumbling to the side (t) and the relation of t to the motor activity (k) while traversing an open-field matrix, (t/k), improved considerably, as did manoeuvring on a slanted wire mesh, but keeping balance on a wooden bench did not to the same degree. Locomotor activity alone improved in some animals. In wildtypes no significant changes occurred after operation, with the exception of a strong reduction in locomotor activity. The experiments demonstrate that the motor performance in Weaver mutant mice benefits from removal of their cerebellum.

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.

Mutant Huntingtin Gene-Dose Impacts on Aggregate Deposition, DARPP32 Expression and Neuroinflammation in HdhQ150 Mice

PubMed Central

Young, Douglas; Mayer, Franziska; Vidotto, Nella; Schweizer, Tatjana; Berth, Ramon; Abramowski, Dorothee; Shimshek, Derya R.; van der Putten, P. Herman; Schmid, Peter

2013-01-01

Huntington's disease (HD) is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (∼150 CAGs) in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i) an increase of neuronal intra-nuclear inclusions (NIIs) in brain, (ii) an increase of extra-nuclear aggregates in dentate gyrus, (iii) a decrease of DARPP32 protein and (iv) an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model. PMID:24086450

Transgenic mice expressing mutant Pinin exhibit muscular dystrophy, nebulin deficiency and elevated expression of slow-type muscle fiber genes

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

Wu, Hsu-Pin; Hsu, Shu-Yuan; Wu, Wen-Ai

Highlights: •Pnn CCD domain functions as a dominant negative mutant regulating Pnn expression and function. •Pnn CCD mutant Tg mice have a muscle wasting phenotype during development and show dystrophic histological features. •Pnn mutant muscles are susceptible to slow fiber type gene transition and NEB reduction. •The Tg mouse generated by overexpression of the Pnn CCD domain displays many characteristics resembling NEB{sup +/−} mice. -- Abstract: Pinin (Pnn) is a nuclear speckle-associated SR-like protein. The N-terminal region of the Pnn protein sequence is highly conserved from mammals to insects, but the C-terminal RS domain-containing region is absent in lower species.more » The N-terminal coiled-coil domain (CCD) is, therefore, of interest not only from a functional point of view, but also from an evolutionarily standpoint. To explore the biological role of the Pnn CCD in a physiological context, we generated transgenic mice overexpressing Pnn mutant in skeletal muscle. We found that overexpression of the CCD reduces endogenous Pnn expression in cultured cell lines as well as in transgenic skeletal muscle fibers. Pnn mutant mice exhibited reduced body mass and impaired muscle function during development. Mutant skeletal muscles show dystrophic histological features with muscle fibers heavily loaded with centrally located myonuclei. Expression profiling and pathway analysis identified over-representation of genes in gene categories associated with muscle contraction, specifically those related to slow type fiber. In addition nebulin (NEB) expression level is repressed in Pnn mutant skeletal muscle. We conclude that Pnn downregulation in skeletal muscle causes a muscular dystrophic phenotype associated with NEB deficiency and the CCD domain is incapable of replacing full length Pnn in terms of functional capacity.« less

Deficient Gene Expression in Protein Kinase Inhibitor α Null Mutant Mice

PubMed Central

Gangolli, Esha A.; Belyamani, Mouna; Muchinsky, Sara; Narula, Anita; Burton, Kimberly A.; McKnight, G. Stanley; Uhler, Michael D.; Idzerda, Rejean L.

2000-01-01

Protein kinase inhibitor (PKI) is a potent endogenous inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKA). It functions by binding the free catalytic (C) subunit with a high affinity and is also known to export nuclear C subunit to the cytoplasm. The significance of these actions with respect to PKI's physiological role is not well understood. To address this, we have generated by homologous recombination mutant mice that are deficient in PKIα, one of the three isoforms of PKI. The mice completely lack PKI activity in skeletal muscle and, surprisingly, show decreased basal and isoproterenol-induced gene expression in muscle. Further examination revealed reduced levels of the phosphorylated (active) form of the transcription factor CREB (cAMP response element binding protein) in the knockouts. This phenomenon stems, at least in part, from lower basal PKA activity levels in the mutants, arising from a compensatory increase in the level of the RIα subunit of PKA. The deficit in gene induction, however, is not easily explained by current models of PKI function and suggests that PKI may play an as yet undescribed role in PKA signaling. PMID:10779334

Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines.

PubMed

Horsch, Marion; Aguilar-Pimentel, Juan Antonio; Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T; Lund, Anders H; Lee, Icksoo; Grossman, Lawrence I; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; de Angelis, Martin Hrabĕ; Beckers, Johannes

2015-01-01

We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype-envirotype interactions for other diseases.

Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines

PubMed Central

Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T.; Lund, Anders H.; Lee, Icksoo; Grossman, Lawrence I.; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; Hrabĕ de Angelis, Martin; Beckers, Johannes

2015-01-01

We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype – envirotype interactions for other diseases. PMID:26263558

Hypoxia-activated cytotoxic agent tirapazamine enhances hepatic artery ligation-induced killing of liver tumor in HBx transgenic mice

PubMed Central

Lin, Wei-Hsiang; Yeh, Shiou-Hwei; Yeh, Kun-Huei; Chen, Kai-Wei; Cheng, Ya-Wen; Su, Tung-Hung; Jao, Ping; Ni, Lin-Chun; Chen, Pei-Jer; Chen, Ding-Shinn

2016-01-01

Transarterial chemoembolization (TACE) is the main treatment for intermediate stage hepatocellular carcinoma (HCC) with Barcelona Clinic Liver Cancer classification because of its exclusive arterial blood supply. Although TACE achieves substantial necrosis of the tumor, complete tumor necrosis is uncommon, and the residual tumor generally rapidly recurs. We combined tirapazamine (TPZ), a hypoxia-activated cytotoxic agent, with hepatic artery ligation (HAL), which recapitulates transarterial embolization in mouse models, to enhance the efficacy of TACE. The effectiveness of this combination treatment was examined in HCC that spontaneously developed in hepatitis B virus X protein (HBx) transgenic mice. We proved that the tumor blood flow in this model was exclusively supplied by the hepatic artery, in contrast to conventional orthotopic HCC xenografts that receive both arterial and venous blood supplies. At levels below the threshold oxygen levels created by HAL, TPZ was activated and killed the hypoxic cells, but spared the normoxic cells. This combination treatment clearly limited the toxicity of TPZ to HCC, which caused the rapid and near-complete necrosis of HCC. In conclusion, the combination of TPZ and HAL showed a synergistic tumor killing activity that was specific for HCC in HBx transgenic mice. This preclinical study forms the basis for the ongoing clinical program for the TPZ-TACE regimen in HCC treatment. PMID:27702890

Searching for biomarkers of CDKL5 disorder: early-onset visual impairment in CDKL5 mutant mice

PubMed Central

Mazziotti, Raffaele; Lupori, Leonardo; Sagona, Giulia; Gennaro, Mariangela; Della Sala, Grazia; Putignano, Elena

2017-01-01

Abstract CDKL5 disorder is a neurodevelopmental disorder still without a cure. Murine models of CDKL5 disorder have been recently generated raising the possibility of preclinical testing of treatments. However, unbiased, quantitative biomarkers of high translational value to monitor brain function are still missing. Moreover, the analysis of treatment is hindered by the challenge of repeatedly and non-invasively testing neuronal function. We analyzed the development of visual responses in a mouse model of CDKL5 disorder to introduce visually evoked responses as a quantitative method to assess cortical circuit function. Cortical visual responses were assessed in CDKL5 null male mice, heterozygous females, and their respective control wild-type littermates by repeated transcranial optical imaging from P27 until P32. No difference between wild-type and mutant mice was present at P25-P26 whereas defective responses appeared from P27-P28 both in heterozygous and homozygous CDKL5 mutant mice. These results were confirmed by visually evoked potentials (VEPs) recorded from the visual cortex of a different cohort. The previously imaged mice were also analyzed at P60–80 using VEPs, revealing a persistent reduction of response amplitude, reduced visual acuity and defective contrast function. The level of adult impairment was significantly correlated with the reduction in visual responses observed during development. Support vector machine showed that multi-dimensional visual assessment can be used to automatically classify mutant and wt mice with high reliability. Thus, monitoring visual responses represents a promising biomarker for preclinical and clinical studies on CDKL5 disorder. PMID:28369421

DISRUPTION OF THE PACAP GENE PROMOTES MEDULLOBLASTOMA IN PTC1 MUTANT MICE

PubMed Central

Lelievre, Vincent; Seksenyan, Akop; Nobuta, Hiroko; Yong, William H.; Chhith, Seririthanar; Niewiadomski, Pawel; Cohen, Joseph R.; Dong, Hongmei; Flores, Avegail; Liau, Linda M.; Kornblum, Harley I.; Scott, Matthew P.; Waschek, James A.

2008-01-01

Hedgehog (Hh) proteins and cAMP-dependent protein kinase A (PKA) generally play opposing roles in developmental patterning events. Humans and mice heterozygous for mutations in the Sonic hedgehog (Shh) receptor gene patched-1 (ptc1) have an increased incidence of certain types of cancer, including medulloblastoma (MB), a highly aggressive tumor of the cerebellum. Despite the importance of PKA in Hh signaling, little is known about how PKA activity is regulated in the context of Hh signaling, or the consequences of improper regulation. One molecule that can influence PKA activity is pituitary adenylyl cyclase activating peptide (PACAP), which has been shown to regulate cerebellar granule precursor proliferation in vitro, a cell population thought to give rise to MB. To test for a PACAP/Hh interaction in the initiation or propagation of these tumors, we introduced a PACAP mutation into ptc1 mutant mice. Deletion of a single copy of PACAP increased MB incidence approximate 2.5-fold, to 66%, thereby demonstrating that PACAP exerts a powerful inhibitory action on the induction, growth or survival of these tumors. Tumors from PACAP/ptc1 mutant mice retained PACAP receptor gene expression, and exhibited superinduction of Hh target genes compared to those from ptc1+/− mice. Moreover, PACAP inhibited proliferation of cell lines derived from tumors in a PKA-dependent manner, and inhibited expression of the Hh target gene gli1. The results provide genetic evidence that PACAP acts as a physiological factor that regulates the pathogenesis of Hh pathway-associated MB tumors. PMID:18036580

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.

Purkinje cells from RyR2 mutant mice are highly arrhythmogenic but responsive to targeted therapy.

PubMed

Kang, Guoxin; Giovannone, Steven F; Liu, Nian; Liu, Fang-Yu; Zhang, Jie; Priori, Silvia G; Fishman, Glenn I

2010-08-20

The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca(2+) release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking. We sought to determine the frequency and severity of spontaneous Ca(2+) release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2(R4496C/+) CPVT mutant mice and littermate controls. We crossed RyR2(R4496C/+) knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP(-)) and Purkinje cells (EGFP(+)) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) dynamics recorded by microfluorimetry. Both wild-type and RyR2(R4496C/+) mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and tau(decay) in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2(R4496C/+) mutant Purkinje cells were also most likely to develop spontaneous Ca(2+) release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca(2+(i)) events, the RyR2(R4496C/+) Purkinje cells responded with the most profound abnormalities in intracellular Ca(2+) handling, including a significant increase in the frequency of unstimulated Ca(2+(i)) events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca(2+) release events with flecainide, whereas in RyR2(R

Suppression of intestinal carcinogenesis in Apc-mutant mice by limonin.

PubMed

Shimizu, Satomi; Miyamoto, Shingo; Fujii, Gen; Nakanishi, Ruri; Onuma, Wakana; Ozaki, Yoshihiko; Fujimoto, Kyoko; Yano, Tomohiro; Mutoh, Michihiro

2015-07-01

Limonoids in citrus fruits are known to possess multiple biological functions, such as anti-proliferative functions in human cancer cell lines. Therefore, we aimed to investigate the suppressive effect of limonin on intestinal polyp development in Apc-mutant Min mice. Five-week-old female Min mice were fed a basal diet or a diet containing 250 or 500 ppm limonin for 8 weeks. The total number of polyps in mice treated with 500 ppm limonin decreased to 74% of the untreated control value. Neoplastic cell proliferation in the polyp parts was assessed by counting PCNA positive cells, and a tendency of reduction was obtained by limonin treatment. Moreover, expression levels of c-Myc and MCP-1 mRNA in the polyp part were reduced by administration of limonin. We finally confirmed the effects of limonin on β-catenin signaling, and found limonin significantly inhibited T-cell factor/lymphocyte enhancer factor-dependent transcriptional activity in a dose-dependent manner in the Caco-2 human colon cancer cell line. Our results suggest that limonin might be a candidate chemopreventive agent against intestinal carcinogenesis.

Searching for biomarkers of CDKL5 disorder: early-onset visual impairment in CDKL5 mutant mice.

PubMed

Mazziotti, Raffaele; Lupori, Leonardo; Sagona, Giulia; Gennaro, Mariangela; Della Sala, Grazia; Putignano, Elena; Pizzorusso, Tommaso

2017-06-15

CDKL5 disorder is a neurodevelopmental disorder still without a cure. Murine models of CDKL5 disorder have been recently generated raising the possibility of preclinical testing of treatments. However, unbiased, quantitative biomarkers of high translational value to monitor brain function are still missing. Moreover, the analysis of treatment is hindered by the challenge of repeatedly and non-invasively testing neuronal function. We analyzed the development of visual responses in a mouse model of CDKL5 disorder to introduce visually evoked responses as a quantitative method to assess cortical circuit function. Cortical visual responses were assessed in CDKL5 null male mice, heterozygous females, and their respective control wild-type littermates by repeated transcranial optical imaging from P27 until P32. No difference between wild-type and mutant mice was present at P25-P26 whereas defective responses appeared from P27-P28 both in heterozygous and homozygous CDKL5 mutant mice. These results were confirmed by visually evoked potentials (VEPs) recorded from the visual cortex of a different cohort. The previously imaged mice were also analyzed at P60-80 using VEPs, revealing a persistent reduction of response amplitude, reduced visual acuity and defective contrast function. The level of adult impairment was significantly correlated with the reduction in visual responses observed during development. Support vector machine showed that multi-dimensional visual assessment can be used to automatically classify mutant and wt mice with high reliability. Thus, monitoring visual responses represents a promising biomarker for preclinical and clinical studies on CDKL5 disorder. © The Author 2017. Published by Oxford University Press.

Reduced heart size and increased myocardial fuel substrate oxidation in ACC2 mutant mice

PubMed Central

Essop, M. Faadiel; Camp, Heidi S.; Choi, Cheol Soo; Sharma, Saumya; Fryer, Ryan M.; Reinhart, Glenn A.; Guthrie, Patrick H.; Bentebibel, Assia; Gu, Zeiwei; Shulman, Gerald I.; Taegtmeyer, Heinrich; Wakil, Salih J.; Abu-Elheiga, Lutfi

2008-01-01

The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC2) is a key regulator of mitochondrial fatty acid (FA) uptake via carnitine palmitoyltransferase 1 (CPT1). To test the hypothesis that oxidative metabolism is upregulated in hearts from animals lacking ACC2 (employing a transgenic Acc2-mutant mouse), we assessed cardiac function in vivo and determined rates of myocardial substrate oxidation ex vivo. When examined by echocardiography, there was no difference in systolic function, but left ventricular mass of the Acc2-mutant (MUT) mouse was significantly reduced (∼25%) compared with wild-types (WT). Reduced activation of the mammalian target of rapamycin (mTOR) and its downstream target p70S6K was found in MUT hearts. Exogenous oxidation rates of oleate were increased ∼22%, and, unexpectedly, exogenous glucose oxidation rates were also increased in MUT hearts. Using a hyperinsulinemic-euglycemic clamp, we found that glucose uptake in MUT hearts was increased by ∼83%. Myocardial triglyceride levels were significantly reduced in MUT vs. WT while glycogen content was the same. In parallel, transcript levels of PPARα and its target genes, pyruvate dehydrogenase kinase-4 (PDK-4), malonyl-CoA decarboxylase (MCD), and mCPT1, were downregulated in MUT mice. In summary, we report that 1) Acc2-mutant hearts exhibit a marked preference for the oxidation of both glucose and FAs coupled with greater utilization of endogenous fuel substrates (triglycerides), 2) attenuated mTOR signaling may result in reduced heart sizes observed in Acc2-mutant mice, and 3) Acc2-mutant hearts displayed normal functional parameters despite a significant decrease in size. PMID:18487439

Radiation Segmentectomy versus TACE Combined with Microwave Ablation for Unresectable Solitary Hepatocellular Carcinoma Up to 3 cm: A Propensity Score Matching Study.

PubMed

Biederman, Derek M; Titano, Joseph J; Bishay, Vivian L; Durrani, Raisa J; Dayan, Etan; Tabori, Nora; Patel, Rahul S; Nowakowski, Francis S; Fischman, Aaron M; Kim, Edward

2017-06-01

Purpose To compare the outcomes of radiation segmentectomy (RS) and transarterial chemoembolization (TACE) combined with microwave ablation (MWA) in the treatment of unresectable solitary hepatocellular carcinoma (HCC) up to 3 cm. Materials and Methods This retrospective study was approved by the institutional review board, and the requirement to obtain informed consent was waived. From January 2010 to June 2015, a total of 417 and 235 consecutive patients with HCC underwent RS and TACE MWA, respectively. A cohort of 121 patients who had not previously undergone local-regional therapy (RS, 41; TACE MWA, 80; mean age, 65.4 years; 84 men [69.4%]) and who had solitary HCC up to 3 cm without vascular invasion or metastasis was retrospectively identified. Outcomes analyzed included procedure-related complications, laboratory toxicity levels, imaging response, time to progression (TTP), 90-day mortality, and survival. Propensity score matching was conducted by using a nearest-neighbor algorithm (1:1) to account for pretreatment clinical, laboratory, and imaging covariates. Postmatching statistical analysis was performed with conditional logistic regression for binary outcomes and the stratified log-rank test for time-dependent outcomes. Results Before matching, the complication rate was 8.9% and 4.9% in the TACE MWA and RS groups, respectively (P = .46). The overall complete response (CR) rate was 82.9% for RS and 82.5% for TACE MWA (odds ratio, 1.0; 95% confidence interval [CI]: 0.4, 2.8; P = .95). There were 41 (RS, 11; TACE MWA, 30) instances of progression occurring after an initial CR, of which 10 (24%) were classified as target progression (RS, one; TACE MWA, nine). Median overall TTP was 11.1 months (95% CI: 8.8 months, 25.6 months) in the RS group and 12.1 months (95% CI: 7.7 months, 19.1 months) in the TACE MWA group (P > .99). After matching, the overall CR rate (P = .94), TTP (P = .83), and overall survival (P > .99) were not significantly different between

A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.

PubMed

Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, Nicolas

2014-05-21

Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions. Copyright © 2014 the authors 0270-6474/14/347203-13$15.00/0.

Shank3 mutant mice display autistic-like behaviours and striatal dysfunction

PubMed Central

Peça, João; Feliciano, Cátia; Ting, Jonathan T.; Wang, Wenting; Wells, Michael F.; Venkatraman, Talaignair N.; Lascola, Christopher D.; Fu, Zhanyan; Feng, Guoping

2011-01-01

Autism spectrum disorders (ASDs) comprise a range of disorders that share a core of neurobehavioural deficits characterized by widespread abnormalities in social interactions, deficits in communication as well as restricted interests and repetitive behaviours. The neurological basis and circuitry mechanisms underlying these abnormal behaviours are poorly understood. Shank3 is a postsynaptic protein, whose disruption at the genetic level is thought to be responsible for development of 22q13 deletion syndrome (Phelan-McDermid Syndrome) and other non-syndromic ASDs. Here we show that mice with Shank3 gene deletions exhibit self-injurious repetitive grooming and deficits in social interaction. Cellular, electrophysiological and biochemical analyses uncovered defects at striatal synapses and cortico-striatal circuits in Shank3 mutant mice. Our findings demonstrate a critical role for Shank3 in the normal development of neuronal connectivity and establish causality between a disruption in the Shank3 gene and the genesis of autistic like-behaviours in mice. PMID:21423165

Diet-induced obesity increases the frequency of Pig-a mutant erythrocytes in male C57BL/6J mice.

PubMed

Wickliffe, Jeffrey K; Dertinger, Stephen D; Torous, Dorothea K; Avlasevich, Svetlana L; Simon-Friedt, Bridget R; Wilson, Mark J

2016-12-01

Obesity increases the risk of a number of chronic diseases in humans including several cancers. Biological mechanisms responsible for such increased risks are not well understood at present. Increases in systemic inflammation and oxidative stress, endogenous production of mutagenic metabolites, altered signaling in proliferative pathways, and increased sensitivity to exogenous mutagens and carcinogens are some of the potential contributing factors. We hypothesize that obesity creates an endogenously mutagenic environment in addition to increasing the sensitivity to environmental mutagens. To test this hypothesis, we examined two in vivo genotoxicity endpoints. Pig-a mutant frequencies and micronucleus frequencies were determined in blood cells in two independent experiments in 30-week old male mice reared on either a high-fat diet (60% calories from fat) that exhibit an obese phenotype or a normal-fat diet (10% calories from fat) that do not exhibit an obese phenotype. Mice were assayed again at 52 weeks of age in one of the experiments. N-ethyl-N-nitrosourea (ENU) was used as a positive mutation control in one experiment. ENU induced a robust Pig-a mutant and micronucleus response in both phenotypes. Obese, otherwise untreated mice, did not differ from non-obese mice with respect to Pig-a mutant frequencies in reticulocytes or micronucleus frequencies. However, such mice, had significantly higher and sustained Pig-a mutant frequencies (increased 2.5-3.7-fold, p < 0.02) in erythrocytes as compared to non-obese mice (based on measurements collected at 30 weeks or 30 and 52 weeks of age). This suggests that obesity, in the absence of exposure to an exogenous mutagen, is itself mutagenic. Environ. Mol. Mutagen. 57:668-677, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

p53 deficiency alters the yield and spectrum of radiation-induced lacZ mutants in the brain of transgenic mice

NASA Technical Reports Server (NTRS)

Chang, P. Y.; Kanazawa, N.; Lutze-Mann, L.; Winegar, R. A.

2001-01-01

Exposure to heavy particle radiation in the galacto-cosmic environment poses a significant risk in space exploration and the evaluation of radiation-induced genetic damage in tissues, especially in the central nervous system, is an important consideration in long-term manned space missions. We used a plasmid-based transgenic mouse model system, with the pUR288 lacZ transgene integrated in the genome of every cell of C57Bl/6(lacZ) mice, to evaluate the genetic damage induced by iron particle radiation. In order to examine the importance of genetic background on the radiation sensitivity of individuals, we cross-bred p53 wild-type lacZ transgenic mice with p53 nullizygous mice, producing lacZ transgenic mice that were either hemizygous or nullizygous for the p53 tumor suppressor gene. Animals were exposed to an acute dose of 1 Gy of iron particles and the lacZ mutation frequency (MF) in the brain was measured at time intervals from 1 to 16 weeks post-irradiation. Our results suggest that iron particles induced an increase in lacZ MF (2.4-fold increase in p53+/+ mice, 1.3-fold increase in p53+/- mice and 2.1-fold increase in p53-/- mice) and that this induction is both temporally regulated and p53 genotype dependent. Characterization of mutants based on their restriction patterns showed that the majority of the mutants arising spontaneously are derived from point mutations or small deletions in all three genotypes. Radiation induced alterations in the spectrum of deletion mutants and reorganization of the genome, as evidenced by the selection of mutants containing mouse genomic DNA. These observations are unique in that mutations in brain tissue after particle radiation exposure have never before been reported owing to technical limitations in most other mutation assays.

Mutant PFN1 causes ALS phenotypes and progressive motor neuron degeneration in mice by a gain of toxicity

PubMed Central

Yang, Chunxing; Danielson, Eric W.; Qiao, Tao; Metterville, Jake; Brown, Robert H.; Landers, John E.; Xu, Zuoshang

2016-01-01

Mutations in the profilin 1 (PFN1) gene cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease caused by the loss of motor neurons leading to paralysis and eventually death. PFN1 is a small actin-binding protein that promotes formin-based actin polymerization and regulates numerous cellular functions, but how the mutations in PFN1 cause ALS is unclear. To investigate this problem, we have generated transgenic mice expressing either the ALS-associated mutant (C71G) or wild-type protein. Here, we report that mice expressing the mutant, but not the wild-type, protein had relentless progression of motor neuron loss with concomitant progressive muscle weakness ending in paralysis and death. Furthermore, mutant, but not wild-type, PFN1 forms insoluble aggregates, disrupts cytoskeletal structure, and elevates ubiquitin and p62/SQSTM levels in motor neurons. Unexpectedly, the acceleration of motor neuron degeneration precedes the accumulation of mutant PFN1 aggregates. These results suggest that although mutant PFN1 aggregation may contribute to neurodegeneration, it does not trigger its onset. Importantly, these experiments establish a progressive disease model that can contribute toward identifying the mechanisms of ALS pathogenesis and the development of therapeutic treatments. PMID:27681617

Changes in the striatal proteome of YAC128Q mice exhibit gene-environment interactions between mutant huntingtin and manganese.

PubMed

Wegrzynowicz, Michal; Holt, Hunter K; Friedman, David B; Bowman, Aaron B

2012-02-03

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat within the Huntingtin (HTT) gene, though the clinical presentation of disease and age-of-onset are strongly influenced by ill-defined environmental factors. We recently reported a gene-environment interaction wherein expression of mutant HTT is associated with neuroprotection against manganese (Mn) toxicity. Here, we are testing the hypothesis that this interaction may be manifested by altered protein expression patterns in striatum, a primary target of both neurodegeneration in HD and neurotoxicity of Mn. To this end, we compared striatal proteomes of wild-type and HD (YAC128Q) mice exposed to vehicle or Mn. Principal component analysis of proteomic data revealed that Mn exposure disrupted a segregation of WT versus mutant proteomes by the major principal component observed in vehicle-exposed mice. Identification of altered proteins revealed novel markers of Mn toxicity, particularly proteins involved in glycolysis, excitotoxicity, and cytoskeletal dynamics. In addition, YAC128Q-dependent changes suggest that axonal pathology may be an early feature in HD pathogenesis. Finally, for several proteins, genotype-specific responses to Mn were observed. These differences include increased sensitivity to exposure in YAC128Q mice (UBQLN1) and amelioration of some mutant HTT-induced alterations (SAE1, ENO1). We conclude that the interaction of Mn and mutant HTT may suppress proteomic phenotypes of YAC128Q mice, which could reveal potential targets in novel treatment strategies for HD.

Somatotroph hypoplasia and dwarfism in transgenic mice expressing a non-phosphorylatable CREB mutant.

PubMed

Struthers, R S; Vale, W W; Arias, C; Sawchenko, P E; Montminy, M R

1991-04-18

Most of the transcriptional effects of cyclic AMP are mediated by the cAMP response element binding protein (CREB). After activation of cAMP-dependent protein kinase A, the catalytic subunits of this enzyme apparently mediate the phosphorylation and activation of CREB. As cAMP serves as a mitogenic signal for anterior pituitary somatotrophic cells, we investigated whether CREB similarly regulates proliferation of these cells. We prepared transgenic mice expressing a transcriptionally inactive mutant of CREB (CREBM1), which cannot be phosphorylated, in cells of the anterior pituitary. If CREB activity is required for proliferation, the overexpressed mutant protein would effectively compete with wild-type CREB activity and thereby block the response to cAMP. As predicted, the CREBM1 transgenic mice exhibited a dwarf phenotype with atrophied pituitary glands markedly deficient in somatotroph but not other cell types. We conclude that transcriptional activation of CREB is necessary for the normal development of a highly restricted cell type, and that environmental cues, possibly provided by the hypothalamic growth hormone-releasing factor, are necessary for population of the pituitary by somatotrophic cells.

A Yersinia pestis lpxM-mutant live vaccine induces enhanced immunity against bubonic plague in mice and guinea pigs.

PubMed

Feodorova, V A; Pan'kina, L N; Savostina, E P; Sayapina, L V; Motin, V L; Dentovskaya, S V; Shaikhutdinova, R Z; Ivanov, S A; Lindner, B; Kondakova, A N; Bystrova, O V; Kocharova, N A; Senchenkova, S N; Holst, O; Pier, G B; Knirel, Y A; Anisimov, A P

2007-11-01

The lpxM mutant of the live vaccine Yersinia pestis EV NIIEG strain synthesising a less toxic penta-acylated lipopolysaccharide was found to be avirulent in mice and guinea pigs, notably showing no measurable virulence in Balb/c mice which do retain some susceptibility to the parental strain itself. Twenty-one days after a single injection of the lpxM-mutant, 85-100% protection was achieved in outbred mice and guinea pigs, whereas a 43% protection rate was achieved in Balb/c mice given single low doses (10(3) to 2.5 x 10(4) CFU) of this vaccine. A subcutaneous challenge with 2000 median lethal doses (equal to 20,000 CFU) of fully virulent Y. pestis 231 strain, is a 6-10-fold higher dose than that which the EV NIIEG itself can protect against.

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.

Mutant prenyltransferase-like mitochondrial protein (PLMP) and mitochondrial abnormalities in kd/kd mice

PubMed Central

Peng, Min; Jarett, Leonard; Meade, Ray; Madaio, Michael P.; Hancock, Wayne W.; George, Alfred L.; Neilson, Eric G.; Gasser, David L.

2008-01-01

Background Mice that are homozygous for the kidney disease (kd) mutation are apparently healthy for the first 8 weeks of life, but spontaneously develop a severe form of interstitial nephritis that progresses to end-stage renal disease (ESRD) by 4 to 8 months of age. By testing for linkage to microsatellite markers, we previously localized the kd gene to a YAC/BAC contig. Methods The sequence of the entire critical region was examined, and candidate genes were identified. These candidate genes were sequenced in both mutant (kd/kd) mice and normal controls. The phenotype was further characterized by immunohistochemistry and electron microscopy. Transgenic mice were constructed that carried the wild-type allele of the prime candidate gene, and this transgene was transferred to a kd/kd background by breeding. Results We have obtained evidence that kd is a mutant allele of a novel gene for a prenyltransferase-like mitochondrial protein (PLMP). This gene is alternatively spliced, with the larger gene product having one domain that resembles transprenyltransferase and another that is similar to geranylgeranyl pyrophosphate synthase. The smaller gene product includes only the first domain. An antiserum to PLMP localizes to mitochondria, and ultrastructural defects are present in the mitochondria of renal tubular epithelial cells, and to a lesser extent, hepatocytes and heart cells from kd/kd mice. In a line of kd/kd mice that carried the wild-type PLMP allele as a transgene, only 1 out of 13 animals expressed the disease by 120 days of age. Conclusion The kd allele codes for a novel protein that localizes to the mitochondria, and the kd/kd mouse has dysmorphic mitochondria in the renal tubular epithelial cells. This mouse is therefore a unique animal model for studying mechanisms that lead to tubulointerstitial nephritis. PMID:15200409

Enamel protein regulation and dental and periodontal physiopathology in MSX2 mutant mice.

PubMed

Molla, Muriel; Descroix, Vianney; Aïoub, Muhanad; Simon, Stéphane; Castañeda, Beatriz; Hotton, Dominique; Bolaños, Alba; Simon, Yohann; Lezot, Frédéric; Goubin, Gérard; Berdal, Ariane

2010-11-01

Signaling pathways that underlie postnatal dental and periodontal physiopathology are less studied than those of early tooth development. Members of the muscle segment homeobox gene (Msx) family encode homeoproteins that show functional redundancy during development and are known to be involved in epithelial-mesenchymal interactions that lead to crown morphogenesis and ameloblast cell differentiation. This study analyzed the MSX2 protein during mouse postnatal growth as well as in the adult. The analysis focused on enamel and periodontal defects and enamel proteins in Msx2-null mutant mice. In the epithelial lifecycle, the levels of MSX2 expression and enamel protein secretion were inversely related. Msx2+/- mice showed increased amelogenin expression, enamel thickness, and rod size. Msx2-/- mice displayed compound phenotypic characteristics of enamel defects, related to both enamel-specific gene mutations (amelogenin and enamelin) in isolated amelogenesis imperfecta, and cell-cell junction elements (laminin 5 and cytokeratin 5) in other syndromes. These effects were also related to ameloblast disappearance, which differed between incisors and molars. In Msx2-/- roots, Malassez cells formed giant islands that overexpressed amelogenin and ameloblastin that grew over months. Aberrant expression of enamel proteins is proposed to underlie the regional osteopetrosis and hyperproduction of cellular cementum. These enamel and periodontal phenotypes of Msx2 mutants constitute the first case report of structural and signaling defects associated with enamel protein overexpression in a postnatal context.

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.

Serial transmission in rodents of neurodegeneration from transgenic mice expressing mutant prion protein.

PubMed Central

Hsiao, K K; Groth, D; Scott, M; Yang, S L; Serban, H; Rapp, D; Foster, D; Torchia, M; Dearmond, S J; Prusiner, S B

1994-01-01

Two lines of transgenic (Tg) mice expressing high (H) levels of the mutant P101L prion protein (PrP) developed a neurologic illness and central nervous system pathology indistinguishable from experimental murine scrapie; these mice were designated Tg(MoPrP-P101L)H. Brain homogenates from Tg(MoPrP-P101L)H mice were inoculated intracerebrally into CD-1 Swiss mice, Syrian hamsters, and Tg196 mice, Tg mice expressing the MoPrP-P101L transgene at low levels. None of the CD-1 mice developed central nervous system dysfunction, whereas approximately 10% of hamsters and approximately 40% of the Tg196 mice manifested neurologic signs between 117 and 639 days after inoculation. Serial transmission of neurodegeneration in Tg196 mice and Syrian hamsters was initiated with brain extracts, producing incubation times of approximately 400 and approximately 75 days, respectively. Although the Tg(MoPrP-P101L)H mice appear to accumulate only low levels of infections prions in their brains, the serial transmission of disease to inoculated recipients argues that prion formation occurs de novo in the brains of these uninoculated animals. These Tg mouse studies, taken together with similar findings in humans dying of inherited prion diseases, provide additional evidence that prions lack a foreign nucleic acid. Images PMID:7916462

Medulloblastomas derived from Cxcr6 mutant mice respond to treatment with a smoothened inhibitor.

PubMed

Sasai, Ken; Romer, Justyna T; Kimura, Hiromichi; Eberhart, Derek E; Rice, Dennis S; Curran, Tom

2007-04-15

The sonic hedgehog (Shh) pathway is activated in approximately 30% of human medulloblastoma resulting in increased expression of downstream target genes. In about half of these cases, this has been shown to be a consequence of mutations in regulatory genes within the pathway, including Ptc1, Smo, and Sufu. However, for some tumors, no mutations have been detected in known pathway genes. This suggests that either mutations in other genes promote tumorigenesis or that epigenetic alterations increase pathway activity in these tumors. Here, we report that 3% to 4% of mice lacking either one or both functional copies of Cxcr6 develop medulloblastoma. Although CXCR6 is not known to be involved in Shh signaling, tumors derived from Cxcr6 mutant mice expressed Shh pathway target genes including Gli1, Gli2, Ptc2, and Sfrp1, indicating elevated pathway activity. Interestingly, the level of Ptc1 expression was decreased in tumor cells although two normal copies of Ptc1 were retained. This implies that reduced CXCR6 function leads to suppression of Ptc1 thereby increasing Smoothened function and promoting tumorigenesis. We used a direct transplant model to test the sensitivity of medulloblastoma arising in Cxcr6 mutant mice to a small-molecule inhibitor of Smoothened (HhAntag). We found that transplanted tumors were dramatically inhibited in mice treated for only 4 days with HhAntag. These findings suggest that HhAntag may be effective against tumors lacking mutations in known Shh pathway genes.

Trans-arterial chemoembolization (TACE) in patients with unresectable Hepatocellular carcinoma: Experience from a tertiary care centre in India

PubMed Central

Paul, Shashi Bala; Gamanagatti, Shivanand; Sreenivas, Vishnubhatla; Chandrashekhara, Sheragaru Hanumanhtappa; Mukund, Amar; Gulati, Manpreet Singh; Gupta, Arun Kumar; Acharya, Subrat Kumar

2011-01-01

Aims: To evaluate the outcome following transarterial chemoembolization (TACE) and to identify the predictors of survival in patients with unresectable hepatocellular carcinoma (HCC). Material and Methods: HCC patients reporting to our hospital (2001-2007) were subjected to clinical, biochemical, and radiological examination. TACE was performed in those who fulfilled the inclusion criteria. Follow-up assessment was done with multiphase CT scan of the liver at 1, 3, and 6 months. Tumor response and survival rate were estimated. Univariate and multivariate analyses were done for determinants of survival. Results: A total of 73 patients (69 males, 4 females; mean age 49±13.4 years) were subjected to 123 sessions of TACE. The Child's classification was: A – 56 patients and B – 17 patients. Barcelona Clinic staging was: A – 20 patients, B – 38 patients, and C – 15 patients. Tumor size was ≤5cm in 28 (38%) patients, >5–10 cm in 28 (38%) patients, and >10 cm in 17 (23%) patients. Median follow-up was for 12 months (range: 1–77 months). No significant postprocedure complications were encountered. Overall survival rate was 66%, 47%, and 36.4% at 1, 2, and 3 years, respectively. Tumor size emerged as an important predictor of survival. Conclusion: TACE offers a reasonable palliative therapy for HCC. Initial tumor size is an independent predictor of survival. PMID:21799594

Brucella abortus Cyclic β-1,2-Glucan Mutants Have Reduced Virulence in Mice and Are Defective in Intracellular Replication in HeLa Cells

PubMed Central

Briones, Gabriel; Iñón de Iannino, Nora; Roset, Mara; Vigliocco, Ana; Paulo, Patricia Silva; Ugalde, Rodolfo A.

2001-01-01

Null cyclic β-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic β-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response. PMID:11401996

Drug discrimination and neurochemical studies in alpha7 null mutant mice: tests for the role of nicotinic alpha7 receptors in dopamine release.

PubMed

Quarta, Davide; Naylor, Christopher G; Barik, Jacques; Fernandes, Cathy; Wonnacott, Susan; Stolerman, Ian P

2009-04-01

The nicotine discriminative stimulus has been linked to beta2-containing (beta2*) nicotinic receptors, with little evidence of a role for alpha7 nicotinic receptors, because nicotine discrimination was very weak in beta2 null mutant mice but normal in alpha7 mutants. As both alpha7 and beta2* nicotinic receptors have been implicated in nicotine-stimulated dopamine overflow, this study focused on the dopamine-mediated element in the nicotine stimulus by examining cross-generalisation between amphetamine and nicotine. Male alpha7 nicotinic receptor null mutant mice and wild-type controls were bred in-house and trained to discriminate nicotine (0.8 mg/kg) or (+)-amphetamine (0.6 mg/kg) from saline in a two-lever procedure with a tandem VI-30 FR-10 schedule of food reinforcement. Dopamine release from striatal slices was determined in parallel experiments. An alpha7 nicotinic receptor-mediated component of dopamine release was demonstrated in tissue from wild-type mice using choline as a selective agonist. This response was absent in tissue from null mutant animals. The mutation did not influence acquisition of drug discriminations but subtly affected the results of cross-generalisation tests. In mice trained to discriminate nicotine or amphetamine, there was partial cross-generalisation in wild-type mice and, at certain doses, these effects were attenuated in mutants. Further support for an alpha7 nicotinic receptor-mediated component was provided by the ability of the alpha7 nicotinic receptor antagonist methyllycaconitine to attenuate responses to nicotine and amphetamine in wild-type mice. These findings support the concept of an alpha7 nicotinic receptor-mediated dopaminergic element in nicotine discrimination, warranting further tests with selective dopamine agonists.

Enhancement of DEN-induced liver tumorigenesis in heme oxygenase-1 G143H mutant transgenic mice

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

Jin, Jianfeng; Wang, Dayong; Xiao, Haifeng

Heme oxygenase (HO) is the rate-limiting enzyme in heme metabolism. HO-1 exhibits anti-oxidative and anti-inflammatory function via the actions of its metabolite, respectively. A growing body of evidence demonstrates that HO-1 is implicated in the pathogenesis and progression of several types of cancer. However, whether HO-1 takes part in healthy-premalignant-malignant transformation is still undefined. In this study, we took advantage of transgenic mice which over-expressed HO-1 dominant negative mutant (HO-1 G143H) and observed its susceptibility to DEN-induced hepatocarcinogenesis. Our results indicate that HO-1 G143H mutant accelerates the progression of tumorigenesis and tumor growth. The mechanism is closely related to enhancementmore » of ROS production which induce more hepatocytes death and secretion of inflammatory cytokines, proliferation of surviving hepatocytes. Our result provides the direct evidence that HO-1 plays an important protective role in liver carcinogenesis. Alternatively, we suggest the possible explanation on effect of HO-1 promoter polymorphism which involved in tumorigenesis. - Highlights: • Enhancement of DEN-induced hepatocarcinogenesis in HO-1 G143H Tg mice. • HO-1G143H mutant enhanced DEN-induced ROS production and liver injury. • HO-1G143H mutant aggravated DEN-induced changes of inflammatory factors and cell proliferation.« less

Enamel Protein Regulation and Dental and Periodontal Physiopathology in Msx2 Mutant Mice

PubMed Central

Molla, Muriel; Descroix, Vianney; Aïoub, Muhanad; Simon, Stéphane; Castañeda, Beatriz; Hotton, Dominique; Bolaños, Alba; Simon, Yohann; Lezot, Frédéric; Goubin, Gérard; Berdal, Ariane

2010-01-01

Signaling pathways that underlie postnatal dental and periodontal physiopathology are less studied than those of early tooth development. Members of the muscle segment homeobox gene (Msx) family encode homeoproteins that show functional redundancy during development and are known to be involved in epithelial-mesenchymal interactions that lead to crown morphogenesis and ameloblast cell differentiation. This study analyzed the MSX2 protein during mouse postnatal growth as well as in the adult. The analysis focused on enamel and periodontal defects and enamel proteins in Msx2-null mutant mice. In the epithelial lifecycle, the levels of MSX2 expression and enamel protein secretion were inversely related. Msx2+/− mice showed increased amelogenin expression, enamel thickness, and rod size. Msx2−/− mice displayed compound phenotypic characteristics of enamel defects, related to both enamel-specific gene mutations (amelogenin and enamelin) in isolated amelogenesis imperfecta, and cell-cell junction elements (laminin 5 and cytokeratin 5) in other syndromes. These effects were also related to ameloblast disappearance, which differed between incisors and molars. In Msx2−/− roots, Malassez cells formed giant islands that overexpressed amelogenin and ameloblastin that grew over months. Aberrant expression of enamel proteins is proposed to underlie the regional osteopetrosis and hyperproduction of cellular cementum. These enamel and periodontal phenotypes of Msx2 mutants constitute the first case report of structural and signaling defects associated with enamel protein overexpression in a postnatal context. PMID:20934968

Global gene profiling of aging lungs in Atp8b1 mutant mice.

PubMed

Soundararajan, Ramani; Stearns, Timothy M; Czachor, Alexander; Fukumoto, Jutaro; Turn, Christina; Westermann-Clark, Emma; Breitzig, Mason; Tan, Lee; Lockey, Richard F; King, Benjamin L; Kolliputi, Narasaiah

2016-09-29

Recent studies implicate cardiolipin oxidation in several age-related diseases. Atp8b1 encoding Type 4 P-type ATPases is a cardiolipin transporter. Mutation in Atp8b1 gene or inflammation of the lungs impairs the capacity of Atp8b1 to clear cardiolipin from lung fluid. However, the link between Atp8b1 mutation and age-related gene alteration is unknown. Therefore, we investigated how Atp8b1 mutation alters age-related genes. We performed Affymetrix gene profiling of lungs isolated from young (7-9 wks, n=6) and aged (14 months, 14 M, n=6) C57BL/6 and Atp8b1 mutant mice. In addition, Ingenuity Pathway Analysis (IPA) was performed. Differentially expressed genes were validated by quantitative real-time PCR (qRT-PCR). Global transcriptome analysis revealed 532 differentially expressed genes in Atp8b1 lungs, 157 differentially expressed genes in C57BL/6 lungs, and 37 overlapping genes. IPA of age-related genes in Atp8b1 lungs showed enrichment of Xenobiotic metabolism and Nrf2-mediated signaling pathways. The increase in Adamts2 and Mmp13 transcripts in aged Atp8b1 lungs was validated by qRT-PCR. Similarly, the decrease in Col1a1 and increase in Cxcr6 transcripts was confirmed in both Atp8b1 mutant and C57BL/6 lungs. Based on transcriptome profiling, our study indicates that Atp8b1 mutant mice may be susceptible to age-related lung diseases.

Hypolipidemic effects of starch and γ-oryzanol from wx/ae double-mutant rice on BALB/c.KOR-Apoe(shl) mice.

PubMed

Nakaya, Makoto; Shojo, Aiko; Hirai, Hiroaki; Matsumoto, Kenji; Kitamura, Shinichi

2013-01-01

waxy/amylose-extender (wx/ae) double-mutant japonica rice (Oryza sativa L.) produces resistant starch (RS) and a large amount of γ-oryzanol. Our previous study has shown the hypolipidemic effect of wx/ae brown rice on mice. To identify the functional constituents of the hypolipidemic activity in wx/ae rice, we prepared pure wx/ae starch and γ-oryzanol from wx/ae rice and investigated their effect on the lipid metabolism in BALB/c.KOR/Stm Slc-Apoe(shl) mice. The mice were fed for 3 weeks a diet containing non-mutant rice starch, non-mutant rice starch plus γ-oryzanol, wx/ae starch, or wx/ae starch plus γ-oryzanol. γ-Oryzanol by itself had no effect on the lipid metabolism, and wx/ae starch prevented an accumulation of triacylglycerol (TAG) in the liver. Interestingly, the combination of wx/ae starch plus γ-oryzanol not only prevented a TAG accumulation in the liver, but also partially suppressed the rise in plasma TAG concentration, indicating that wx/ae starch and γ-oryzanol could have a synergistic effect on the lipid metabolism.

Ectopic norrin induces growth of ocular capillaries and restores normal retinal angiogenesis in Norrie disease mutant mice.

PubMed

Ohlmann, Andreas; Scholz, Michael; Goldwich, Andreas; Chauhan, Bharesh K; Hudl, Kristiane; Ohlmann, Anne V; Zrenner, Eberhart; Berger, Wolfgang; Cvekl, Ales; Seeliger, Mathias W; Tamm, Ernst R

2005-02-16

Norrie disease is an X-linked retinal dysplasia that presents with congenital blindness, sensorineural deafness, and mental retardation. Norrin, the protein product of the Norrie disease gene (NDP), is a secreted protein of unknown biochemical function. Norrie disease (Ndp(y/-)) mutant mice that are deficient in norrin develop blindness, show a distinct failure in retinal angiogenesis, and completely lack the deep capillary layers of the retina. We show here that the transgenic expression of ectopic norrin under control of a lens-specific promoter restores the formation of a normal retinal vascular network in Ndp(y/-) mutant mice. The improvement in structure correlates with restoration of neuronal function in the retina. In addition, lenses of transgenic mice with ectopic expression of norrin show significantly more capillaries in the hyaloid vasculature that surrounds the lens during development. In vitro, lenses of transgenic mice in coculture with microvascular endothelial cells induce proliferation of the cells. Transgenic mice with ectopic expression of norrin show more bromodeoxyuridine-labeled retinal progenitor cells at embryonic day 14.5 and thicker retinas at postnatal life than wild-type littermates, indicating a putative direct neurotrophic effect of norrin. These data provide direct evidence that norrin induces growth of ocular capillaries and that pharmacologic modulation of norrin might be used for treatment of the vascular abnormalities associated with Norrie disease or other vascular disorders of the retina.

Impaired discrimination learning in interneuronal NMDAR-GluN2B mutant mice.

PubMed

Brigman, Jonathan L; Daut, Rachel A; Saksida, Lisa; Bussey, Timothy J; Nakazawa, Kazu; Holmes, Andrew

2015-06-17

Previous studies have established a role for N-methyl-D-aspartate receptor (NMDAR) containing the GluN2B subunit in efficient learning behavior on a variety of tasks. Recent findings have suggested that NMDAR on GABAergic interneurons may underlie the modulation of striatal function necessary to balance efficient action with cortical excitatory input. Here we investigated how loss of GluN2B-containing NMDAR on GABAergic interneurons altered corticostriatal-mediated associative learning. Mutant mice (floxed-GluN2B×Ppp1r2-Cre) were generated to produce loss of GluN2B on forebrain interneurons and phenotyped on a touchscreen-based pairwise visual learning paradigm. We found that the mutants showed normal performance during Pavlovian and instrumental pretraining, but were significantly impaired on a discrimination learning task. Detailed analysis of the microstructure of discrimination performance revealed reduced win→stay behavior in the mutants. These results further support the role of NMDAR, and GluN2B in particular, on modulation of striatal function necessary for efficient choice behavior and suggest that NMDAR on interneurons may play a critical role in associative learning.

Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging

PubMed Central

Park, Seok-Joo; Shin, Eun-Joo; Min, Sun Seek; An, Jihua; Li, Zhengyi; Hee Chung, Yoon; Hoon Jeong, Ji; Bach, Jae-Hyung; Nah, Seung-Yeol; Kim, Won-Ki; Jang, Choon-Gon; Kim, Yong-Sun; Nabeshima, Yo-ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2013-01-01

We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice. PMID:23389690

Identification and targeting of a TACE-dependent autocrine loopwhich predicts poor prognosis in breast cancer

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

Kenny, Paraic A.; Bissell, Mina J.

2005-06-15

The ability to proliferate independently of signals from other cell types is a fundamental characteristic of tumor cells. Using a 3D culture model of human breast cancer progression, we have delineated a protease-dependent autocrine loop which provides an oncogenic stimulus in the absence of proto-oncogene mutation. Inhibition of this protease, TACE/ADAM17, reverts the malignant phenotype by preventing mobilization of two crucial growth factors, Amphiregulin and TGF{alpha}. We show further that the efficacy of EGFR inhibitors is overcome by physiological levels of growth factors and that successful EGFR inhibition is dependent on reducing ligand bioavailability. Using existing patient outcome data, wemore » demonstrate a strong correlation between TACE and TGF{alpha} expression in human breast cancers that is predictive of poor prognosis.« less

Three Herpes Simplex Virus Type 1 Latency-Associated Transcript Mutants with Distinct and Asymmetric Effects on Virulence in Mice Compared with Rabbits

PubMed Central

Perng, Guey-Chuen; Esmaili, Daniel; Slanina, Susan M.; Yukht, Ada; Ghiasi, Homayon; Osorio, Nelson; Mott, Kevin R.; Maguen, Barak; Jin, Ling; Nesburn, Anthony B.; Wechsler, Steven L.

2001-01-01

Herpes simplex virus type 1 latency-associated transcript (LAT)-null mutants have decreased reactivation but normal virulence in rabbits and mice. We report here on dLAT1.5, a mutant with LAT nucleotides 76 to 1667 deleted. Following ocular infection of rabbits, dLAT1.5 reactivated at a lower rate than its wild-type parent McKrae (6.1 versus 11.8%; P = 0.0025 [chi-square test]). Reactivation was restored in the marker-rescued virus dLAT1.5R (12.6%; P = 0.53 versus wild type), confirming the importance of the deleted region in spontaneous reactivation. Compared with wild-type or marker-rescued virus, dLAT1.5 had similar or slightly reduced virulence in rabbits (based on survival following ocular infection). In contrast, in mice, dLAT1.5 had increased virulence (P < 0.0001). Thus, deletion of LAT nucleotides 76 to 1667 increased viral virulence in mice but not in rabbits. In contrast, we also report here that LAT2.9A, a LAT mutant that we previously reported to have increased virulence in rabbits (G. C. Perng, S. M. Slanina, A. Yuhkt, B. S. Drolet, W. J. Keleher, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 73:920–929, 1999), had decreased virulence in mice (P = 0.03). In addition, we also found that dLAT371, a LAT mutant that we previously reported to have wild-type virulence in rabbits (G. C. Perng, S. M. Slanina, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 70:2014–2018, 1996), had decreased virulence in mice (P < 0.05). Thus, these three mutants, each of which encodes a different LAT RNA, have different virulence phenotypes. dLAT1.5 had wild-type virulence in rabbits but increased virulence in mice. In contrast, LAT2.9A had increased virulence in rabbits but decreased virulence in mice, and dLAT371 had wild-type virulence in rabbits but decreased virulence in mice. Taken together, these results suggest that (i) the 5′ end of LAT and/or a gene that overlaps part of this region is involved in viral virulence, (ii) this virulence appears

Expression of Mutant Human DISC1 in Mice Supports Abnormalities in Differentiation of Oligodendrocytes

PubMed Central

Katsel, Pavel; Tan, Weilun; Abazyan, Bagrat; Davis, Kenneth L; Ross, Christopher; Pletnikov, Mikhail V; Haroutunian, Vahram

2011-01-01

Abnormalities in oligodendrocyte (OLG) differentiation and OLG gene expression deficit have been described in schizophrenia (SZ). Recent studies revealed a critical requirement for Disrupted-in-Schizophrenia 1 (DISC1) in neural development. Transgenic mice with forebrain restricted expression of mutant human DISC1 (ΔhDISC1) are characterized by neuroanatomical and behavioral abnormalities reminiscent of some features of SZ. We sought to determine whether the expression of ΔhDISC1 may influence the development of OLGs in this mouse model. OLG- and cell cycle-associated gene and protein expression were characterized in the forebrain of ΔhDISC1 mice during different stages of neurodevelopment (E15 and P1 days) and in adulthood. The results suggest that the expression of ΔhDISC1 exerts a significant influence on oligodendrocyte differentiation and function, evidenced by premature OLG differentiation and increased proliferation of their progenitors. Additional findings showed that neuregulin 1 and its receptors may be contributing factors to the observed upregulation of OLG genes. Thus, OLG function may be perturbed by mutant hDISC1 in a model system that provides new avenues for studying aspects of the pathogenesis of SZ. PMID:21605958

Hyperactivity and Learning Deficits in Transgenic Mice Bearing a Human Mutant Thyroid Hormone β1 Receptor Gene

PubMed Central

McDonald, Michael P.; Wong, Rosemary; Goldstein, Gregory; Weintraub, Bruce; Cheng, Sheue-yann; Crawley, Jacqueline N.

1998-01-01

Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor β (TRβ) gene on chromosome 3, representing a mutation of the ligandbinding domain of the TRβ gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutant TRβ gene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant human TRβ gene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD. PMID:10454355

Hyperactivity and learning deficits in transgenic mice bearing a human mutant thyroid hormone beta1 receptor gene.

PubMed

McDonald, M P; Wong, R; Goldstein, G; Weintraub, B; Cheng, S Y; Crawley, J N

1998-01-01

Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor beta (TRbeta) gene on chromosome 3, representing a mutation of the ligand-binding domain of the TRbeta gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutant TRbeta gene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant human TRbeta gene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD.

Peroxisome proliferator-activated receptor gamma agonist troglitazone induces colon tumors in normal C57BL/6J mice and enhances colonic carcinogenesis in Apc1638 N/+ Mlh1+/- double mutant mice.

PubMed

Yang, Kan; Fan, Kun-Hua; Lamprecht, Sergio A; Edelmann, Winfried; Kopelovich, Levy; Kucherlapati, Raju; Lipkin, Martin

2005-09-10

The role of the nuclear peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in colon tumorigenesis remains controversial. Notwithstanding evidence that PPAR-gamma ligands impede murine colorectal carcinogenesis, PPAR-gamma agonists have been shown to enhance in vivo tumor formation in mouse models of human colon cancer. Our study was designed to determine whether troglitazone (TGZ) induces colonic tumor formation in normal C57BL/6J mice and enhances colorectal carcinogenesis in double mutant Apc1638N/+ Mlh1+/- mice fed a standard AIN-76A diet. We report herein that not only does TGZ enhance carcinogenesis in the large intestine of mutant mice predisposed to intestinal carcinogenesis but TGZ also induces colonic tumors in normal mice without gene targeting or carcinogen administration. This observation indicates that preexisting mutational events are not necessary for induction of colonic tumors by activated PPAR-gamma in vivo. (c) 2005 Wiley-Liss, Inc.

Overexpression of survival motor neuron improves neuromuscular function and motor neuron survival in mutant SOD1 mice.

PubMed

Turner, Bradley J; Alfazema, Neza; Sheean, Rebecca K; Sleigh, James N; Davies, Kay E; Horne, Malcolm K; Talbot, Kevin

2014-04-01

Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1(G93A) mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1(G93A) mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1(G93A) mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS. Copyright © 2014 Elsevier Inc. All rights reserved.

Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

PubMed Central

Filippov, Andrey A.; Sergueev, Kirill V.; He, Yunxiu; Huang, Xiao-Zhe; Gnade, Bryan T.; Mueller, Allen J.; Fernandez-Prada, Carmen M.; Nikolich, Mikeljon P.

2011-01-01

Background Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. Methodology/Principal Findings The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. Conclusions/Significance We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis. PMID:21980477

Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice.

PubMed

Filippov, Andrey A; Sergueev, Kirill V; He, Yunxiu; Huang, Xiao-Zhe; Gnade, Bryan T; Mueller, Allen J; Fernandez-Prada, Carmen M; Nikolich, Mikeljon P

2011-01-01

Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.

A Yersinia pestis YscN ATPase mutant functions as a live attenuated vaccine against bubonic plague in mice.

PubMed

Bozue, Joel; Cote, Christopher K; Webster, Wendy; Bassett, Anthony; Tobery, Steven; Little, Stephen; Swietnicki, Wieslaw

2012-07-01

Yersinia pestis is the causative agent responsible for bubonic and pneumonic plague. The bacterium uses the pLcr plasmid-encoded type III secretion system to deliver virulence factors into host cells. Delivery requires ATP hydrolysis by the YscN ATPase encoded by the yscN gene also on pLcr. A yscN mutant was constructed in the fully virulent CO92 strain containing a nonpolar, in-frame internal deletion within the gene. We demonstrate that CO92 with a yscN mutation was not able to secrete the LcrV protein (V-Antigen) and attenuated in a subcutaneous model of plague demonstrating that the YscN ATPase was essential for virulence. However, if the yscN mutant was complemented with a functional yscN gene in trans, virulence was restored. To evaluate the mutant as a live vaccine, Swiss-Webster mice were vaccinated twice with the ΔyscN mutant at varying doses and were protected against bubonic plague in a dose-dependent manner. Antibodies to F1 capsule but not to LcrV were detected in sera from the vaccinated mice. These preliminary results suggest a proof-of-concept for an attenuated, genetically engineered, live vaccine effective against bubonic plague. Published 2012. This article is a US Government work and is in the public domain in the USA.

Enriching the Environment of [alpha]CaMKII[superscript T286A] Mutant Mice Reveals that LTD Occurs in Memory Processing but Must be Subsequently Reversed by LTP

ERIC Educational Resources Information Center

Soto, Florentina; Giese, K. Peter; Edwards, Frances A.; Parsley, Stephanie L.; Pilgram, Sara M.

2007-01-01

[alpha]CaMKII[superscript T286A] mutant mice lack long-term potentiation (LTP) in the hippocampal CA1 region and are impaired in spatial learning. In situ hybridization confirms that the mutant mice show the same developmental expression of [alpha]CaMKII as their wild-type littermates. A simple hypothesis would suggest that if LTP is a substrate…

An Antibody to Notch2 Reverses the Osteopenic Phenotype of Hajdu-Cheney Mutant Male Mice

PubMed Central

Sanjay, Archana; Yu, Jungeun; Zanotti, Stefano

2017-01-01

Notch receptors play a central role in skeletal development and bone remodeling. Hajdu-Cheney syndrome (HCS), a disease characterized by osteoporosis and fractures, is associated with gain-of-NOTCH2 function mutations. To study HCS, we created a mouse model harboring a point 6955C>T mutation in the Notch2 locus upstream of the proline, glutamic acid, serine, and threonine domain, leading to a Q2319X change at the amino acid level. Notch2Q2319X heterozygous mutants exhibited cancellous and cortical bone osteopenia. Microcomputed tomography demonstrated that the cancellous and cortical osteopenic phenotype was reversed by the administration of antibodies generated against the negative regulatory region (NRR) of Notch2, previously shown to neutralize Notch2 activity. Bone histomorphometry revealed that anti-Notch2 NRR antibodies decreased the osteoclast number and eroded surface in cancellous bone of Notch2Q2319X mice. An increase in osteoclasts on the endocortical surface of Notch2Q2319X mice was not observed in the presence of anti-Notch2 NRR antibodies. The anti-Notch2 NRR antibody decreased the induction of Notch target genes and Tnfsf11 messenger RNA levels in bone extracts and osteoblasts from Notch2Q2319X mice. In vitro experiments demonstrated increased osteoclastogenesis in Notch2Q2319X mutants in response to macrophage colony-stimulating factor and receptor activator of nuclear factor–κB ligand, and these effects were suppressed by the anti-Notch2 NRR. In conclusion, Notch2Q2319X mice exhibit cancellous and cortical bone osteopenia that can be corrected by the administration of anti-Notch2 NRR antibodies. PMID:28323963

Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice.

PubMed

Zhang, Honghao; Kamiya, Nobuhiro; Tsuji, Takehito; Takeda, Haruko; Scott, Greg; Rajderkar, Sudha; Ray, Manas K; Mochida, Yoshiyuki; Allen, Benjamin; Lefebvre, Veronique; Hung, Irene H; Ornitz, David M; Kunieda, Tetsuo; Mishina, Yuji

2016-12-01

Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome.

Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice

PubMed Central

Zhang, Honghao; Kamiya, Nobuhiro; Tsuji, Takehito; Takeda, Haruko; Scott, Greg; Ray, Manas K.; Mochida, Yoshiyuki; Lefebvre, Veronique; Hung, Irene H.; Kunieda, Tetsuo; Mishina, Yuji

2016-01-01

Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome. PMID:28027321

Genetic variations in IDH gene as prognosis predictors in TACE-treated hepatocellular carcinoma patients.

PubMed

Zhang, Huiqing; Guo, Xu; Dai, Jingyao; Wu, Yousheng; Ge, Naijian; Yang, Yefa; Ji, Jiansong; Zhang, Hongxin

2014-11-01

Metabolic reprogramming is an important hallmark of cancer cells, including the alterations of activity and expression in tricarboxylic acid (TCA) cycle key enzymes. Previous studies have reported the associations between tumor formation and three core enzymes involved in the TCA cycle. However, the association between functional single nucleotide polymorphisms (SNPs) in one of TCA cycle key gene isocitrate dehydrogenase (IDH) and the overall survival of hepatocellular carcinoma (HCC) patients treated with transcatheter arterial chemoembolization (TACE) has never been investigated. Five functional SNPs in IDH1 and IDH2 genes were genotyped using the Sequenom iPLEX genotyping system in a cohort of 419 unresectable Chinese HCC patients treated with TACE. Multivariate Cox proportional hazards model and Kaplan-Meier curve were used for the prognosis analysis. We found that SNPs rs12478635 in IDH1 and rs11632348 in IDH2 gene exhibited significant associations with death risk in HCC patients in the dominant model (HR 1.33; 95 % CI 1.02-1.73; P = 0.037) and in recessive model (HR 1.87; 95 % CI 1.27-2.75; P = 0.001), respectively. Moreover, we observed a cumulative effect of these two SNPs on HCC overall survival, indicating a significant trend of death risk increase with increasing number of unfavorable genotypes (P for trend = 0.001). Additionally, our data suggest that unfavorable genotypes of two SNPs may be used as an independent prognostic marker in those with advanced stage and patients with serum AFP <200 μg/L. Our results for the first time suggest that IDH gene polymorphisms may serve as an independent prognostic marker for HCC patients treated with TACE.

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

PubMed Central

Ding, Jianqiang; Yannam, Govardhana R.; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I.; Wong, Ronald J.; Avsar, Yesim; Guha, Chandan; Perlmutter, David H.; Fox, Ira J.; Roy-Chowdhury, Jayanta

2011-01-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z–expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%–98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z–expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals. PMID:21505264

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes.

PubMed

Ding, Jianqiang; Yannam, Govardhana R; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I; Wong, Ronald J; Avsar, Yesim; Guha, Chandan; Perlmutter, David H; Fox, Ira J; Roy-Chowdhury, Jayanta

2011-05-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

Metabolic roles of the M3 muscarinic acetylcholine receptor studied with M3 receptor mutant mice: a review.

PubMed

Gautam, Dinesh; Jeon, Jongrye; Li, Jian Hua; Han, Sung-Jun; Hamdan, Fadi F; Cui, Yinghong; Lu, Huiyan; Deng, Chuxia; Gavrilova, Oksana; Wess, Jürgen

2008-01-01

The M(3) muscarinic acetylcholine (ACh) receptor (M(3) mAChR) is expressed in many central and peripheral tissues. It is a prototypic member of the superfamily of G protein-coupled receptors and preferentially activates G proteins of the G(q) family. Recent studies involving the use of newly generated mAChR mutant mice have revealed that the M(3) mAChR plays a key role in regulating many important metabolic functions. Phenotypic analyses of mutant mice that either selectively lacked or overexpressed M(3) receptors in pancreatic beta -cells indicated that beta -cell M(3) mAChRs are essential for maintaining proper insulin release and glucose homeostasis. The experimental data also suggested that strategies aimed at enhancing signaling through beta -cell M(3) mAChRs might be beneficial for the treatment of type 2 diabetes. Recent studies with whole body M(3) mAChR knockout mice showed that the absence of M(3) receptors protected mice against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. Under all experimental conditions tested, M(3) receptor-deficient mice showed greatly ameliorated impairments in glucose homeostasis and insulin sensitivity, reduced food intake, and a significant elevation in basal and total energy expenditure, most likely due to increased central sympathetic outflow and increased rate of fatty acid oxidation. These findings are of potential interest for the development of novel therapeutic approaches for the treatment of obesity and associated metabolic disorders.

Spontaneous Pneumocystis carinii pneumonia in immunodeficient mutant scid mice. Natural history and pathobiology.

PubMed Central

Roths, J. B.; Marshall, J. D.; Allen, R. D.; Carlson, G. A.; Sidman, C. L.

1990-01-01

The opportunistic pathogen Pneumocystis carinii (Pc) poses a major clinical health problem in individuals with immune deficiency, including those patients with human immunodeficiency (HIV)-associated acquired immune deficiency disease (AIDS). Heretofore, in vivo investigations of the biology of Pc and pathogenesis of pneumocystosis have generally employed steroid-induced immune suppression with antibiotic prophylaxis and protein deprivation. This approach has many drawbacks, chief among them being the widespread, multiple interacting effects caused by the inducing agents. Athymic (nude) mice and rats have been used, but are less than ideal, as the immune defect primarily affects T lymphocytes. This article describes the natural history, pathobiology, and environmental effects on Pc pneumonitis in nonaxenically housed mice homozygous for the autosomal recessive mutation 'severe combined immunodeficiency' (scid), which almost totally lack both cell-mediated and antibody-mediated immune functions. The predictability, unequivocal expression, high morbidity, and well-defined genetic basis make scid/scid mutant mice the model of choice for in vivo studies of spontaneous pneumocystosis. Images Figure 3 Figure 6 PMID:2349968

Early-onset lymphoma and extensive embryonic apoptosis in two domain-specific Fen1 mice mutants.

PubMed

Larsen, Elisabeth; Kleppa, Liv; Meza, Trine J; Meza-Zepeda, Leonardo A; Rada, Christina; Castellanos, Cesilie G; Lien, Guro F; Nesse, Gaute J; Neuberger, Michael S; Laerdahl, Jon K; William Doughty, Richard; Klungland, Arne

2008-06-15

Flap endonuclease 1 (FEN1) processes Okazaki fragments in lagging strand DNA synthesis, and FEN1 is involved in several DNA repair pathways. The interaction of FEN1 with the proliferating cell nuclear antigen (PCNA) processivity factor is central to the function of FEN1 in both DNA replication and repair. Here we present two gene-targeted mice with mutations in FEN1. The first mutant mouse carries a single amino acid point mutation in the active site of the nuclease domain of FEN1 (Fen1(E160D/E160D)), and the second mutant mouse contains two amino acid substitutions in the highly conserved PCNA interaction domain of FEN1 (Fen1(DeltaPCNA/DeltaPCNA)). Fen1(E160D/E160D) mice develop a considerably elevated incidence of B-cell lymphomas beginning at 6 months of age, particularly in females. By 16 months of age, more than 90% of the Fen1(E160D/E160D) females have tumors, primarily lymphomas. By contrast, Fen1(DeltaPCNA/DeltaPCNA) mouse embryos show extensive apoptosis in the forebrain and vertebrae area and die around stage E9.5 to E11.5.

Identification of biochemical adaptations in hyper- or hypocontractile hearts from phospholamban mutant mice by expression proteomics.

PubMed

Pan, Yan; Kislinger, Thomas; Gramolini, Anthony O; Zvaritch, Elena; Kranias, Evangelia G; MacLennan, David H; Emili, Andrew

2004-02-24

Phospholamban (PLN) is a critical regulator of cardiac contractility through its binding to and regulation of the activity of the sarco(endo)plasmic reticulum Ca2+ ATPase. To uncover biochemical adaptations associated with extremes of cardiac muscle contractility, we used high-throughput gel-free tandem MS to monitor differences in the relative abundance of membrane proteins in standard microsomal fractions isolated from the hearts of PLN-null mice (PLN-KO) with high contractility and from transgenic mice overexpressing a superinhibitory PLN mutant in a PLN-null background (I40A-KO) with diminished contractility. Significant differential expression was detected for a subset of the 782 proteins identified, including known membrane-associated biomarkers, components of signaling pathways, and previously uninvestigated proteins. Proteins involved in fat and carbohydrate metabolism and proteins linked to G protein-signaling pathways activating protein kinase C were enriched in I40A-KO cardiac muscle, whereas proteins linked to enhanced contractile function were enriched in PLN-KO mutant hearts. These data demonstrate that Ca2+ dysregulation, leading to elevated or depressed cardiac contractility, induces compensatory biochemical responses.

Dominant negative DISC1 mutant mice display specific social behaviour deficits and aberration in BDNF and cannabinoid receptor expression.

PubMed

Kaminitz, Ayelet; Barzilay, Ran; Segal, Hadar; Taler, Michal; Offen, Daniel; Gil-Ad, Irit; Mechoulam, Raphael; Weizman, Abraham

2014-01-01

OBJECTIVES. Disrupted in schizophrenia 1 (DISC1) is considered the most prominent candidate gene for schizophrenia. In this study, we aimed to characterize behavioural and brain biochemical traits in a mouse expressing a dominant negative DISC1mutant (DN-DISC1). DN-DISC1 mice underwent behavioural tests to evaluate object recognition, social preference and social novelty seeking. ELISA was conducted on brain tissue to evaluate BDNF levels. Western blot was employed to measure BDNF receptor (TrkB) and cannabinoid receptor CB1. The mutant DISC1 mice displayed deficits in preference to social novelty while both social preference and object recognition were intact. Biochemical analysis of prefrontal cortex and hippocampus revealed a modest reduction in cortical TrkB protein levels of male mice while no differences in BDNF levels were observed. We found sex dependent differences in the expression of cannabinoid-1 receptors. We describe novel behavioural and biochemical abnormalities in the DN-DISC1 mouse model of schizophrenia. The data shows for the first time a possible link between DISC1 mutation and the cannabinoid system.

Serotonin hyperinnervation and upregulated 5-HT2A receptor expression and motor-stimulating function in nigrostriatal dopamine-deficient Pitx3 mutant mice.

PubMed

Li, Li; Qiu, Guozhen; Ding, Shengyuan; Zhou, Fu-Ming

2013-01-23

The striatum receives serotonin (5-hydroxytryptamine, 5-HT) innervation and expresses 5-HT2A receptors (5-HT2ARs) and other 5-HT receptors, raising the possibility that the striatal 5-HT system may undergo adaptive changes after chronic severe dopamine (DA) loss and contribute to the function and dysfunction of the striatum. Here we show that in transcription factor Pitx3 gene mutant mice with a selective, severe DA loss in the dorsal striatum mimicking the DA denervation in late Parkinson's disease (PD), both the 5-HT innervation and the 5-HT2AR mRNA expression were increased in the dorsal striatum. Functionally, while having no detectable motor effect in wild type mice, the 5-HT2R agonist 2,5-dimethoxy-4-iodoamphetamine increased both the baseline and l-dopa-induced normal ambulatory and dyskinetic movements in Pitx3 mutant mice, whereas the selective 5-HT2AR blocker volinanserin had the opposite effects. These results demonstrate that Pitx3 mutant mice are a convenient and valid mouse model to study the compensatory 5-HT upregulation following the loss of the nigrostriatal DA projection and that the upregulated 5-HT2AR function in the DA deficient dorsal striatum may enhance both normal and dyskinetic movements. Copyright © 2012 Elsevier B.V. All rights reserved.

Irxl1 mutant mice show reduced tendon differentiation and no patterning defects in musculoskeletal system development.

PubMed

Kimura, Wataru; Machii, Masashi; Xue, XiaoDong; Sultana, Nishat; Hikosaka, Keisuke; Sharkar, Mohammad T K; Uezato, Tadayoshi; Matsuda, Masashi; Koseki, Haruhiko; Miura, Naoyuki

2011-01-01

Irxl1 (Iroquois-related homeobox like-1) is a newly identified three amino-acid loop extension (TALE) homeobox gene, which is expressed in various mesoderm-derived tissues, particularly in the progenitors of the musculoskeletal system. To analyze the roles of Irxl1 during embryonic development, we generated mice carrying a null allele of Irxl1. Mice homozygous for the targeted allele were viable, fertile, and showed reduced tendon differentiation. Skeletal morphology and skeletal muscle weight in Irxl1-knockout mice appeared normal. Expression patterns of several marker genes for cartilage, tendon, and muscle progenitors in homozygous mutant embryos were unchanged. These results suggest that Irxl1 is required for the tendon differentiation but dispensable for the patterning of the musculoskeletal system in development. Copyright © 2010 Wiley-Liss, Inc.

Visceral adipose tissue macrophage-targeted TACE silencing to treat obesity-induced type 2 diabetes.

PubMed

Yong, Seok-Beom; Song, Yoonsung; Kim, Yong-Hee

2017-12-01

Obesity is an increasingly prevalent global health problem. Due to its close relations with metabolic diseases and cancer, new therapeutic approaches for treating obesity and obesity-induced metabolic diseases are required. Visceral white adipose tissue (WAT) has been closely associated with obesity-induced inflammation and adipose tissue macrophages (ATMs) are responsible for obesity-induced inflammation by releasing inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6. TNF-α converting enzyme (TACE) is a transmembrane enzyme that induces the enzymatic cleavage and release of inflammatory cytokines. In this study, we developed a nonviral gene delivery system consisting of an oligopeptide (ATS-9R) that can selectively target visceral ATMs. In here we shows visceral adipose tissue-dominant inflammatory gene over-expressions in obese mouse and our strategy enabled the preferential delivery of therapeutic genes to visceral ATMs and successfully achieved ATM-targeted gene silencing. Finally, ATS-9R-mediated TACE gene silencing in visceral ATMs alleviated visceral fat inflammation and improved type 2 diabetes by reducing whole body inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Otx1 null mutant mice show partial segregation of sensory epithelia comparable to lamprey ears

NASA Technical Reports Server (NTRS)

Fritzsch, B.; Signore, M.; Simeone, A.

2001-01-01

We investigated the development of inner ear innervation in Otx1 null mutants, which lack a horizontal canal, between embryonic day 12 (E12) and postnatal day 7 (P7) with DiI and immunostaining for acetylated tubulin. Comparable to control animals, horizontal crista-like fibers were found to cross over the utricle in Otx1 null mice. In mutants these fibers extend toward an area near the endolymphatic duct, not to a horizontal crista. Most Otx1 null mutants had a small patch of sensory hair cells at this position. Measurement of the area of the utricular macula suggested it to be enlarged in Otx1 null mutants. We suggest that parts of the horizontal canal crista remain incorporated in the utricular sensory epithelium in Otx1 null mutants. Other parts of the horizontal crista appear to be variably segregated to form the isolated patch of hair cells identifiable by the unique fiber trajectory as representing the horizontal canal crista. Comparison with lamprey ear innervation reveals similarities in the pattern of innervation with the dorsal macula, a sensory patch of unknown function. SEM data confirm that all foramina are less constricted in Otx1 null mutants. We propose that Otx1 is not directly involved in sensory hair cell formation of the horizontal canal but affects the segregation of the horizontal canal crista from the utricle. It also affects constriction of the two main foramina in the ear, but not their initial formation. Otx1 is thus causally related to horizontal canal morphogenesis as well as morphogenesis of these foramina.

Mutant SOD1 in cell types other than motor neurons and oligodendrocytes accelerates onset of disease in ALS mice

PubMed Central

Yamanaka, Koji; Boillee, Severine; Roberts, Elizabeth A.; Garcia, Michael L.; McAlonis-Downes, Melissa; Mikse, Oliver R.; Cleveland, Don W.; Goldstein, Lawrence S. B.

2008-01-01

Dominant mutations in ubiquitously expressed superoxide dismutase (SOD1) cause familial ALS by provoking premature death of adult motor neurons. To test whether mutant damage to cell types beyond motor neurons is required for the onset of motor neuron disease, we generated chimeric mice in which all motor neurons and oligodendrocytes expressed mutant SOD1 at a level sufficient to cause fatal, early-onset motor neuron disease when expressed ubiquitously, but did so in a cellular environment containing variable numbers of non-mutant, non-motor neurons. Despite high-level mutant expression within 100% of motor neurons and oligodendrocytes, in most of these chimeras, the presence of WT non-motor neurons substantially delayed onset of motor neuron degeneration, increasing disease-free life by 50%. Disease onset is therefore non-cell autonomous, and mutant SOD1 damage within cell types other than motor neurons and oligodendrocytes is a central contributor to initiation of motor neuron degeneration. PMID:18492803

The Tennessee Mouse Genome Consortium: Identification of ocular mutants

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

Jablonski, Monica M.; Wang, Xiaofei; Lu, Lu

2005-06-01

The Tennessee Mouse Genome Consortium (TMGC) is in its fifth year of a ethylnitrosourea (ENU)-based mutagenesis screen to detect recessive mutations that affect the eye and brain. Each pedigree is tested by various phenotyping domains including the eye, neurohistology, behavior, aging, ethanol, drug, social behavior, auditory, and epilepsy domains. The utilization of a highly efficient breeding protocol and coordination of various universities across Tennessee makes it possible for mice with ENU-induced mutations to be evaluated by nine distinct phenotyping domains within this large-scale project known as the TMGC. Our goal is to create mutant lines that model human diseases andmore » disease syndromes and to make the mutant mice available to the scientific research community. Within the eye domain, mice are screened for anterior and posterior segment abnormalities using slit-lamp biomicroscopy, indirect ophthalmoscopy, fundus photography, eye weight, histology, and immunohistochemistry. As of January 2005, we have screened 958 pedigrees and 4800 mice, excluding those used in mapping studies. We have thus far identified seven pedigrees with primary ocular abnormalities. Six of the mutant pedigrees have retinal or subretinal aberrations, while the remaining pedigree presents with an abnormal eye size. Continued characterization of these mutant mice should in most cases lead to the identification of the mutated gene, as well as provide insight into the function of each gene. Mice from each of these pedigrees of mutant mice are available for distribution to researchers for independent study.« less

Maternal nicotine exposure effects on adolescent learning and memory are abolished in alpha(α)2* nicotinic acetylcholine receptor-null mutant mice.

PubMed

Mojica, Celina; Bai, Yu; Lotfipour, Shahrdad

2018-06-01

The objective of the current study is to test the hypothesis that the deletion of alpha(α)2* nicotinic acetylcholine receptors (nAChRs) (encoded by the Chrna2 gene) ablate maternal nicotine-induced learning and memory deficits in adolescent mice. We use a pre-exposure-dependent contextual fear conditioning behavioral paradigm that is highly hippocampus-dependent. Adolescent wild type and α2-null mutant offspring are exposed to vehicle or maternal nicotine exposure (200 μg/ml, expressed as base) in the drinking water throughout pregnancy until weaning. Adolescent male offspring mice are tested for alterations in growth and development characteristics as well as modifications in locomotion, anxiety, shock-reactivity and learning and memory. As expected, maternal nicotine exposure has no effects on pup number, weight gain and only modestly reduces fluid intake by 19%. Behaviorally, maternal nicotine exposure impedes extinction learning in adolescent wild type mice, a consequence that is abolished in α2-null mutant mice. The effects on learning and memory are not confounded by alternations in stereotypy, locomotion, anxiety or sensory shock reactivity. Overall, the findings highlight that the deletion of α2* nAChRs eliminate the effects of maternal nicotine exposure on learning and memory in adolescent mice. Copyright © 2018 Elsevier Ltd. All rights reserved.

Intraprocedural C-Arm Dual-Phase Cone-Beam CT: Can It Be Used to Predict Short-term Response to TACE with Drug-eluting Beads in Patients with Hepatocellular Carcinoma?

PubMed Central

Loffroy, Romaric; Lin, MingDe; Yenokyan, Gayane; Rao, Pramod P.; Bhagat, Nikhil; Noordhoek, Niels; Radaelli, Alessandro; Blijd, Järl; Liapi, Eleni

2013-01-01

Purpose: To investigate whether C-arm dual-phase cone-beam computed tomography (CT) performed during transcatheter arterial chemoembolization (TACE) with doxorubicin-eluting beads can help predict tumor response at 1-month follow-up in patients with hepatocellular carcinoma (HCC). Materials and Methods: This prospective study was compliant with HIPAA and approved by the institutional review board and animal care and use committee. Analysis was performed retrospectively on 50 targeted HCC lesions in 29 patients (16 men, 13 women; mean age, 61.9 years ± 10.7) treated with TACE with drug-eluting beads. Magnetic resonance (MR) imaging was performed at baseline and 1 month after TACE. Dual-phase cone-beam CT was performed before and after TACE. Tumor enhancement at dual-phase cone-beam CT in early arterial and delayed venous phases was assessed retrospectively with blinding to MR findings. Tumor response at MR imaging was assessed according to European Association for the Study of the Liver (EASL) guidelines. Two patients were excluded from analysis because dual-phase cone-beam CT scans were not interpretable. Logistic regression models for correlated data were used to compare changes in tumor enhancement between modalities. The radiation dose with dual-phase cone-beam CT was measured in one pig. Results: At 1-month MR imaging follow-up, complete and/or partial tumor response was seen in 74% and 76% of lesions in the arterial and venous phases, respectively. Paired t tests used to compare images obtained before and after TACE showed a significant reduction in tumor enhancement with both modalities (P < .0001). The decrease in tumor enhancement seen with dual-phase cone-beam CT after TACE showed a linear correlation with MR findings. Estimated correlation coefficients were excellent for first (R = 0.89) and second (R = 0.82) phases. A significant relationship between tumor enhancement at cone-beam CT after TACE and complete and/or partial tumor response at MR imaging

Hip1-related mutant mice grow and develop normally but have accelerated spinal abnormalities and dwarfism in the absence of HIP1.

PubMed

Hyun, Teresa S; Li, Lina; Oravecz-Wilson, Katherine I; Bradley, Sarah V; Provot, Melissa M; Munaco, Anthony J; Mizukami, Ikuko F; Sun, Hanshi; Ross, Theodora S

2004-05-01

In mice and humans, there are two known members of the Huntingtin interacting protein 1 (HIP1) family, HIP1 and HIP1-related (HIP1r). Based on structural and functional data, these proteins participate in the clathrin trafficking network. The inactivation of Hip1 in mice leads to spinal, hematopoietic, and testicular defects. To investigate the biological function of HIP1r, we generated a Hip1r mutant allele in mice. Hip1r homozygous mutant mice are viable and fertile without obvious morphological abnormalities. In addition, embryonic fibroblasts derived from these mice do not have gross abnormalities in survival, proliferation, or clathrin trafficking pathways. Altogether, this demonstrates that HIP1r is not necessary for normal development of the embryo or for normal adulthood and suggests that HIP1 or other functionally related members of the clathrin trafficking network can compensate for HIP1r absence. To test the latter, we generated mice deficient in both HIP1 and HIP1r. These mice have accelerated development of abnormalities seen in Hip1 -deficient mice, including kypholordosis and growth defects. The severity of the Hip1r/Hip1 double-knockout phenotype compared to the Hip1 knockout indicates that HIP1r partially compensates for HIP1 function in the absence of HIP1 expression, providing strong evidence that HIP1 and HIP1r have overlapping roles in vivo.

Vaccination with plasmid DNA encoding a mutant toxic shock syndrome toxin-1 ameliorates toxin-induced lethal shock in mice.

PubMed

Feng, Mao-Hui; Cui, Jing-Chun; Nakane, Akio; Hu, Dong-Liang

2013-09-01

Staphylococcal toxic shock syndrome toxin-1 (TSST-1), a superantigenic toxin produced by Staphylococcus (S.) aureus, is a major cause of septic shock and toxic shock syndrome. To investigate whether vaccination with a plasmid DNA encoding a non-toxic mutant TSST-1 (mTSST-1) can protect mice against wild-type TSST-1-induced lethal shock, the mice were intranasally immunized with the plasmid DNA (named pcDNA-mTSST-1) plus a mucosal adjuvant, a non-toxic mutant labile toxin (mLT). After the immunization, the mice were challenged with TSST-1 and lipopolysaccharide (LPS). The survival rate of mice immunized with pcDNA-mTSST-1 plus mLT was higher than that of the control mice immunized with PBS alone, mLT alone, pcDNA-mTSST-1 alone, or a parent plasmid plus mLT. The titers of interferon-γ (IFN-γ) in the sera of mice immunized with pcDNA-mTSST-1 plus mLT were significantly lower than those of the mLT control mice. Immunization with pcDNA-mTSST-1 plus mLT increased the serum levels of TSST-1-specific antibodies, especially immunoglobulin G1 (IgG1) and IgG2a subclasses. Furthermore, the sera obtained from mice immunized with pcDNA-mTSST-1 plus mLT significantly inhibited the TSST-1-induced secretion of IFN-γ and tumor necrosis factor-α (TNF-α) in murine spleen cells in vitro. These results indicate that immunization with pcDNA-mTSST-1 plus mLT provides protection against the lethal toxic shock of mice induced by wild-type TSST-1. The protective effect could be due to TSST-1-specific neutralizing antibodies as well as the inhibition of IFN-γ and TNF-α secretions. Since TSST-1 is commonly released by invasive S. aureus, the pcDNA-mTSST-1 should be useful in preventing toxin-induced shock resulting from S. aureus infection.

Etiology of a genetically complex seizure disorder in Celf4 mutant mice

PubMed Central

Wagnon, Jacy L.; Mahaffey, Connie L.; Sun, Wenzhi; Yang, Yan; Chao, Hsiao-Tuan; Frankel, Wayne N.

2011-01-01

Mice deficient for the gene encoding the RNA-binding protein CELF4 (CUGBP, ELAV-like family member 4) have a complex seizure phenotype that includes both convulsive and non-convulsive seizures, depending upon gene dosage and strain background, modeling genetically complex epilepsy. Invertebrate CELF is associated with translational control in fruit fly ovary epithelium and with neurogenesis and neuronal function in the nematode. Mammalian CELF4 is expressed widely during early development, but is restricted to the central nervous system in adult. To better understand the etiology of the seizure disorder of Celf4 deficient mice, we studied seizure incidence with spatial and temporal conditional knockout Celf4 alleles. For convulsive seizure phenotypes, it is sufficient to delete Celf4 in adulthood at seven weeks of age. This timing is in contrast to absence-like non-convulsive seizures, which require deletion before the end of the first postnatal week. Interestingly, selective deletion of Celf4 from cerebral cortex and hippocampus excitatory neurons, but not from inhibitory neurons, is sufficient to lower seizure threshold and to promote spontaneous convulsions. Correspondingly, Celf4 deficient mice have altered excitatory, but not inhibitory, neurotransmission as measured by patch-clamp recordings of cortical layer V pyramidal neurons. Finally, immunostaining in conjunction with an inhibitory neuron-specific reporter shows that CELF4 is expressed predominantly in excitatory neurons. Our results suggest that CELF4 plays a specific role in regulating excitatory neurotransmission. We posit that altered excitatory neurotransmission resulting from Celf4 deficiency underlies the complex seizure disorder in Celf4 mutant mice. PMID:21745337

The cleft lip and palate defects in Dancer mutant mice result from gain of function of the Tbx10 gene

PubMed Central

Bush, Jeffrey O.; Lan, Yu; Jiang, Rulang

2004-01-01

Cleft lip and palate (CL/P) is a common disfiguring birth defect with complex, poorly understood etiology. Mice carrying a spontaneous mutation, Dancer (Dc), exhibit CL/P in homozygotes and show significantly increased susceptibility to CL/P in heterozygotes [Deol, M. S. & Lane, P. W. (1966) J. Embryol. Exp. Morphol. 16, 543–558 and Trasler, D. G., Kemp, D. & Trasler, T. A. (1984) Teratology 29, 101–104], providing an animal model for understanding the molecular pathogenesis of CL/P. We genetically mapped Dc to within a 1-cM region near the centromere of chromosome 19. In situ hybridization analysis showed that one positional candidate gene, Tbx10, is ectopically expressed in Dc mutant embryos. Positional cloning of the Dc locus revealed an insertion of a 3.3-kb sequence containing the 5′ region of the p23 gene into the first intron of Tbx10, which causes ectopic expression of a p23-Tbx10 chimeric transcript encoding a protein product identical to a normal variant of the Tbx10 protein. Furthermore, we show that ectopic expression of Tbx10 in transgenic mice recapitulates the Dc mutant phenotype, indicating that CL/Pin Dc mutant mice results from the p23 insertion-induced ectopic Tbx10 expression. These results identify gain of function of a T-box transcription factor gene as a mechanism underlying CL/P pathogenesis. PMID:15118109

Serine 421 regulates mutant huntingtin toxicity and clearance in mice

PubMed Central

Kratter, Ian H.; Zahed, Hengameh; Lau, Alice; Daub, Aaron C.; Weiberth, Kurt F.; Gu, Xiaofeng; Humbert, Sandrine; Yang, X. William; Osmand, Alex; Steffan, Joan S.; Masliah, Eliezer

2016-01-01

Huntington’s disease (HD) is a progressive, adult-onset neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminal region of the protein huntingtin (HTT). There are no cures or disease-modifying therapies for HD. HTT has a highly conserved Akt phosphorylation site at serine 421, and prior work in HD models found that phosphorylation at S421 (S421-P) diminishes the toxicity of mutant HTT (mHTT) fragments in neuronal cultures. However, whether S421-P affects the toxicity of mHTT in vivo remains unknown. In this work, we used murine models to investigate the role of S421-P in HTT-induced neurodegeneration. Specifically, we mutated the human mHTT gene within a BAC to express either an aspartic acid or an alanine at position 421, mimicking tonic phosphorylation (mHTT-S421D mice) or preventing phosphorylation (mHTT-S421A mice), respectively. Mimicking HTT phosphorylation strongly ameliorated mHTT-induced behavioral dysfunction and striatal neurodegeneration, whereas neuronal dysfunction persisted when S421 phosphorylation was blocked. We found that S421 phosphorylation mitigates neurodegeneration by increasing proteasome-dependent turnover of mHTT and reducing the presence of a toxic mHTT conformer. These data indicate that S421 is a potent modifier of mHTT toxicity and offer in vivo validation for S421 as a therapeutic target in HD. PMID:27525439

Production and characterization of streptomycin dependent mutants of Pasteurella multocida from bovine haemorrhagic septicaemia.

PubMed Central

de Alwis, M C; Carter, G R; Chengappa, M M

1980-01-01

A large number of streptomycin dependent mutants were produced from bovine haemorrhagic septicaemia strains of Pasteurella multocida. The mutants required a minimum concentration of 25-50 microgram/mL streptomycin for growth and tolerated a concentration of 200 mg/mL. These mutants were avirulent to mice, when inoculated alone, but some mutants killed mice when inoculated with streptomycin. Biochemically all mutants were uniform and similar to the wild type. Most mutants were stable, but a few produced streptomycin independent revertants. The rate of reversion varied with each mutant. Most revertants were highly virulent for mice, some totally avirulant and a few relatively avirulent. PMID:6778598

Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

PubMed Central

Gey, Manuel; Wanner, Renate; Schilling, Corinna; Pedro, Maria T.; Sinske, Daniela

2016-01-01

Axon injury in the peripheral nervous system (PNS) induces a regeneration-associated gene (RAG) response. Atf3 (activating transcription factor 3) is such a RAG and ATF3's transcriptional activity might induce ‘effector’ RAGs (e.g. small proline rich protein 1a (Sprr1a), Galanin (Gal), growth-associated protein 43 (Gap43)) facilitating peripheral axon regeneration. We provide a first analysis of Atf3 mouse mutants in peripheral nerve regeneration. In Atf3 mutant mice, facial nerve regeneration and neurite outgrowth of adult ATF3-deficient primary dorsal root ganglia neurons was decreased. Using genome-wide transcriptomics, we identified a neuropeptide-encoding RAG cluster (vasoactive intestinal peptide (Vip), Ngf, Grp, Gal, Pacap) regulated by ATF3. Exogenous administration of neuropeptides enhanced neurite growth of Atf3 mutant mice suggesting that these molecules might be effector RAGs of ATF3's pro-regenerative function. In addition to the induction of growth-promoting molecules, we present data that ATF3 suppresses growth-inhibiting molecules such as chemokine (C-C motif) ligand 2. In summary, we show a pro-regenerative ATF3 function during PNS nerve regeneration involving transcriptional activation of a neuropeptide-encoding RAG cluster. ATF3 is a general injury-inducible factor, therefore ATF3-mediated mechanisms identified herein might apply to other cell and injury types. PMID:27581653

Validation of the Risk Prediction Models STATE-Score and START-Strategy to Guide TACE Treatment in Patients with Hepatocellular Carcinoma.

PubMed

Mähringer-Kunz, Aline; Kloeckner, Roman; Pitton, Michael B; Düber, Christoph; Schmidtmann, Irene; Galle, Peter R; Koch, Sandra; Weinmann, Arndt

2017-07-01

Several scoring systems that guide patients' treatment regimen for transarterial chemoembolization (TACE) of hepatocellular carcinoma (HCC) have been introduced, but none have gained widespread acceptance in clinical practice. The purpose of this study is to externally validate the Selection for TrAnsarterial chemoembolization TrEatment (STATE)-score and START-strategy [i.e., sequential use of the STATE-score and Assessment for Retreatment with TACE (ART)-score]. From January 2000 to September 2015, 933 patients with HCC underwent TACE at our institution. All variables needed to calculate the STATE-score and implement the START-strategy were determined. STATE comprised serum albumin, up-to-seven criteria, and C-reactive protein (CRP). ART comprised an increase in aspartate aminotransferase, the Child-Pugh score, and a radiological tumor response. Overall survival was calculated, and multivariate analysis performed. In addition, the STATE-score and START-strategy were validated using the Harrell's C-index and integrated Brier score (IBS). The STATE-score was calculated in 228 patients. Low and high STATE-scores corresponded to median survival of 14.3 and 20.2 months, respectively. Harrell's C was 0.558 and IBS 0.133. For the STATE-score, significant predictors of survival were up-to-seven criteria (p = 0.006) and albumin (p = 0.022). CRP values were not predictive (p = 0.367). The ART-score was calculated in 207 patients. Combining the STATE-score and ART-score led to a Harrell's C of 0.580 and IBS of 0.132. The STATE-score was unable to reliably determine the suitability for initial TACE. The START-strategy only slightly improved the predictive ability compared to the ART-score alone. Therefore, neither the STATE-score nor START-strategy alone provides sufficient certainty for clear-cut clinical decisions.

Structural abnormalities of corpus callosum and cortical axonal tracts accompanied by decreased anxiety-like behavior and lowered sociability in spock3- mutant mice.

PubMed

Yamamoto, Ayako; Uchiyama, Koji; Nara, Tomoka; Nishimura, Naomichi; Hayasaka, Michiko; Hanaoka, Kazunori; Yamamoto, Tatsuro

2014-01-01

Spock3/Testican-3 is a nervous system-expressed heparan sulfate proteoglycan belonging to a subgroup of the BM-40/SPARC/osteonectin family, the role of which in brain development is unclear. Because Spock1, a member of the Spock family, inhibits their attachment to substrates and the neurite outgrowth of cultured neuronal cells, Spock3 is also thought to be similarly involved in the neuronal development. In the present study, we established a Spock3-mutant mouse harboring a deletion extending from the presumptive upstream regulatory region to exon 4 of the Spock3 locus and performed histological and behavioral studies on these mutant mice. In wild-type (WT) mice, all Spock members were clearly expressed during brain development. In adults, intense Spock1 and Spock2 expressions were observed throughout the entire brain; whereas, Spock3 expression was no longer visible except in the thalamic nuclei. Thus, Spock3 expression is mostly confined to the developmental stage of the brain. In adult mutant mice, the cells of all cortical layers were swollen. The corpus callosum was narrowed around the central region along the rostral-caudal axis and many small spaces were observed without myelin sheaths throughout the entire corpus callosum. In addition, the cortical input and output fibers did not form into thick bundled fibers as well as the WT counterparts did. Moreover, a subpopulation of corticospinal axonal fibers penetrated into the dorsal striatum with moderately altered orientations. Consistent with these modifications of brain structures, the mutant mice exhibited decreased anxiety-like behavior and lowered sociability. Together, these results demonstrate that Spock3 plays an important role in the formation or maintenance of major neuronal structures in the brain. © 2014 S. Karger AG, Basel.

Primary Coenzyme Q Deficiency in Pdss2 Mutant Mice Causes Isolated Renal Disease

PubMed Central

Haase, Volker H.; King, Rhonda; Polyak, Erzsebet; Selak, Mary; Yudkoff, Marc; Hancock, Wayne W.; Meade, Ray; Saiki, Ryoichi; Lunceford, Adam L.; Clarke, Catherine F.; Gasser, David L.

2008-01-01

Coenzyme Q (CoQ) is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2kd/kd genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2kd/kd mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2loxP/loxP knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2loxP/loxP knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment. PMID:18437205

Hip1-related Mutant Mice Grow and Develop Normally but Have Accelerated Spinal Abnormalities and Dwarfism in the Absence of HIP1†

PubMed Central

Hyun, Teresa S.; Li, Lina; Oravecz-Wilson, Katherine I.; Bradley, Sarah V.; Provot, Melissa M.; Munaco, Anthony J.; Mizukami, Ikuko F.; Sun, Hanshi; Ross, Theodora S.

2004-01-01

In mice and humans, there are two known members of the Huntingtin interacting protein 1 (HIP1) family, HIP1 and HIP1-related (HIP1r). Based on structural and functional data, these proteins participate in the clathrin trafficking network. The inactivation of Hip1 in mice leads to spinal, hematopoietic, and testicular defects. To investigate the biological function of HIP1r, we generated a Hip1r mutant allele in mice. Hip1r homozygous mutant mice are viable and fertile without obvious morphological abnormalities. In addition, embryonic fibroblasts derived from these mice do not have gross abnormalities in survival, proliferation, or clathrin trafficking pathways. Altogether, this demonstrates that HIP1r is not necessary for normal development of the embryo or for normal adulthood and suggests that HIP1 or other functionally related members of the clathrin trafficking network can compensate for HIP1r absence. To test the latter, we generated mice deficient in both HIP1 and HIP1r. These mice have accelerated development of abnormalities seen in Hip1 -deficient mice, including kypholordosis and growth defects. The severity of the Hip1r/Hip1 double-knockout phenotype compared to the Hip1 knockout indicates that HIP1r partially compensates for HIP1 function in the absence of HIP1 expression, providing strong evidence that HIP1 and HIP1r have overlapping roles in vivo. PMID:15121852

Effects of dietary restriction on the expression of insulin-signaling-related genes in long-lived mutant mice.

PubMed

Bartke, Andrzej; Masternak, Michal M; Al-Regaiey, Khalid A; Bonkowski, Michael S

2007-01-01

Hypopituitary Ames dwarf mice and growth-hormone-resistant (growth hormone receptor knockout, GHRKO) mice have reduced plasma levels of insulin-like growth factor 1 and insulin, enhanced insulin sensitivity and a remarkably increased life span. This resembles the phenotypic characteristics of genetically normal animals subjected to dietary restriction (DR). Interestingly, DR leads to further increases in insulin sensitivity and longevity in Ames dwarfs but not in GHRKO mice. It was therefore of interest to examine the effects of DR on the expression of insulin-related genes in these two types of long-lived mutant mice. The effects of DR partially overlapped but did not duplicate the effects of Ames dwarfism or GHR deletion on the expression of genes related to insulin signaling and cell responsiveness to insulin. Moreover, the effects of DR on the expression of the examined genes in different insulin target organs were not identical. Some of the insulin-related genes were similarly affected by DR in both GHRKO and normal mice, some were affected only in GHRKO mice and some only in normal animals. This last category is of particular interest since genes affected in normal but not GHRKO mice may be related to mechanisms by which DR extends longevity.

Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice.

PubMed

Ma, Dezun; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Xiao, Gaojun; Cui, Wentao

2015-08-24

Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity.

AAV-Mediated Administration of Myostatin Pro-Peptide Mutant in Adult Ldlr Null Mice Reduces Diet-Induced Hepatosteatosis and Arteriosclerosis

PubMed Central

Guo, Wen; Wong, Siu; Bhasin, Shalender

2013-01-01

Genetic disruption of myostatin or its related signaling is known to cause strong protection against diet-induced metabolic disorders. The translational value of these prior findings, however, is dependent on whether such metabolically favorable phenotype can be reproduced when myostatin blockade begins at an adult age. Here, we reported that AAV-mediated delivery of a myostatin pro-peptide D76A mutant in adult mice attenuates the development of hepatic steatosis and arteriosclerosis, two common diet-induced metabolic diseases. A single dose of AAV-D76A in adult Ldlr null mice resulted in sustained expression of myostatin pro-peptide in the liver. Compared to vehicle-treated mice, D76A-treated mice gained similar amount of lean and fat mass when fed a high fat diet. However, D76A-treated mice displayed significantly reduced aortic lesions and liver fat, in association with a reduction in hepatic expression of lipogenic genes and improvement in liver insulin sensitivity. This suggests that muscle and fat may not be the primary targets of treatment under our experimental condition. In support to this argument, we show that myostatin directly up-regulated lipogenic genes and increased fat accumulation in cultured liver cells. We also show that both myostatin and its receptor were abundantly expressed in mouse aorta. Cultured aortic endothelial cells responded to myostatin with a reduction in eNOS phosphorylation and an increase in ICAM-1 and VCAM-1 expression. Conclusions: AAV-mediated expression of myostatin pro-peptide D76A mutant in adult Ldlr null mice sustained metabolic protection without remarkable impacts on body lean and fat mass. Further investigations are needed to determine whether direct impact of myostatin on liver and aortic endothelium may contribute to the related metabolic phenotypes. PMID:23936482

Dissociation of spatial navigation and visual guidance performance in Purkinje cell degeneration (pcd) mutant mice.

PubMed

Goodlett, C R; Hamre, K M; West, J R

1992-04-10

Spatial learning in rodents requires normal functioning of hippocampal and cortical structures. Recent data suggest that the cerebellum may also be essential. Neurological mutant mice with dysgenesis of the cerebellum provide useful models to examine the effects of abnormal cerebellar function. Mice with one such mutation, Purkinje cell degeneration (pcd), in which Purkinje cells degenerate between the third and fourth postnatal weeks, were evaluated for performance of spatial navigation learning and visual guidance learning in the Morris maze swim-escape task. Unaffected littermates and C57BL/6J mice served as controls. Separate groups of pcd and control mice were tested at 30, 50 and 110 days of age. At all ages, pcd mice had severe deficits in distal-cue (spatial) navigation, failing to decrease path lengths over training and failing to express appropriate spatial biases on probe trials. On the proximal-cue (visual guidance) task, whenever performance differences between groups did occur, they were limited to the initial trials. The ability of the pcd mice to perform the proximal-cue but not the distal-cue task indicates that the massive spatial navigation deficit was not due simply to motor dysfunction. Histological evaluations confirmed that the pcd mutation resulted in Purkinje cell loss without significant depletion of cells in the hippocampal formation. These data provide further evidence that the cerebellum is vital for the expression of behavior directed by spatial cognitive processes.

Down regulation of miR-124 in both Werner syndrome DNA helicase mutant mice and mutant Caenorhabditis elegans wrn-1 reveals the importance of this microRNA in accelerated aging

PubMed Central

Dallaire, Alexandra; Garand, Chantal; Paquet, Eric R.; Mitchell, Sarah J.; de Cabo, Rafael; Simard, Martin J.

2012-01-01

Small non-coding microRNAs are believed to be involved in the mechanism of aging but nothing is known on the impact of microRNAs in the progeroid disorder Werner syndrome (WS). WS is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN ortholog exhibit many phenotypic features of WS, including a pro-oxidant status and a shorter mean life span. Caenorhabditis elegans (C. elegans) with a nonfunctional wrn-1 DNA helicase also exhibit a shorter life span. Thus, both models are relevant to study the expression of microRNAs involved in WS. In this study, we show that miR-124 expression is lost in the liver of Wrn helicase mutant mice. Interestingly, the expression of this conserved miR-124 in whole wrn-1 mutant worms is also significantly reduced. The loss of mir-124 in C. elegans increases reactive oxygen species formation and accumulation of the aging marker lipofuscin, reduces whole body ATP levels and results in a reduction in life span. Finally, supplementation of vitamin C normalizes the median life span of wrn-1 and mir-124 mutant worms. These results suggest that biological pathways involving WRN and miR-124 are conserved in the aging process across different species. PMID:23075628

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.

Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: A sequestration mechanism in metabolic regulation

PubMed Central

Farrelly, Dennis; Brown, Karen S.; Tieman, Aaron; Ren, Jianming; Lira, Sergio A.; Hagan, Deborah; Gregg, Richard; Mookhtiar, Kasim A.; Hariharan, Narayanan

1999-01-01

The importance of glucokinase (GK; EC 2.7.1.12) in glucose homeostasis has been demonstrated by the association of GK mutations with diabetes mellitus in humans and by alterations in glucose metabolism in transgenic and gene knockout mice. Liver GK activity in humans and rodents is allosterically inhibited by GK regulatory protein (GKRP). To further understand the role of GKRP in GK regulation, the mouse GKRP gene was inactivated. With the knockout of the GKRP gene, there was a parallel loss of GK protein and activity in mutant mouse liver. The loss was primarily because of posttranscriptional regulation of GK, indicating a positive regulatory role for GKRP in maintaining GK levels and activity. As in rat hepatocytes, both GK and GKRP were localized in the nuclei of mouse hepatocytes cultured in low-glucose-containing medium. In the presence of fructose or high concentrations of glucose, conditions known to relieve GK inhibition by GKRP in vitro, only GK was translocated into the cytoplasm. In the GKRP-mutant hepatocytes, GK was not found in the nucleus under any tested conditions. We propose that GKRP functions as an anchor to sequester and inhibit GK in the hepatocyte nucleus, where it is protected from degradation. This ensures that glucose phosphorylation is minimal when the liver is in the fasting, glucose-producing phase. This also enables the hepatocytes to rapidly mobilize GK into the cytoplasm to phosphorylate and store or metabolize glucose after the ingestion of dietary glucose. In GKRP-mutant mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control. PMID:10588736

Mice mutant for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and defects in craniofacial development.

PubMed

Rossel, M; Capecchi, M R

1999-11-01

The analysis of mice mutant for both Hoxa1 and Hoxb1 suggests that these two genes function together to pattern the hindbrain. Separately, mutations in Hoxa1 and Hoxb1 have profoundly different effects on hindbrain development. Hoxa1 mutations disrupt the rhombomeric organization of the hindbrain, whereas Hoxb1 mutations do not alter the rhombomeric pattern, but instead influence the fate of cells originating in rhombomere 4. We suggest that these differences are not the consequences of different functional roles for these gene products, but rather reflect differences in the kinetics of Hoxa1 and Hoxb1 gene expression. In strong support of the idea that Hoxa1 and Hoxb1 have overlapping functions, Hoxa1/Hoxb1 double mutant homozygotes exhibit a plethora of defects either not seen, or seen only in a very mild form, in mice mutant for only Hoxa1 or Hoxb1. Examples include: the loss of both rhombomeres 4 and 5, the selective loss of the 2(nd) branchial arch, and the loss of most, but not all, 2(nd) branchial arch-derived tissues. We suggest that the early role for both of these genes in hindbrain development is specification of rhombomere identities and that the aberrant development of the hindbrain in Hoxa1/Hoxb1 double mutants proceeds through two phases, the misspecification of rhombomeres within the hindbrain, followed subsequently by size regulation of the misspecified hindbrain through induction of apoptosis.

Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague.

PubMed

Tiner, Bethany L; Sha, Jian; Ponnusamy, Duraisamy; Baze, Wallace B; Fitts, Eric C; Popov, Vsevolod L; van Lier, Christina J; Erova, Tatiana E; Chopra, Ashok K

2015-12-01

Earlier, we showed that the Δlpp ΔmsbB Δail triple mutant of Yersinia pestis CO92 with deleted genes encoding Braun lipoprotein (Lpp), an acyltransferase (MsbB), and the attachment invasion locus (Ail), respectively, was avirulent in a mouse model of pneumonic plague. In this study, we further evaluated the immunogenic potential of the Δlpp ΔmsbB Δail triple mutant and its derivative by different routes of vaccination. Mice were immunized via the subcutaneous (s.c.) or the intramuscular (i.m.) route with two doses (2 × 10(6) CFU/dose) of the above-mentioned triple mutant with 100% survivability of the animals. Upon subsequent pneumonic challenge with 70 to 92 50% lethal doses (LD(50)) of wild-type (WT) strain CO92, all of the mice survived when immunization occurred by the i.m. route. Since Ail has virulence and immunogenic potential, a mutated version of Ail devoid of its virulence properties was created, and the genetically modified ail replaced the native ail gene on the chromosome of the Δlpp ΔmsbB double mutant, creating a Δlpp ΔmsbB::ailL2 vaccine strain. This newly generated mutant was attenuated similarly to the Δlpp ΔmsbB Δail triple mutant when administered by the i.m. route and provided 100% protection to animals against subsequent pneumonic challenge. Not only were the two above-mentioned mutants cleared rapidly from the initial i.m. site of injection in animals with no histopathological lesions, the immunized mice did not exhibit any disease symptoms during immunization or after subsequent exposure to WT CO92. These two mutants triggered balanced Th1- and Th2-based antibody responses and cell-mediated immunity. A substantial increase in interleukin-17 (IL-17) from the T cells of vaccinated mice, a cytokine of the Th17 cells, further augmented their vaccine potential. Thus, the Δlpp ΔmsbB Δail and Δlpp ΔmsbB::ailL2 mutants represent excellent vaccine candidates for plague, with the latter mutant still retaining Ail immunogenicity but

Choline kinase β mutant mice exhibit reduced phosphocholine, elevated osteoclast activity, and low bone mass.

PubMed

Kular, Jasreen; Tickner, Jennifer C; Pavlos, Nathan J; Viola, Helena M; Abel, Tamara; Lim, Bay Sie; Yang, Xiaohong; Chen, Honghui; Cook, Robert; Hool, Livia C; Zheng, Ming Hao; Xu, Jiake

2015-01-16

The maintenance of bone homeostasis requires tight coupling between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the precise molecular mechanism(s) underlying the differentiation and activities of these specialized cells are still largely unknown. Here, we identify choline kinase β (CHKB), a kinase involved in the biosynthesis of phosphatidylcholine, as a novel regulator of bone homeostasis. Choline kinase β mutant mice (flp/flp) exhibit a systemic low bone mass phenotype. Consistently, osteoclast numbers and activity are elevated in flp/flp mice. Interestingly, osteoclasts derived from flp/flp mice exhibit reduced sensitivity to excessive levels of extracellular calcium, which could account for the increased bone resorption. Conversely, supplementation of cytidine 5'-diphosphocholine in vivo and in vitro, a regimen that bypasses CHKB deficiency, restores osteoclast numbers to physiological levels. Finally, we demonstrate that, in addition to modulating osteoclast formation and function, loss of CHKB corresponds with a reduction in bone formation by osteoblasts. Taken together, these data posit CHKB as a new modulator of bone homeostasis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

PubMed

Iglesias, Ainhoa; Murga, Matilde; Laresgoiti, Usua; Skoudy, Anouchka; Bernales, Irantzu; Fullaondo, Asier; Moreno, Bernardino; Lloreta, José; Field, Seth J; Real, Francisco X; Zubiaga, Ana M

2004-05-01

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

Functional verification of a porcine myostatin propeptide mutant.

PubMed

Ma, Dezun; Jiang, Shengwang; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Xiao, Gaojun; Yang, Jinzeng; Cui, Wentao

2015-10-01

Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells.

A MEK Inhibitor Abrogates Myeloproliferative Disease in Kras Mutant Mice

PubMed Central

Lyubynska, Natalya; Gorman, Matthew F.; Lauchle, Jennifer O.; Hong, Wan Xing; Akutagawa, Jon K.; Shannon, Kevin; Braun, Benjamin S.

2012-01-01

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are aggressive myeloproliferative neoplasms that are incurable with conventional chemotherapy. Mutations that deregulate Ras signaling play a central pathogenic role in both disorders, and Mx1-Cre, KrasLSL-G12D mice that express the Kras oncogene develop a fatal disease that closely mimics these two leukemias in humans. Activated Ras controls multiple downstream effectors, but the specific pathways that mediate the leukemogenic effects of hyperactive Ras are unknown. We used PD0325901, a highly selective pharmacological inhibitor of mitogen-activated protein kinase kinase (MEK), a downstream component of the Ras signaling network, to address how deregulated Raf/MEK/ERK signaling drives neoplasm formation in Mx1-Cre, KrasLSL-G12D mice. PD0325901 treatment induced a rapid and sustained reduction in leukocyte counts, enhanced erythropoiesis, prolonged mouse survival, and corrected the aberrant proliferation and differentiation of bone marrow progenitor cells. These responses were due to direct effects of PD0325901 on Kras mutant cells rather than to stimulation of normal hematopoietic cell proliferation. Consistent with the in vivo response, inhibition of MEK reversed the cytokine hypersensitivity characteristic of KrasG12D hematopoietic progenitor cells in vitro. Our data demonstrate that deregulated Raf/MEK/ERK signaling is integral to the growth of Kras-mediated myeloproliferative neoplasias, and further suggest that MEK inhibition could be a useful way to ameliorate functional hematologic abnormalities in patients with CMML and JMML. PMID:21451123

Treadmill performance of mice with cerebellar lesions: 1. Purkinje cell degeneration mutant mice.

PubMed

Le Marec, N; Lalonde, R

1998-02-01

The purpose of this study was to evaluate the sensorimotor skills of a spontaneous mouse mutant, Purkinje cell degeneration (PCD), marked by selective cerebellar cortical atrophy on a treadmill activated at 1 of 2 speeds and at 1 of 3 slopes, requiring forward movements to avoid footshocks. There was no difference in latencies before falling from the belt between PCD mutants and controls during acquisition. However, PCD mutants were impaired on the fast treadmill during retention, implicating the cerebellum in the memory of a motor skill. During acquisition of the slow treadmill task at the 2 lowest slopes of inclination, PCD mutants spent more time walking than controls, an indication of a decreased ability of coordinating whole body movements. The same pattern of higher walking time on the slow treadmill in PCD mutants was evident during retention. These results indicate that the cerebellar cortex is involved in the acquisition and the retention of a task requiring equilibrium.

Auditory processing and morphological anomalies in medial geniculate nucleus of Cntnap2 mutant mice.

PubMed

Truong, Dongnhu T; Rendall, Amanda R; Castelluccio, Brian C; Eigsti, Inge-Marie; Fitch, R Holly

2015-12-01

Genetic epidemiological studies support a role for CNTNAP2 in developmental language disorders such as autism spectrum disorder, specific language impairment, and dyslexia. Atypical language development and function represent a core symptom of autism spectrum disorder (ASD), with evidence suggesting that aberrant auditory processing-including impaired spectrotemporal processing and enhanced pitch perception-may both contribute to an anomalous language phenotype. Investigation of gene-brain-behavior relationships in social and repetitive ASD symptomatology have benefited from experimentation on the Cntnap2 knockout (KO) mouse. However, auditory-processing behavior and effects on neural structures within the central auditory pathway have not been assessed in this model. Thus, this study examined whether auditory-processing abnormalities were associated with mutation of the Cntnap2 gene in mice. Cntnap2 KO mice were assessed on auditory-processing tasks including silent gap detection, embedded tone detection, and pitch discrimination. Cntnap2 knockout mice showed deficits in silent gap detection but a surprising superiority in pitch-related discrimination as compared with controls. Stereological analysis revealed a reduction in the number and density of neurons, as well as a shift in neuronal size distribution toward smaller neurons, in the medial geniculate nucleus of mutant mice. These findings are consistent with a central role for CNTNAP2 in the ontogeny and function of neural systems subserving auditory processing and suggest that developmental disruption of these neural systems could contribute to the atypical language phenotype seen in autism spectrum disorder. (c) 2015 APA, all rights reserved).

Impaired Object Recognition but Normal Social Behavior and Ultrasonic Communication in Cofilin1 Mutant Mice

PubMed Central

Sungur, A. Özge; Stemmler, Lea; Wöhr, Markus; Rust, Marco B.

2018-01-01

Autism spectrum disorder (ASD), schizophrenia (SCZ) and intellectual disability (ID) show a remarkable overlap in symptoms, including impairments in cognition, social behavior and communication. Human genetic studies revealed an enrichment of mutations in actin-related genes for these disorders, and some of the strongest candidate genes control actin dynamics. These findings led to the hypotheses: (i) that ASD, SCZ and ID share common disease mechanisms; and (ii) that, at least in a subgroup of affected individuals, defects in the actin cytoskeleton cause or contribute to their pathologies. Cofilin1 emerged as a key regulator of actin dynamics and we previously demonstrated its critical role for synaptic plasticity and associative learning. Notably, recent studies revealed an over-activation of cofilin1 in mutant mice displaying ASD- or SCZ-like behavioral phenotypes, suggesting that dysregulated cofilin1-dependent actin dynamics contribute to their behavioral abnormalities, such as deficits in social behavior. These findings let us hypothesize: (i) that, apart from cognitive impairments, cofilin1 mutants display additional behavioral deficits with relevance to ASD or SCZ; and (ii) that our cofilin1 mutants represent a valuable tool to study the underlying disease mechanisms. To test our hypotheses, we compared social behavior and ultrasonic communication of juvenile mutants to control littermates, and we did not obtain evidence for impaired direct reciprocal social interaction, social approach or social memory. Moreover, concomitant emission of ultrasonic vocalizations was not affected and time-locked to social activity, supporting the notion that ultrasonic vocalizations serve a pro-social communicative function as social contact calls maintaining social proximity. Finally, cofilin1 mutants did not display abnormal repetitive behaviors. Instead, they performed weaker in novel object recognition, thereby demonstrating that cofilin1 is relevant not only for

Mutant laboratory mice with abnormalities in hair follicle morphogenesis, cycling, and/or structure: an update.

PubMed

Nakamura, Motonobu; Schneider, Marlon R; Schmidt-Ullrich, Ruth; Paus, Ralf

2013-01-01

Human hair disorders comprise a number of different types of alopecia, atrichia, hypotrichosis, distinct hair shaft disorders as well as hirsutism and hypertrichosis. Their causes vary from genodermatoses (e.g. hypotrichoses) via immunological disorders (e.g. alopecia areata, autoimmune cicatrical alopecias) to hormone-dependent abnormalities (e.g. androgenetic alopecia). A large number of spontaneous mouse mutants and genetically engineered mice develop abnormalities in hair follicle morphogenesis, cycling, and/or hair shaft formation, whose analysis has proven invaluable to define the molecular regulation of hair growth, ranging from hair follicle development, and cycling to hair shaft formation and stem cell biology. Also, the accumulating reports on hair phenotypes of mouse strains provide important pointers to better understand the molecular mechanisms underlying human hair growth disorders. Since numerous new mouse mutants with a hair phenotype have been reported since the publication of our earlier review on this matter a decade ago, we present here an updated, tabulated mini-review. The updated annotated tables list a wide selection of mouse mutants with hair growth abnormalities, classified into four categories: Mutations that affect hair follicle (1) morphogenesis, (2) cycling, (3) structure, and (4) mutations that induce extrafollicular events (for example immune system defects) resulting in secondary hair growth abnormalities. This synthesis is intended to provide a useful source of reference when studying the molecular controls of hair follicle growth and differentiation, and whenever the hair phenotypes of a newly generated mouse mutant need to be compared with existing ones. Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Potent, selective, orally bioavailable inhibitors of tumor necrosis factor-alpha converting enzyme (TACE): discovery of indole, benzofuran, imidazopyridine and pyrazolopyridine P1' substituents.

PubMed

Lu, Zhonghui; Ott, Gregory R; Anand, Rajan; Liu, Rui-Qin; Covington, Maryanne B; Vaddi, Krishna; Qian, Mingxin; Newton, Robert C; Christ, David D; Trzaskos, James; Duan, James J-W

2008-03-15

Potent and selective inhibitors of tumor necrosis factor-alpha converting enzyme (TACE) were discovered with several new heterocyclic P1' groups in conjunction with cyclic beta-amino hydroxamic acid scaffolds. Among them, the pyrazolopyridine provided the best overall profile when combined with tetrahydropyran beta-amino hydroxamic acid scaffold. Specifically, inhibitor 49 showed IC(50) value of 1 nM against porcine TACE and 170 nM in the suppression of LPS-induced TNF-alpha of human whole blood. Compound 49 also displayed excellent selectivity over a wide panel of MMPs as well as excellent oral bioavailability (F%>90%) in rat n-in-1 PK studies.

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

Optimizing catecholaminergic polymorphic ventricular tachycardia therapy in calsequestrin-mutant mice

PubMed Central

Katz, Guy; Khoury, Assad; Kurtzwald, Efrat; Hochhauser, Edith; Porat, Eyal; Shainberg, Asher; Seidman, Jonathan G.; Seidman, Christine E.; Lorber, Abraham; Eldar, Michael; Arad, Michael

2014-01-01

BACKGROUND Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal arrhythmia provoked by physical or emotional stress and mediated by spontaneous Ca2+ release and delayed after-depolarizations. Beta-adrenergic blockers are the therapy of choice but fail to control arrhythmia in up to 50% of patients. OBJECTIVE To optimize antiarrhythmic therapy in recessively inherited CPVT caused by calsequestrin (CASQ2) mutations. METHODS Murine heart rhythm telemetry was obtained at rest, during treadmill exercise, and after injection of epinephrine. The protocol was repeated after injection of different antiarrhythmic drugs. Results were then validated in human patients. RESULTS Adult CASQ2 mutant mice had complex ventricular arrhythmia at rest and developed bidirectional and polymorphic ventricular tachycardia on exertion. Class I antiarrhythmic agents (procainamide, lidocaine, flecainide) were ineffective in controlling arrhythmia. Propranolol and sotalol attenuated arrhythmia at rest but failed to prevent VT during sympathetic stimulation. The calcium channel blocker verapamil showed a dose-dependent protection against CPVT. Verapamil was more effective than the dihydropyridine L-type Ca2+ channel blocker nifedipine, and its activity was markedly enhanced when combined with propranolol. Human patients homozygous for CASQ2D307H mutation, remaining symptomatic despite chronic β-blocker therapy, underwent exercise testing according to the Bruce protocol with continuous electrocardiogram recording. Verapamil was combined with propranolol at maximum tolerated doses. Adding verapamil attenuated ventricular arrhythmia and prolonged exercise duration in five of 11 patients. CONCLUSION Verapamil is highly effective against catecholamine-induced arrhythmia in mice with CASQ2 mutations and may potentiate the antiarrhythmic activity of β-blockers in humans with CPVT2. PMID:20620233

Responses of flocculus and vestibular nuclei neurons in Weaver mutant mice (B6CBA wv/wv) to combined head and body rotation.

PubMed

Grüsser-Cornehls, U

1995-01-01

The responses of vestibular nuclei (Vn) neurons and floccular Purkinje (P) cells to natural stimulation of the horizontal canals were recorded in paralyzed Weaver mutant mice. The Weaver mice suffer from an almost complete postnatal degeneration of granule cells and a portion of the P cells (Sidman et al. 1965). Parallel fibers are never elaborated (Bradley and Berry 1978). Recording sites were localized by means of small, iontophoretically applied HRP markings. Phase and sensitivity were analyzed by a Fourier analysis and a "best sine fitting" program. As in the normal "control" mice (Grüsser-Cornehls et al. 1995), the "simple spike" discharges of Vn and P cells in Weaver mutant mice are modulated sinusoidally upon sinusoidal stimulation. The neuronal response amplitude at fundamental frequency (determined from peristimulus time histograms, PSTHs increased with frequency (0.05-0.5 Hz) for both Vn and floccular neurons. The stimulus frequency/response amplitude and sensitivity (re velocity) curves for floccular neurons are distinctly lower in magnitude than those of Vn neurons (P < 0.01). In our sample of neurons, the Vn neurons curves of the mutants display a remarkable be behavior: the mean value curve of type I neurons is shifted upward, indicating a loss of inhibition but that of type II, downward, demonstrating a downregulation in comparison with the control values. The difference between the two curves is statistically significant (P < 0.001). The mean value curve of all mutant Vn neurons depends on the different fractions of type I and type II neurons in the sample investigated. In our investigations, the mean value curves of both type I and type II neurons also exceed those of the normal controls. The phase shift relative to head angular velocity in the midfrequency range in Vn neurons was very similar to that in normal controls, but the phase advance in the range of 0.3-0.5 Hz was somewhat larger and the SD larger over the whole range tested. Concerning

Fragile X-like behaviors and abnormal cortical dendritic spines in cytoplasmic FMR1-interacting protein 2-mutant mice.

PubMed

Han, Kihoon; Chen, Hogmei; Gennarino, Vincenzo A; Richman, Ronald; Lu, Hui-Chen; Zoghbi, Huda Y

2015-04-01

Silencing of fragile X mental retardation 1 (FMR1) gene and loss of fragile X mental retardation protein (FMRP) cause fragile X syndrome (FXS), a genetic disorder characterized by intellectual disability and autistic behaviors. FMRP is an mRNA-binding protein regulating neuronal translation of target mRNAs. Abnormalities in actin-rich dendritic spines are major neuronal features in FXS, but the molecular mechanism and identity of FMRP targets mediating this phenotype remain largely unknown. Cytoplasmic FMR1-interacting protein 2 (Cyfip2) was identified as an interactor of FMRP, and its mRNA is a highly ranked FMRP target in mouse brain. Importantly, Cyfip2 is a component of WAVE regulatory complex, a key regulator of actin cytoskeleton, suggesting that Cyfip2 could be implicated in the dendritic spine phenotype of FXS. Here, we generated and characterized Cyfip2-mutant (Cyfip2(+/-)) mice. We found that Cyfip2(+/-) mice exhibited behavioral phenotypes similar to Fmr1-null (Fmr1(-/y)) mice, an animal model of FXS. Synaptic plasticity and dendritic spines were normal in Cyfip2(+/-) hippocampus. However, dendritic spines were altered in Cyfip2(+/-) cortex, and the dendritic spine phenotype of Fmr1(-/y) cortex was aggravated in Fmr1(-/y); Cyfip2(+/-) double-mutant mice. In addition to the spine changes at basal state, metabotropic glutamate receptor (mGluR)-induced dendritic spine regulation was impaired in both Fmr1(-/y) and Cyfip2(+/-) cortical neurons. Mechanistically, mGluR activation induced mRNA translation-dependent increase of Cyfip2 in wild-type cortical neurons, but not in Fmr1(-/y) or Cyfip2(+/-) neurons. These results suggest that misregulation of Cyfip2 function and its mGluR-induced expression contribute to the neurobehavioral phenotypes of FXS. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Transgenic mice overexpressing tyrosine-to-cysteine mutant human alpha-synuclein: a progressive neurodegenerative model of diffuse Lewy body disease.

PubMed

Zhou, Wenbo; Milder, Julie B; Freed, Curt R

2008-04-11

Abnormal aggregation of human alpha-synuclein in Lewy bodies and Lewy neurites is a pathological hallmark of Parkinson disease and dementia with Lewy bodies. Studies have shown that oxidation and nitration of alpha-synuclein lead to the formation of stable dimers and oligomers through dityrosine cross-linking. Previously we have reported that tyrosine-to-cysteine mutations, particularly at the tyrosine 39 residue (Y39C), significantly enhanced alpha-synuclein fibril formation and neurotoxicity. In the current study, we have generated transgenic mice expressing the Y39C mutant human alpha-synuclein gene controlled by the mouse Thy1 promoter. Mutant human alpha-synuclein was widely expressed in transgenic mouse brain, resulting in 150% overexpression relative to endogenous mouse alpha-synuclein. At age 9-12 months, transgenic mice began to display motor dysfunction in rotarod testing. Older animals aged 15-18 months showed progressive accumulation of human alpha-synuclein oligomers, associated with worse motor function and cognitive impairment in the Morris water maze. By age 21-24 months, alpha-synuclein aggregates were further increased, accompanied by severe behavioral deficits. At this age, transgenic mice developed neuropathology, such as Lewy body-like alpha-synuclein and ubiquitin-positive inclusions, phosphorylation at Ser(129) of human alpha-synuclein, and increased apoptotic cell death. In summary, Y39C human alpha-synuclein transgenic mice show age-dependent, progressive neuronal degeneration with motor and cognitive deficits similar to diffuse Lewy body disease. The time course of alpha-synuclein oligomer accumulation coincided with behavioral and pathological changes, indicating that these oligomers may initiate protein aggregation, disrupt cellular function, and eventually lead to neuronal death.

Transforming Growth Factor Beta (TGFβ1, TGFβ2 and TGFβ3) Null-Mutant Phenotypes in Embryonic Gonadal Development

PubMed Central

Memon, Mushtaq A.; Anway, Matthew D.; Covert, Trevor R.; Uzumcu, Mehmet; Skinner, Michael K.

2008-01-01

The role transforming growth factor beta (TGFb) isoforms TGFb1, TGFb2 and TGFb3 have in the regulation of embryonic gonadal development was investigated with the use of null-mutant (i.e. knockout) mice for each of the TGFb isoforms. Late embryonic gonadal development was investigated because homozygote TGFb null-mutant mice generally die around birth, with some embryonic loss as well. In the testis, the TGFb1 null-mutant mice had a decrease in the number of germ cells at birth, postnatal day 0 (P0). In the testis, the TGFb2 null-mutant mice had a decrease in the number of seminiferous cords at embryonic day 15 (E15). In the ovary, the TGFb2 null-mutant mice had an increase in the number of germ cells at P0. TGFb isoforms appear to have a role in gonadal development, but interactions between the isoforms is speculated to compensate in the different TGFb isoform null-mutant mice. PMID:18790002

Efficacy of TACE in TIPS Patients: Comparison of Treatment Response to Chemoembolization for Hepatocellular Carcinoma in Patients With and Without a Transjugular Intrahepatic Portosystemic Shunt

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

Kuo, Yuo-Chen, E-mail: yuo-chen.kuo@ucsf.edu; Kohi, Maureen P., E-mail: maureen.kohi@ucsf.edu; Naeger, David M., E-mail: david.naeger@ucsf.edu

Purpose: To compare treatment response after transarterial chemoembolization (TACE) for hepatocellular carcinoma (HCC) in patients with and without a transjugular intrahepatic portosystemic shunt (TIPS). Materials and Methods: A retrospective review of patients who underwent conventional TACE for HCC between January 2005 and December 2009 identified 10 patients with patent TIPS. From the same time period, 23 patients without TIPS were selected to control for comparable Model for End-Stage Liver Disease and Child-Pugh-Turcotte scores. The two groups showed similar distribution of Barcelona Clinic Liver Cancer and United Network of Organ Sharing stages. Target HCC lesions were evaluated according to the modifiedmore » response evaluation criteria in solid tumors (mRECIST) guidelines. Transplantation rate, time to tumor progression, and overall survival (OS) were documented. Results: After TACE, the rate of complete response was significantly greater in non-TIPS patients compared with TIPS patients (74 vs. 30 %, p = 0.03). Objective response rate (complete and partial response) trended greater in the non-TIPS group (83 vs. 50 %, p = 0.09). The liver transplantation rate was 80 and 74 % in the TIPS and non-TIPS groups, respectively (p = 1.0). Time to tumor progression was similar (p = 0.47) between the two groups. OS favored the non-TIPS group (p = 0.01) when censored for liver transplantation. Conclusion: TACE is less effective in achieving complete or partial response using mRECIST criteria in TIPS patients compared with those without a TIPS. Nevertheless, similar clinical outcomes may be achieved, particularly in TIPS patients who are liver-transplantation candidates.« less

Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function.

PubMed

Won, Hyejung; Lee, Hye-Ryeon; Gee, Heon Yung; Mah, Won; Kim, Jae-Ick; Lee, Jiseok; Ha, Seungmin; Chung, Changuk; Jung, Eun Suk; Cho, Yi Sul; Park, Sae-Geun; Lee, Jung-Soo; Lee, Kyungmin; Kim, Daesoo; Bae, Yong Chul; Kaang, Bong-Kiun; Lee, Min Goo; Kim, Eunjoon

2012-06-13

Autism spectrum disorder (ASD) is a group of conditions characterized by impaired social interaction and communication, and restricted and repetitive behaviours. ASD is a highly heritable disorder involving various genetic determinants. Shank2 (also known as ProSAP1) is a multi-domain scaffolding protein and signalling adaptor enriched at excitatory neuronal synapses, and mutations in the human SHANK2 gene have recently been associated with ASD and intellectual disability. Although ASD-associated genes are being increasingly identified and studied using various approaches, including mouse genetics, further efforts are required to delineate important causal mechanisms with the potential for therapeutic application. Here we show that Shank2-mutant (Shank2(-/-)) mice carrying a mutation identical to the ASD-associated microdeletion in the human SHANK2 gene exhibit ASD-like behaviours including reduced social interaction, reduced social communication by ultrasonic vocalizations, and repetitive jumping. These mice show a marked decrease in NMDA (N-methyl-D-aspartate) glutamate receptor (NMDAR) function. Direct stimulation of NMDARs with D-cycloserine, a partial agonist of NMDARs, normalizes NMDAR function and improves social interaction in Shank2(-/-) mice. Furthermore, treatment of Shank2(-/-) mice with a positive allosteric modulator of metabotropic glutamate receptor 5 (mGluR5), which enhances NMDAR function via mGluR5 activation, also normalizes NMDAR function and markedly enhances social interaction. These results suggest that reduced NMDAR function may contribute to the development of ASD-like phenotypes in Shank2(-/-) mice, and mGluR modulation of NMDARs offers a potential strategy to treat ASD.

Insights into wild-type and mutant p53 functions provided by genetically engineered mice.

PubMed

Donehower, Lawrence A

2014-06-01

Recent whole-exome sequencing studies of numerous human cancers have now conclusively shown that the TP53 tumor-suppressor gene is the most frequently mutated gene in human cancers. Despite extensive studies of the TP53 gene and its encoded protein (p53), our understanding of how TP53 mutations contribute to cancer initiation and progression remain incomplete. Genetically engineered mice with germline or inducible Trp53 somatic mutations have provided important insights into the mechanisms by which different types of p53 mutation influence cancer development. Trp53 germline mutations that alter specific p53 structural domains or posttranslation modification sites have benefitted our understanding of wild-type p53 functions in a whole organism context. Moreover, genetic approaches to reestablish functional wild-type p53 to p53-deficient tissues and tumors have increased our understanding of the therapeutic potential of restoring functional p53 signaling to cancers. This review outlines many of the key insights provided by the various categories of Trp53 mutant mice that have been generated by multiple genetic engineering approaches. © 2014 WILEY PERIODICALS, INC.

Retarded axonal transport of R406W mutant tau in transgenic mice with a neurodegenerative tauopathy.

PubMed

Zhang, Bin; Higuchi, Makoto; Yoshiyama, Yasumasa; Ishihara, Takeshi; Forman, Mark S; Martinez, Dan; Joyce, Sonali; Trojanowski, John Q; Lee, Virginia M-Y

2004-05-12

Intracellular accumulations of filamentous tau inclusions are neuropathological hallmarks of neurodegenerative diseases known as tauopathies. The discovery of multiple pathogenic tau gene mutations in many kindreds with familial frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) unequivocally confirmed the central role of tau abnormalities in the etiology of neurodegenerative disorders. To examine the effects of tau gene mutations and the role of tau abnormalities in neurodegenerative tauopathies, transgenic (Tg) mice were engineered to express the longest human tau isoform (T40) with or without the R406W mutation (RW and hWT Tg mice, respectively) that is pathogenic for FTDP-17 in several kindreds. RW but not hWT tau Tg mice developed an age-dependent accumulation of insoluble filamentous tau aggregates in neuronal perikarya of the cerebral cortex, hippocampus, cerebellum, and spinal cord. Significantly, CNS axons in RW mice contained reduced levels of tau when compared with hWT mice, and this was linked to retarded axonal transport and increased accumulation of an insoluble pool of RW but not hWT tau. Furthermore, RW but not hWT mice demonstrated neurodegeneration and a reduced lifespan. These data indicate that the R406W mutation causes reduced binding of this mutant tau to microtubules, resulting in slower axonal transport. This altered tau function caused by the RW mutation leads to increased accumulation and reduced solubility of RW tau in an age-dependent manner, culminating in the formation of filamentous intraneuronal tau aggregates similar to that observed in tauopathy patients.

Probucol ameliorates renal and metabolic sequelae of primary CoQ deficiency in Pdss2 mutant mice.

PubMed

Falk, Marni J; Polyak, Erzsebet; Zhang, Zhe; Peng, Min; King, Rhonda; Maltzman, Jonathan S; Okwuego, Ezinne; Horyn, Oksana; Nakamaru-Ogiso, Eiko; Ostrovsky, Julian; Xie, Letian X; Chen, Jia Yan; Marbois, Beth; Nissim, Itzhak; Clarke, Catherine F; Gasser, David L

2011-07-01

Therapy of mitochondrial respiratory chain diseases is complicated by limited understanding of cellular mechanisms that cause the widely variable clinical findings. Here, we show that focal segmental glomerulopathy-like kidney disease in Pdss2 mutant animals with primary coenzyme Q (CoQ) deficiency is significantly ameliorated by oral treatment with probucol (1% w/w). Preventative effects in missense mutant mice are similar whether fed probucol from weaning or for 3 weeks prior to typical nephritis onset. Furthermore, treating symptomatic animals for 2 weeks with probucol significantly reduces albuminuria. Probucol has a more pronounced health benefit than high-dose CoQ(10) supplementation and uniquely restores CoQ(9) content in mutant kidney. Probucol substantially mitigates transcriptional alterations across many intermediary metabolic domains, including peroxisome proliferator-activated receptor (PPAR) pathway signaling. Probucol's beneficial effects on the renal and metabolic manifestations of Pdss2 disease occur despite modest induction of oxidant stress and appear independent of its hypolipidemic effects. Rather, decreased CoQ(9) content and altered PPAR pathway signaling appear, respectively, to orchestrate the glomerular and global metabolic consequences of primary CoQ deficiency, which are both preventable and treatable with oral probucol therapy. Copyright © 2011 EMBO Molecular Medicine.

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

Reanalysis of parabiosis of obesity mutants in the age of leptin.

PubMed

Zeng, Wenwen; Lu, Yi-Hsueh; Lee, Jonah; Friedman, Jeffrey M

2015-07-21

In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin(-/-) double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure.

Reanalysis of parabiosis of obesity mutants in the age of leptin

PubMed Central

Zeng, Wenwen; Lu, Yi-Hsueh; Lee, Jonah; Friedman, Jeffrey M.

2015-01-01

In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin−/− double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure. PMID:26150485

Immunogenicity and protective efficacy of the Mycobacterium tuberculosis fadD26 mutant

PubMed Central

Infante, E; Aguilar, L D; Gicquel, B; Pando, R Hernandez

2005-01-01

The Mycobacterium tuberculosis fadD26 mutant has impaired synthesis of phthiocerol dimycocerosates (DIM) and is attenuated in BALB/c mice. Survival analysis following direct intratracheal infection confirmed the attenuation: 60% survival at 4 months post-infection versus 100% mortality at 9 weeks post-infection with the wild-type strain. The fadD26 mutant induced less pneumonia and larger DTH reactions. It induced lower but progressive production of interferon (IFN)-γ, interleukin (IL)-4 and tumour necrosis factor (TNF)-α. Used as a subcutaneous vaccine 60 days before intratracheal challenge with a hypervirulent strain of M. tuberculosis (Beijing code 9501000), the mutant induced a higher level of protection than did Bacille Calmette–Guérin (BCG). Seventy per cent of the mice vaccinated with the fadD26 mutant survived at 16 weeks after challenge compared to 30% of those vaccinated with BCG. Similarly, there was less tissue damage (pneumonia) and lower colony-forming units (CFU) in the mice vaccinated with the fadD26 mutant compared to the findings in mice vaccinated with BCG. These data suggest that DIM synthesis is important for the pathogenicity of M. tuberculosis, and that inactivation of DIM synthesis can increase the immunogenicity of live vaccines, and increase their ability to protect against tuberculosis. PMID:15958066

Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

PubMed

Züst, Roland; Dong, Hongping; Li, Xiao-Feng; Chang, David C; Zhang, Bo; Balakrishnan, Thavamalar; Toh, Ying-Xiu; Jiang, Tao; Li, Shi-Hua; Deng, Yong-Qiang; Ellis, Brett R; Ellis, Esther M; Poidinger, Michael; Zolezzi, Francesca; Qin, Cheng-Feng; Shi, Pei-Yong; Fink, Katja

2013-01-01

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

Abnormal iron metabolism and oxidative stress in mice expressing a mutant form of the ferritin light polypeptide gene

PubMed Central

Barbeito, Ana G.; Garringer, Holly J.; Baraibar, Martin A.; Gao, Xiaoying; Arredondo, Miguel; Núñez, Marco T.; Smith, Mark A.; Ghetti, Bernardino; Vidal, Ruben

2009-01-01

Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498-499InsTC cDNA. Compared with wild-type mice, brain extracts from Tg (FTL-Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor-1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone–protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL-Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates in the brain. PMID:19519778

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

Intact interval timing in circadian CLOCK mutants.

PubMed

Cordes, Sara; Gallistel, C R

2008-08-28

While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval-timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/- and -/- mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing.

Haemolysis and Perturbations in the Systemic Iron Metabolism of Suckling, Copper-Deficient Mosaic Mutant Mice – An Animal Model of Menkes Disease

PubMed Central

Lenartowicz, Małgorzata; Starzyński, Rafał R.; Krzeptowski, Wojciech; Grzmil, Paweł; Bednarz, Aleksandra; Ogórek, Mateusz; Pierzchała, Olga; Staroń, Robert; Gajowiak, Anna; Lipiński, Paweł

2014-01-01

The biological interaction between copper and iron is best exemplified by the decreased activity of multicopper ferroxidases under conditions of copper deficiency that limits the availability of iron for erythropoiesis. However, little is known about how copper deficiency affects iron homeostasis through alteration of the activity of other copper-containing proteins, not directly connected with iron metabolism, such as superoxide dismutase 1 (SOD1). This antioxidant enzyme scavenges the superoxide anion, a reactive oxygen species contributing to the toxicity of iron via the Fenton reaction. Here, we analyzed changes in the systemic iron metabolism using an animal model of Menkes disease: copper-deficient mosaic mutant mice with dysfunction of the ATP7A copper transporter. We found that the erythrocytes of these mutants are copper-deficient, display decreased SOD1 activity/expression and have cell membrane abnormalities. In consequence, the mosaic mice show evidence of haemolysis accompanied by haptoglobin-dependent elimination of haemoglobin (Hb) from the circulation, as well as the induction of haem oxygenase 1 (HO1) in the liver and kidney. Moreover, the hepcidin-ferroportin regulatory axis is strongly affected in mosaic mice. These findings indicate that haemolysis is an additional pathogenic factor in a mouse model of Menkes diseases and provides evidence of a new indirect connection between copper deficiency and iron metabolism. PMID:25247420

Loss of Either Rac1 or Rac3 GTPase Differentially Affects the Behavior of Mutant Mice and the Development of Functional GABAergic Networks

PubMed Central

Pennucci, Roberta; Talpo, Francesca; Astro, Veronica; Montinaro, Valentina; Morè, Lorenzo; Cursi, Marco; Castoldi, Valerio; Chiaretti, Sara; Bianchi, Veronica; Marenna, Silvia; Cambiaghi, Marco; Tonoli, Diletta; Leocani, Letizia; Biella, Gerardo; D'Adamo, Patrizia; de Curtis, Ivan

2016-01-01

Rac GTPases regulate the development of cortical/hippocampal GABAergic interneurons by affecting the early development and migration of GABAergic precursors. We have addressed the function of Rac1 and Rac3 proteins during the late maturation of hippocampal interneurons. We observed specific phenotypic differences between conditional Rac1 and full Rac3 knockout mice. Rac1 deletion caused greater generalized hyperactivity and cognitive impairment compared with Rac3 deletion. This phenotype matched with a more evident functional impairment of the inhibitory circuits in Rac1 mutants, showing higher excitability and reduced spontaneous inhibitory currents in the CA hippocampal pyramidal neurons. Morphological analysis confirmed a differential modification of the inhibitory circuits: deletion of either Rac caused a similar reduction of parvalbumin-positive inhibitory terminals in the pyramidal layer. Intriguingly, cannabinoid receptor-1-positive terminals were strongly increased only in the CA1 of Rac1-depleted mice. This increase may underlie the stronger electrophysiological defects in this mutant. Accordingly, incubation with an antagonist for cannabinoid receptors partially rescued the reduction of spontaneous inhibitory currents in the pyramidal cells of Rac1 mutants. Our results show that Rac1 and Rac3 have independent roles in the formation of GABAergic circuits, as highlighted by the differential effects of their deletion on the late maturation of specific populations of interneurons. PMID:26582364

Age-dependent synapse withdrawal at axotomised neuromuscular junctions in Wlds mutant and Ube4b/Nmnat transgenic mice

PubMed Central

Gillingwater, Thomas H; Thomson, Derek; Mack, Till G A; Soffin, Ellen M; Mattison, Richard J; Coleman, Michael P; Ribchester, Richard R

2002-01-01

Axons in WldS mutant mice are protected from Wallerian degeneration by overexpression of a chimeric Ube4b/Nmnat (Wld) gene. Expression of Wld protein was independent of age in these mice. However we identified two distinct neuromuscular synaptic responses to axotomy. In young adult Wlds mice, axotomy induced progressive, asynchronous synapse withdrawal from motor endplates, strongly resembling neonatal synapse elimination. Thus, five days after axotomy, 50–90 % of endplates were still partially or fully occupied and expressed endplate potentials (EPPs). By 10 days, fewer than 20 % of endplates still showed evidence of synaptic activity. Recordings from partially occupied junctions indicated a progressive decrease in quantal content in inverse proportion to endplate occupancy. In Wlds mice aged > 7 months, axons were still protected from axotomy but synapses degenerated rapidly, in wild-type fashion: within three days less than 5 % of endplates contained vestiges of nerve terminals. The axotomy-induced synaptic withdrawal phenotype decayed with a time constant of ∼30 days. Regenerated synapses in mature Wlds mice recapitulated the juvenile phenotype. Within 4–6 days of axotomy 30–50 % of regenerated nerve terminals still occupied motor endplates. Age-dependent synapse withdrawal was also seen in transgenic mice expressing the Wld gene. Co-expression of Wld protein and cyan fluorescent protein (CFP) in axons and neuromuscular synapses did not interfere with the protection from axotomy conferred by the Wld gene. Thus, Wld expression unmasks age-dependent, compartmentally organised programmes of synapse withdrawal and degeneration. PMID:12231635

Genetic background effects in Neuroligin-3 mutant mice: Minimal behavioral abnormalities on C57 background.

PubMed

Jaramillo, Thomas C; Escamilla, Christine Ochoa; Liu, Shunan; Peca, Lauren; Birnbaum, Shari G; Powell, Craig M

2018-02-01

Neuroligin-3 (NLGN3) is a postsynaptic cell adhesion protein that interacts with presynaptic ligands including neurexin-1 (NRXN1) [Ichtchenko et al., Journal of Biological Chemistry, 271, 2676-2682, 1996]. Mice harboring a mutation in the NLGN3 gene (NL3R451C) mimicking a mutation found in two brothers with autism spectrum disorder (ASD) were previously generated and behaviorally phenotyped for autism-related behaviors. In these NL3R451C mice generated and tested on a hybrid C57BL6J/129S2/SvPasCrl background, we observed enhanced spatial memory and reduced social interaction [Tabuchi et al., Science, 318, 71-76, 2007]. Curiously, an independently generated second line of mice harboring the same mutation on a C57BL6J background exhibited minimal aberrant behavior, thereby providing apparently discrepant results. To investigate the origin of the discrepancy, we previously replicated the original findings of Tabuchi et al. by studying the same NL3R451C mutation on a pure 129S2/SvPasCrl genetic background. Here we complete the behavioral characterization of the NL3R451C mutation on a pure C57BL6J genetic background to determine if background genetics play a role in the discrepant behavioral outcomes involving NL3R451C mice. NL3R451C mutant mice on a pure C57BL6J background did not display spatial memory enhancements or social interaction deficits. We only observed a decreased startle response and mildly increased locomotor activity in these mice suggesting that background genetics influences behavioral outcomes involving the NL3R451C mutation. Autism Res 2018, 11: 234-244. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. Behavioral symptoms of autism can be highly variable, even in cases that involve identical genetic mutations. Previous studies in mice with a mutation of the Neuroligin-3 gene showed enhanced learning and social deficits. We replicated these findings on the same and different genetic backgrounds. In this study, however, the

Mutant soluble ectodomain of fibroblast growth factor receptor-2 IIIc attenuates bleomycin-induced pulmonary fibrosis in mice.

PubMed

Yu, Zhi-hong; Wang, Ding-ding; Zhou, Zhi-you; He, Shui-lian; Chen, An-an; Wang, Ju

2012-01-01

We have developed a strong inhibitor (S252W mutant soluble ectodomain of fibroblast growth factor recptor-2 IIIc, msFGFR2) that binds FGFs strongly and blocks the activation of FGFRs. In vitro, msFGFR2 could inhibit the promoting effect of transforming growth factor (TGF)-β1 on the proliferation of primary lung fibroblasts. In vivo, msFGFR2 alleviated lung fibrosis through inhibiting the expression of α-smooth muscle actin (SMA) and collagen deposit. In Western blotting of the right lung tissues and immunohistochemical assay, we found the level of p-FGFRs, p-mitogen activated protein kinase (MAPK) and p-Smad3 in the mice of bleomycin (BLM) group treated with msFGFR2 was down dramatically compared with the mice of BLM group, which suggested the activations of FGF and TGF-β signals were blocked meanwhile. In summary, msFGFR2 attenuated BLM-induced fibrosis and is an attractive therapeutic candidate for human pulmonary fibrosis.

A novel candidate gene for mouse and human preaxial polydactyly with altered expression in limbs of Hemimelic extra-toes mutant mice.

PubMed

Clark, R M; Marker, P C; Kingsley, D M

2000-07-01

Polydactyly is a common malformation of vertebrate limbs. In humans a major locus for nonsyndromic pre-axial polydactyly (PPD) has been mapped previously to 7q36. The mouse Hemimelic extra-toes (Hx) mutation maps to a homologous chromosome segment and has been proposed to affect a homologous gene. To understand the molecular changes underlying PPD, we used a positional cloning approach to identify the gene or genes disrupted by the Hx mutation and a closely linked limb mutation, Hammertoe (Hm). High resolution genetic mapping identified a small candidate interval for the mouse mutations located 1.2 cM distal to the Shh locus. The nonrecombinant interval was completely cloned in bacterial artificial chromosomes and searched for genes using a combination of exon trapping, sample sequencing, and mapping of known genes. Two novel genes, Lmbr1 and Lmbr2, are entirely within the candidate interval we defined genetically. The open reading frame of both genes is intact in mutant mice, but the expression of the Lmbr1 gene is dramatically altered in developing limbs of Hx mutant mice. The correspondence between the spatial and temporal changes in Lmbr1 expression and the embryonic onset of the Hx mutant phenotype suggests that the mouse Hx mutation may be a regulatory allele of Lmbr1. The human ortholog of Lmbr1 maps within the recently described interval for human PPD, strengthening the possibility that both mouse and human limb abnormalities are due to defects in the same highly conserved gene.

Are Sema5a mutant mice a good model of autism? A behavioral analysis of sensory systems, emotionality and cognition

PubMed Central

Gunn, Rhian K.; Huentelman, Matthew J.; Brown, Richard E.

2011-01-01

Semaphorin 5A (Sema5A) expression is reduced in the brain of individuals with autism, thus mice with reduced Sema5A levels may serve as a model of this neurodevelopmental disorder. We tested male and female Sema5a knockout mice (B6.129P2SEMA5A/J) and C57BL/6J controls for emotionality, visual ability, prepulse inhibition, motor learning and cognition. Overall, there were only two genotype differences in emotionality: Sema5a mutant mice had more stretch-attend postures in the elevated plus-maze and more defecations in the open field. All mice could see, but Sema5a mice had better visual ability than C57BL/6J mice. There were no genotype differences in sensory-motor gating. Sema5a mice showed higher levels of activity in the elevated plus-maze and light/dark transition box, and there were sex by genotype differences in the Rotarod, suggesting a sex difference in balance and coordination differentially affected by Sema5a. There were no genotype effects on cognition: Sema5a mice did not differ from C57BL/6J in the Morris water maze, set-shifting or cued and contextual fear conditioning. In the social recognition test, all mice preferred social stimuli, but there was no preference for social novelty, thus the Sema5A mice do not have a deficit in social behavior. Overall, there were a number of sex differences, with females showing greater activity and males performing better in tests of spatial learning and memory, but no deficits in the behavior of Sema5A mice. We conclude that the Sema5a mice do not meet the behavioral criteria for a mouse model of autism. PMID:21777623

The subcutaneous inoculation of pH 6 antigen mutants of Yersinia pestis does not affect virulence and immune response in mice.

PubMed

Anisimov, Andrey P; Bakhteeva, Irina V; Panfertsev, Evgeniy A; Svetoch, Tat'yana E; Kravchenko, Tat'yana B; Platonov, Mikhail E; Titareva, Galina M; Kombarova, Tat'yana I; Ivanov, Sergey A; Rakin, Alexander V; Amoako, Kingsley K; Dentovskaya, Svetlana V

2009-01-01

Two isogenic sets of Yersinia pestis strains were generated, composed of wild-type strains 231 and I-1996, their non-polar pH 6(-) mutants with deletions in the psaA gene that codes for its structural subunit or the whole operon, as well as strains with restored ability for temperature- and pH-dependent synthesis of adhesion pili or constitutive production of pH 6 antigen. The mutants were generated by site-directed mutagenesis of the psa operon and subsequent complementation in trans. It was shown that the loss of synthesis or constitutive production of pH 6 antigen did not influence Y. pestis virulence or the average survival time of subcutaneously inoculated BALB/c naïve mice or animals immunized with this antigen.

Intact Interval Timing in Circadian CLOCK Mutants

PubMed Central

Cordes, Sara; Gallistel, C. R.

2008-01-01

While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/− and −/− mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing. PMID:18602902

The clock gene Period1 regulates innate routine behaviour in mice.

PubMed

Bechstein, Philipp; Rehbach, Nils-Jörn; Yuhasingham, Gowzekan; Schürmann, Christoph; Göpfert, Melanie; Kössl, Manfred; Maronde, Erik

2014-04-22

Laboratory mice are well capable of performing innate routine behaviour programmes necessary for courtship, nest-building and exploratory activities although housed for decades in animal facilities. We found that in mice inactivation of the clock gene Period1 profoundly changes innate routine behaviour programmes like those necessary for courtship, nest building, exploration and learning. These results in wild-type and Period1 mutant mice, together with earlier findings on courtship behaviour in wild-type and period-mutant Drosophila melanogaster, suggest a conserved role of Period-genes on innate routine behaviour. Additionally, both per-mutant flies and Period1-mutant mice display spatial learning and memory deficits. The profound influence of Period1 on routine behaviour programmes in mice, including female partner choice, may be independent of its function as a circadian clock gene, since Period1-deficient mice display normal circadian behaviour.

Paternal Aging Affects Behavior in Pax6 Mutant Mice: A Gene/Environment Interaction in Understanding Neurodevelopmental Disorders.

PubMed

Yoshizaki, Kaichi; Furuse, Tamio; Kimura, Ryuichi; Tucci, Valter; Kaneda, Hideki; Wakana, Shigeharu; Osumi, Noriko

2016-01-01

Neurodevelopmental disorders such as autism spectrum disorder (ASD) and attention deficit and hyperactivity disorder (ADHD) have increased over the last few decades. These neurodevelopmental disorders are characterized by a complex etiology, which involves multiple genes and gene-environmental interactions. Various genes that control specific properties of neural development exert pivotal roles in the occurrence and severity of phenotypes associated with neurodevelopmental disorders. Moreover, paternal aging has been reported as one of the factors that contribute to the risk of ASD and ADHD. Here we report, for the first time, that paternal aging has profound effects on the onset of behavioral abnormalities in mice carrying a mutation of Pax6, a gene with neurodevelopmental regulatory functions. We adopted an in vitro fertilization approach to restrict the influence of additional factors. Comprehensive behavioral analyses were performed in Sey/+ mice (i.e., Pax6 mutant heterozygotes) born from in vitro fertilization of sperm taken from young or aged Sey/+ fathers. No body weight changes were found in the four groups, i.e., Sey/+ and wild type (WT) mice born to young or aged father. However, we found important differences in maternal separation-induced ultrasonic vocalizations of Sey/+ mice born from young father and in the level of hyperactivity of Sey/+ mice born from aged fathers in the open-field test, respectively, compared to WT littermates. Phenotypes of anxiety were observed in both genotypes born from aged fathers compared with those born from young fathers. No significant difference was found in social behavior and sensorimotor gating among the four groups. These results indicate that mice with a single genetic risk factor can develop different phenotypes depending on the paternal age. Our study advocates for serious considerations on the role of paternal aging in breeding strategies for animal studies.

The N domain of somatic angiotensin-converting enzyme negatively regulates ectodomain shedding and catalytic activity.

PubMed

Woodman, Zenda L; Schwager, Sylva L U; Redelinghuys, Pierre; Carmona, Adriana K; Ehlers, Mario R W; Sturrock, Edward D

2005-08-01

sACE (somatic angiotensin-converting enzyme) consists of two homologous, N and C domains, whereas the testis isoenzyme [tACE (testis ACE)] consists of a single C domain. Both isoenzymes are shed from the cell surface by a sheddase activity, although sACE is shed much less efficiently than tACE. We hypothesize that the N domain of sACE plays a regulatory role, by occluding a recognition motif on the C domain required for ectodomain shedding and by influencing the catalytic efficiency. To test this, we constructed two mutants: CNdom-ACE and CCdom-ACE. CNdom-ACE was shed less efficiently than sACE, whereas CCdom-ACE was shed as efficiently as tACE. Notably, cleavage occurred both within the stalk and the interdomain bridge in both mutants, suggesting that a sheddase recognition motif resides within the C domain and is capable of directly cleaving at both positions. Analysis of the catalytic properties of the mutants and comparison with sACE and tACE revealed that the k(cat) for sACE and CNdom-ACE was less than or equal to the sum of the kcat values for tACE and the N-domain, suggesting negative co-operativity, whereas the kcat value for the CCdom-ACE suggested positive co-operativity between the two domains. Taken together, the results provide support for (i) the existence of a sheddase recognition motif in the C domain and (ii) molecular flexibility of the N and C domains in sACE, resulting in occlusion of the C-domain recognition motif by the N domain as well as close contact of the two domains during hydrolysis of peptide substrates.

The N domain of somatic angiotensin-converting enzyme negatively regulates ectodomain shedding and catalytic activity

PubMed Central

Woodman, Zenda L.; Schwager, Sylva L. U.; Redelinghuys, Pierre; Carmona, Adriana K.; Ehlers, Mario R. W.; Sturrock, Edward D.

2005-01-01

sACE (somatic angiotensin-converting enzyme) consists of two homologous, N and C domains, whereas the testis isoenzyme [tACE (testis ACE)] consists of a single C domain. Both isoenzymes are shed from the cell surface by a sheddase activity, although sACE is shed much less efficiently than tACE. We hypothesize that the N domain of sACE plays a regulatory role, by occluding a recognition motif on the C domain required for ectodomain shedding and by influencing the catalytic efficiency. To test this, we constructed two mutants: CNdom-ACE and CCdom-ACE. CNdom-ACE was shed less efficiently than sACE, whereas CCdom-ACE was shed as efficiently as tACE. Notably, cleavage occurred both within the stalk and the interdomain bridge in both mutants, suggesting that a sheddase recognition motif resides within the C domain and is capable of directly cleaving at both positions. Analysis of the catalytic properties of the mutants and comparison with sACE and tACE revealed that the kcat for sACE and CNdom-ACE was less than or equal to the sum of the kcat values for tACE and the N-domain, suggesting negative co-operativity, whereas the kcat value for the CCdom-ACE suggested positive co-operativity between the two domains. Taken together, the results provide support for (i) the existence of a sheddase recognition motif in the C domain and (ii) molecular flexibility of the N and C domains in sACE, resulting in occlusion of the C-domain recognition motif by the N domain as well as close contact of the two domains during hydrolysis of peptide substrates. PMID:15813703

Allele-Specific Silencing of Mutant mRNA Rescues Ultrastructural and Arrhythmic Phenotype in Mice Carriers of the R4496C Mutation in the Ryanodine Receptor Gene (RYR2).

PubMed

Bongianino, Rossana; Denegri, Marco; Mazzanti, Andrea; Lodola, Francesco; Vollero, Alessandra; Boncompagni, Simona; Fasciano, Silvia; Rizzo, Giulia; Mangione, Damiano; Barbaro, Serena; Di Fonso, Alessia; Napolitano, Carlo; Auricchio, Alberto; Protasi, Feliciano; Priori, Silvia G

2017-08-18

Mutations in the cardiac Ryanodine Receptor gene ( RYR2 ) cause dominant catecholaminergic polymorphic ventricular tachycardia (CPVT), a leading cause of sudden death in apparently healthy individuals exposed to emotions or physical exercise. We investigated the efficacy of allele-specific silencing by RNA interference to prevent CPVT phenotypic manifestations in our dominant CPVT mice model carriers of the heterozygous mutation R4496C in RYR2 . We developed an in vitro mRNA and protein-based assays to screen multiple siRNAs for their ability to selectively silence mutant RYR2 -R4496C mRNA over the corresponding wild-type allele. For the most performant of these siRNAs (siRYR2-U10), we evaluated the efficacy of an adeno-associated serotype 9 viral vector (AAV9) expressing miRYR2-U10 in correcting RyR2 (Ryanodine Receptor type 2 protein) function after in vivo delivery by intraperitoneal injection in neonatal and adult RyR2 R4496C/+ (mice heterozygous for the R4496C mutation in the RyR2) heterozygous CPVT mice. Transcriptional analysis showed that after treatment with miRYR2-U10, the ratio between wild-type and mutant RYR2 mRNA was doubled (from 1:1 to 2:1) confirming the ability of miRYR2-U10 to selectively inhibit RYR2 -R4496C mRNA, whereas protein quantification showed that total RyR2 was reduced by 15% in the heart of treated mice. Furthermore, AAV9-miRYR2-U10 effectively (1) reduced isoproterenol-induced delayed afterdepolarizations and triggered activity in infected cells, (2) reduced adrenergically mediated ventricular tachycardia in treated mice, (3) reverted ultrastructural abnormalities of junctional sarcoplasmic reticulum and transverse tubules, and (4) attenuated mitochondrial abnormalities. The study demonstrates that allele-specific silencing with miRYR2-U10 prevents life-threatening arrhythmias in CPVT mice, suggesting that the reduction of mutant RyR2 may be a novel therapeutic approach for CPVT. © 2017 American Heart Association, Inc.

A novel p53 mutational hotspot in skin tumors from UV-irradiated Xpc mutant mice alters transactivation functions.

PubMed

Inga, Alberto; Nahari, Dorit; Velasco-Miguel, Susana; Friedberg, Errol C; Resnick, Michael A

2002-08-22

A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122-->L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc(-/-)) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122-->L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122-->L allele in p53(-/-) mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122-->L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122-->L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122-->L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122-->L mutant allele in the Xpc(-/-) background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.

Reduced alcohol consumption in mice lacking preprodynorphin.

PubMed Central

Blednov, Yuri A.; Walker, Danielle; Martinez, Marni; Harris., R. Adron

2007-01-01

Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the κ-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice of both sexes showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of limited (4 hours) access (light phase of the light/dark cycle), null mutant mice did not show any differences in consumption of saccharin but they showed significantly reduced intake of sucrose. To determine the possible cause for reduction of ethanol preference and intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild type mice in ethanol-induced loss of righting reflex, acute ethanol withdrawal, ethanol-induced conditioned place preference or conditioned taste aversion to ethanol. These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake in female mice, and suggest thath this is caused by decreased orosensory reward of alcohol (sweet taste and/or palatability). PMID:17307643

Reduced alcohol consumption in mice lacking preprodynorphin.

PubMed

Blednov, Yuri A; Walker, Danielle; Martinez, Marni; Harris, R Adron

2006-10-01

Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the kappa-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice of both sexes showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of limited (4 h) access (light phase of the light/dark cycle), null mutant mice did not show any differences in consumption of saccharin, but they showed significantly reduced intake of sucrose. To determine the possible cause for reduction of ethanol preference and intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild-type mice in ethanol-induced loss of righting reflex, acute ethanol withdrawal, ethanol-induced conditioned place preference, or conditioned taste aversion to ethanol. These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake in female mice, and suggest that this is caused by decreased orosensory reward of alcohol (sweet taste and/or palatability).

Lipofuscin accumulation, abnormal electrophysiology, and photoreceptor degeneration in mutant ELOVL4 transgenic mice: a model for macular degeneration.

PubMed

Karan, G; Lillo, C; Yang, Z; Cameron, D J; Locke, K G; Zhao, Y; Thirumalaichary, S; Li, C; Birch, D G; Vollmer-Snarr, H R; Williams, D S; Zhang, K

2005-03-15

Macular degeneration is a heterogeneous group of disorders characterized by photoreceptor degeneration and atrophy of the retinal pigment epithelium (RPE) in the central retina. An autosomal dominant form of Stargardt macular degeneration (STGD) is caused by mutations in ELOVL4, which is predicted to encode an enzyme involved in the elongation of long-chain fatty acids. We generated transgenic mice expressing a mutant form of human ELOVL4 that causes STGD. In these mice, we show that accumulation by the RPE of undigested phagosomes and lipofuscin, including the fluorophore, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hyydroxyethyl)-4-[4-methyl-6-(2,6,6,-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2E) is followed by RPE atrophy. Subsequently, photoreceptor degeneration occurs in the central retina in a pattern closely resembling that of human STGD and age-related macular degeneration. The ELOVL4 transgenic mice thus provide a good model for both STGD and dry age-related macular degeneration, and represent a valuable tool for studies on therapeutic intervention in these forms of blindness.

The clock gene Period1 regulates innate routine behaviour in mice

PubMed Central

Bechstein, Philipp; Rehbach, Nils-Jörn; Yuhasingham, Gowzekan; Schürmann, Christoph; Göpfert, Melanie; Kössl, Manfred; Maronde, Erik

2014-01-01

Laboratory mice are well capable of performing innate routine behaviour programmes necessary for courtship, nest-building and exploratory activities although housed for decades in animal facilities. We found that in mice inactivation of the clock gene Period1 profoundly changes innate routine behaviour programmes like those necessary for courtship, nest building, exploration and learning. These results in wild-type and Period1 mutant mice, together with earlier findings on courtship behaviour in wild-type and period-mutant Drosophila melanogaster, suggest a conserved role of Period-genes on innate routine behaviour. Additionally, both per-mutant flies and Period1-mutant mice display spatial learning and memory deficits. The profound influence of Period1 on routine behaviour programmes in mice, including female partner choice, may be independent of its function as a circadian clock gene, since Period1-deficient mice display normal circadian behaviour. PMID:24598427

The temperature-sensitive mutants of Toxoplasma gondii and ocular toxoplasmosis.

PubMed

Lu, Fangli; Huang, Shiguang; Kasper, Lloyd H

2009-01-22

The risk of blindness caused by ocular toxoplasmosis supports efforts to improve our understanding for control of this disease. In this study, the involvement of CD8(+), CD4(+), B cell, and IL-10 gene in the immune response of primary ocular infection with the temperature-sensitive mutant (ts-4) of the RH Toxoplasma gondii strain, and in the protective immunity of ocular ts-4 vaccination and challenge with RH strain was investigated in murine models utilizing inbred C57BL/6 mice-deficient in CD4(+), CD8(+), B cells (microMT), or IL-10 gene. Compared to naive mice, all WT and mutant mice had different degree of ocular pathological changes after ts-4 ocular infection, in which both CD8 KO and IL-10 KO mice showed the most severe ocular lesions. Immunized by ts-4 intracameral (i.c.) inoculation, all mutant mice had partially decreased vaccine-induced resistance associated with increased ocular parasite burdens after RH strain challenge. A significant increase of the percentages of B cells and CD8(+) T cells in the draining lymph nodes were observed in WT and IL-10 KO mice after either infection or challenge. The levels of specific anti-toxoplasma IgG in both eye fluid and serum from all the mice were significantly increased after ts-4 i.c. immunization, except microMT mice. These results suggest that the avirulent ts-4 of T. gondii inoculated intracamerally can induce both ocular pathology and ocular protective immunity; CD4(+), CD8(+), B cell, and IL-10 gene are all necessary to the vaccine-induced resistance to ocular challenge by virulent RH strain, in which CD8(+) T cells are the most important component.

Rational Design of a Live Attenuated Dengue Vaccine: 2′-O-Methyltransferase Mutants Are Highly Attenuated and Immunogenic in Mice and Macaques

PubMed Central

Chang, David C.; Zhang, Bo; Balakrishnan, Thavamalar; Toh, Ying-Xiu; Jiang, Tao; Li, Shi-Hua; Deng, Yong-Qiang; Ellis, Brett R.; Ellis, Esther M.; Poidinger, Michael; Zolezzi, Francesca; Qin, Cheng-Feng; Shi, Pei-Yong; Fink, Katja

2013-01-01

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2′-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2′-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2′-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response. PMID:23935499

Proteostasis and ageing: insights from long-lived mutant mice.

PubMed

Sands, William A; Page, Melissa M; Selman, Colin

2017-10-15

The global increase in life expectancy is creating significant medical, social and economic challenges to current and future generations. Consequently, there is a need to identify the fundamental mechanisms underlying the ageing process. This knowledge should help develop realistic interventions capable of combatting age-related disease, and thus improving late-life health and vitality. While several mechanisms have been proposed as conserved lifespan determinants, the loss of proteostasis - where proteostasis is defined here as the maintenance of the proteome - appears highly relevant to both ageing and disease. Several studies have shown that multiple proteostatic mechanisms, including the endoplasmic reticulum (ER)-induced unfolded protein response (UPR), the ubiquitin-proteasome system (UPS) and autophagy, appear indispensable for longevity in many long-lived invertebrate mutants. Similarly, interspecific comparisons suggest that proteostasis may be an important lifespan determinant in vertebrates. Over the last 20 years a number of long-lived mouse mutants have been described, many of which carry single-gene mutations within the growth-hormone, insulin/IGF-1 or mTOR signalling pathways. However, we still do not know how these mutations act mechanistically to increase lifespan and healthspan, and accordingly whether mechanistic commonality occurs between different mutants. Recent evidence supports the premise that the successful maintenance of the proteome during ageing may be linked to the increased lifespan and healthspan of long-lived mouse mutants. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Inhibitory effect on natural killer activity of microphthalmia transcription factor encoded by the mutant mi allele of mice.

PubMed

Ito, A; Kataoka, T R; Kim, D K; Koma , Y; Lee, Y M; Kitamura, Y

2001-04-01

The mouse mi locus encodes a basic-helix-loop-helix-leucine zipper-type transcription factor, microphthalmia transcription factor (MITF). Mice of mi/mi genotype express a mutant form of MITF (mi-MITF), whereas mice of tg/tg genotype have a transgene in the 5' flanking region of the mi gene and do not express MITF. Although the mi/mi mouse is deficient in natural killer (NK) activity, it was found that the tg/tg mouse was normal in this respect. To know the cause, spleen cells of both genotypes were compared. Although the proportion of spleen cells expressing an NK cell marker, NK1.1, was comparable in both mice, the proportion of large granular lymphocytes decreased only in mi/mi mice. The difference between mi/mi and tg/tg mice was reproducible in the culture supplemented with interleukin-2. Moreover, the perforin gene expression was reduced in mi/mi-cultured spleen cells. Wild-type (+) MITF transactivated, but mi-MITF suppressed, the perforin gene promoter through the NF-P motif, a strong cis-acting element. However, neither +-MITF nor mi-MITF bound the NF-P motif. Instead, 2 nuclear factors that bound the NF-P motif were retained in the cytoplasm of mi/mi-cultured spleen cells. In addition, overexpression of mi-MITF resulted in cytoplasmic retention of the 2 NF-P motif-binding factors in cytotoxic T lymphocytes. The presence of mi-MITF rather than the absence of +-MITF appeared to lead to poor transactivation of the NF-P motif by intercepting NF-P motif-binding factors. This inhibitory effect of mi-MITF may cause the deficient cytotoxicity of NK cells in mi/mi mice. (Blood. 2001;97:2075-2083)

Alternating Hemiplegia of Childhood-Related Neural and Behavioural Phenotypes in Na+,K+-ATPase α3 Missense Mutant Mice

PubMed Central

Kirshenbaum, Greer S.; Dawson, Neil; Mullins, Jonathan G. L.; Johnston, Tom H.; Drinkhill, Mark J.; Edwards, Ian J.; Fox, Susan H.; Pratt, Judith A.; Brotchie, Jonathan M.; Roder, John C.; Clapcote, Steven J.

2013-01-01

Missense mutations in ATP1A3 encoding Na+,K+-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC), a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na+,K+-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N) that affects the same position in Na+,K+-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na+,K+-ATPase α3, including upon the K+ pore and predicted K+ binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality), directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na+,K+-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC. PMID:23527305

Suppressive Effects of Pelargonidin on Endothelial Protein C Receptor Shedding via the Inhibition of TACE Activity and MAP Kinases.

PubMed

Kang, Hyejin; Lee, Taeho; Bae, Jong-Sup

2016-01-01

Beyond its role in the activation of protein C, the endothelial cell protein C receptor (EPCR) plays an important role in the cytoprotective pathway. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-[Formula: see text] converting enzyme (TACE). Pelargonidin is a well-known red pigment found in plants, and has been reported to have important biological activities that are potentially beneficial to human health. However, little is known about the effects of pelargonidin on EPCR shedding. We investigated this issue by monitoring the effects of pelargonidin on phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-[Formula: see text]-, interleukin (IL)-1β-, and cecal ligation and puncture (CLP)-mediated EPCR shedding and by investigating the underlying mechanism of pelargonidin action. Data demonstrate that pelargonidin induced potent inhibition of PMA-, TNF-[Formula: see text]-, IL-1β-, and CLP-induced EPCR shedding by inhibiting the phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38, janus kinase (JNK), and extracellular signal-regulated kinase (ERK) 1/2. Pelargonidin also inhibited the expression and activity of PMA-induced TACE in endothelial cells. These results demonstrate the potential of pelargonidin as an anti-EPCR shedding reagent against PMA- and CLP-mediated EPCR shedding.

Discovery and process development of a novel TACE inhibitor for the topical treatment of psoriasis.

PubMed

Boiteau, Jean-Guy; Ouvry, Gilles; Arlabosse, Jean-Marie; Astri, Stéphanie; Beillard, Audrey; Bhurruth-Alcor, Yushma; Bonnary, Laetitia; Bouix-Peter, Claire; Bouquet, Karine; Bourotte, Marilyne; Cardinaud, Isabelle; Comino, Catherine; Deprez, Benoît; Duvert, Denis; Féret, Angélique; Hacini-Rachinel, Feriel; Harris, Craig S; Luzy, Anne-Pascale; Mathieu, Arnaud; Millois, Corinne; Orsini, Nicolas; Pascau, Jonathan; Pinto, Artur; Piwnica, David; Polge, Gaëlle; Reitz, Arnaud; Reversé, Kevin; Rodeville, Nicolas; Rossio, Patricia; Spiesse, Delphine; Tabet, Samuel; Taquet, Nathalie; Tomas, Loïc; Vial, Emmanuel; Hennequin, Laurent F

2018-02-15

Targeting the TNFα pathway is a validated approach to the treatment of psoriasis. In this pathway, TACE stands out as a druggable target and has been the focus of in-house research programs. In this article, we present the discovery of clinical candidate 26a. Starting from hits plagued with poor solubility or genotoxicity, 26a was identified through thorough multiparameter optimisation. Showing robust in vivo activity in an oxazolone-mediated inflammation model, the compound was selected for development. Following a polymorph screen, the hydrochloride salt was selected and the synthesis was efficiently developed to yield the API in 47% overall yield. Copyright © 2017. Published by Elsevier Ltd.

Paranodal permeability in `myelin mutants'

PubMed Central

Shroff, S.; Mierzwa, A.; Scherer, S.S.; Peles, E.; Arevalo, J.C.; Chao, M.V.; Rosenbluth, J.

2011-01-01

Fluorescent dextran tracers of varying sizes have been used to assess paranodal permeability in myelinated sciatic nerve fibers from control and three `myelin mutant' mice, Caspr-null, cst-null and shaking. We demonstrate that in all of these the paranode is permeable to small tracers (3kDa, 10kDa), which penetrate most fibers, and to larger tracers (40kDa, 70kDa), which penetrate far fewer fibers and move shorter distances over longer periods of time. Despite gross diminution in transverse bands in the Caspr-null and cst-null mice, the permeability of their paranodal junctions is equivalent to that in controls. Thus, deficiency of transverse bands in these mutants does not increase the permeability of their paranodal junctions to the dextrans we used, moving from the perinodal space through the paranode to the internodal periaxonal space. In addition, we show that the shaking mice, which have thinner myelin and shorter paranodes, show increased permeability to the same tracers despite the presence of transverse bands. We conclude that the extent of penetration of these tracers does not depend on the presence or absence of transverse bands but does depend on the length of the paranode and, in turn, on the length of `pathway 3', the helical extracellular pathway that passes through the paranode parallel to the lateral edge of the myelin sheath. PMID:21618613

HoxB2 binds mutant SOD1 and is altered in transgenic model of ALS.

PubMed

Zhai, Jinbin; Lin, Hong; Canete-Soler, Rafaela; Schlaepfer, William W

2005-09-15

Mutations in Cu/Zn superoxide dismutase (SOD1) cause approximately 20% of familial amyotrophic lateral sclerosis by a toxic gain of function; however, the precise mechanisms remain unclear. Here, we report the identification of HoxB2, a homeodomain-containing transcription factor, as a G93A mutant SOD1 interactive protein in a yeast two-hybrid screen. We show that HoxB2 co-precipitates and co-localizes with mutant SOD1 in neuronal cell lines, as well as in brain and spinal cord of G93A mutant SOD1 transgenic mice. Mutagenesis further shows that this interaction is mediated by the central homeodomain of HoxB2. In motor neuron-like NSC-34 cells, overexpression of HoxB2 or its homeodomain decreases the insolubility of mutant SOD1 and inhibits G93A or G86R mutant SOD1-induced neuronal cell death. In human and mouse tissues, we show that expression of HoxB2 persists in adult spinal cord and is primarily localized in nuclei of motor neurons. In G93A transgenic mice, HoxB2 co-localizes with mutant SOD1 and is redistributed to perikarya and proximal neurites of motor neurons. In addition, there is progressive accumulation of HoxB2 and mutant SOD1 as punctate inclusions in the neuropil surrounding motor neurons. Taken together, our findings demonstrate that interaction of HoxB2 with mutant SOD1 occurs in motor neurons of G93A mutant SOD1 transgenic mice and suggest that this interaction may modulate the neurotoxicity of mutant SOD1.

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

PubMed Central

Jiménez de Bagüés, M P; Elzer, P H; Jones, S M; Blasco, J M; Enright, F M; Schurig, G G; Winter, A J

1994-01-01

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis. Images PMID:7927779

Laser Interferometer Measurements of the Viscoelastic Properties of Tectorial Membrane Mutants

NASA Astrophysics Data System (ADS)

Jones, Gareth; Russell, Ian; Lukashkin, Andrei

2011-11-01

The visco-elastic properties of the tectorial membrane (TM) can be determined by measuring the propagation velocity of travelling waves over a range of frequencies. This study presents a new method using laser interferometry and compares the TM's material properties (sheer storage modulus, G' and viscosity, η) at basal and apical locations in wild-type mice and basal locations of three mutant groups (TectaY1870C/+, Tectb-/- and Otoa-/-). The G' and η values calculated for the wild-type mice are similar to estimates derived using other methods whereas the mutant groups all exhibit slower wave propagation velocities and reduced longitudinal coupling.

Induction of Unconventional T Cells by a Mutant Mycobacterium bovis BCG Strain Formulated in Cationic Liposomes Correlates with Protection against Mycobacterium tuberculosis Infections of Immunocompromised Mice

PubMed Central

Yabe, Idalia; Morris, Sheldon; Cowley, Siobhan

2016-01-01

Earlier studies aimed at defining protective immunity induced by Mycobacterium bovis BCG immunization have largely focused on the induction of antituberculosis CD4+ and CD8+ T cell responses. Here we describe a vaccine consisting of a BCGΔmmaA4 deletion mutant formulated in dimethyl dioctadecyl-ammonium bromide (DDA) with d-(+)-trehalose 6,6′-dibehenate (TDB) (DDA/TDB) adjuvant (A4/Adj) that protected TCRδ−/− mice depleted of CD4+, CD8+, and NK1.1+ T cells against an aerosol challenge with M. tuberculosis. These mice were significantly protected relative to mice immunized with a nonadjuvanted BCGΔmmaA4 (BCG-A4) mutant and nonvaccinated controls at 2 months and 9 months postvaccination. In the absence of all T cells following treatment with anti-Thy1.2 antibody, the immunized mice lost the ability to control the infection. These results indicate that an unconventional T cell population was mediating protection in the absence of CD4+, CD8+, NK1.1+, and TCRγδ T cells and could exhibit memory. Focusing on CD4− CD8− double-negative (DN) T cells, we found that these cells accumulated in the lungs postchallenge significantly more in A4/Adj-immunized mice and induced significantly greater frequencies of pulmonary gamma interferon (IFN-γ)-producing cells than were seen in the nonvaccinated or nonadjuvanted BCG control groups. Moreover, pulmonary DN T cells from the A4/Adj group exhibited significantly higher IFN-γ integrated median fluorescence intensity (iMFI) values than were seen in the control groups. We also showed that enriched DN T cells from mice immunized with A4/Adj could control mycobacterial growth in vitro significantly better than naive whole-spleen cells. These results suggest that formulating BCG in DDA/TDB adjuvant confers superior protection in immunocompromised mice and likely involves the induction of long-lived memory DN T cells. PMID:27226281

Sleep apneas are increased in mice lacking monoamine oxidase A.

PubMed

Real, Caroline; Popa, Daniela; Seif, Isabelle; Callebert, Jacques; Launay, Jean-Marie; Adrien, Joëlle; Escourrou, Pierre

2007-10-01

Alterations in the serotonin (5-HT) system have been suggested as a mechanism of sleep apnea in humans and rodents. The objective is to evaluate the contribution of 5-HT to this disorder. We studied sleep and breathing (whole-body plethysmography) in mutant mice that lack monoamine oxidase A (MAOA) and have increased concentrations of monoamines, including 5-HT. Compared to wild-type mice, the mutants showed similar amounts of slow wave sleep (SWS) and rapid eye movement sleep (REMS), but exhibited a 3-fold increase in SWS and REMS apnea indices. Acute administration of the MAOA inhibitor clorgyline decreased REMS amounts and increased the apnea index in wild-type but not mutant mice. Parachlorophenylalanine, a 5-HT synthesis inhibitor, reduced whole brain concentrations of 5-HT in both strains, and induced a decrease in apnea index in mutant but not wild-type mice. Our results show that MAOA deficiency is associated with increased sleep apnea in mice and suggest that an acute or chronic excess of 5-HT contributes to this phenotype.

BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

PubMed

Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

2010-12-01

During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

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

Cook, Melloni N.; Dunning, Jonathan P; Wiley, Ronald G

2007-01-01

We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsivenessmore » to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.« less

EPHRIN-A5 REGULATES INTER-MALE AGGRESSION IN MICE

PubMed Central

Sheleg, Michal; Yochum, Carrie L.; Richardson, Jason R.; Wagner, George C.; Zhou, Renping

2015-01-01

The Eph family of receptor tyrosine kinases play key roles in both the patterning of the developing nervous system and neural plasticity in the mature brain. To determine functions of ephrin-A5, a GPI-linked ligand to the Eph receptors, in animal behavior regulations, we examined effects of its inactivation on male mouse aggression. When tested in the resident-intruder paradigm for offensive aggression, ephrin-A5-mutant animals (ephrin-A5−/−) exhibited severe reduction in conspecific aggression compared to wild-type controls. On the contrary, defensive aggression in the form of target biting was higher in ephrin-A5−/− mice, indicating that the mutant mice are capable of attacking behavior. In addition, given the critical role of olfaction in aggressive behavior, we examined the ability of the ephrin-A5−/− mice to smell and found no differences between the mutant and control animals. Testosterone levels in the mutant mice were also found to be within the normal range. Taken together, our data reveal a new role of ephrin-A5 in the regulation of aggressive behavior in mice. PMID:25746458

Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities

PubMed Central

Kerr, Emma; Gaude, Edoardo; Turrell, Frances; Frezza, Christian; Martins, Carla P

2016-01-01

Summary The RAS/MAPK-signalling pathway is frequently deregulated in non-small cell lung cancer (NSCLC), often through KRAS activating mutations1-3. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations4-7. We recently showed that advanced lung tumours from KrasG12D/+;p53-null mice frequently exhibit KrasG12D allelic enrichment (KrasG12D/Kraswild-type>1)7, implying that mutant Kras copy gains are positively selected during progression. Through a comprehensive analysis of mutant Kras homozygous and heterozygous MEFs and lung cancer cells we now show that these genotypes are phenotypically distinct. In particular, KrasG12D/G12D cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the TCA cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous NSCLC cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of KrasG12D copy gain), but not in the corresponding early tumours (KrasG12D heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprised of two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated based on their relative mutant allelic content. We also provide the first in vivo evidence of metabolic rewiring during lung cancer malignant progression. PMID:26909577

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 Ikk

Dental and Cranial Pathologies in Mice Lacking the Cl−/H+-Exchanger ClC-7

PubMed Central

WEN, Xin; LACRUZ, Rodrigo S.; PAINE, Michael L.

2015-01-01

ClC-7 is a 2Cl−/1H+-exchanger expressed at late endosomes and lysosomes, as well as the ruffled border of osteoclasts. ClC-7 deficiencies in mice and humans lead to impaired osteoclast function and therefore osteopetrosis. Failure of tooth eruption is also apparent in ClC-7 mutant animals, and this has been attributed to the osteoclast dysfunction and the subsequent defect in alveolar bone resorptive activity surrounding tooth roots. Ameloblasts also express ClC-7, and this study aims to determine the significance of ClC-7 in enamel formation by examining the dentitions of ClC-7 mutant mice. Micro-CT analysis revealed that the molar teeth of 3-week old ClC-7 mutant mice had no roots, and the incisors were smaller than their age-matched controls. Despite these notable developmental differences, the enamel and dentin densities of the mutant mice were comparable to those of the wild type littermates. Scanning electron microscopy (SEM) showed normal enamel crystallite and prismatic organization in the ClC-7 mutant mice, although the enamel was thinner (hypoplastic) than in controls. These results suggested that ClC-7 was not critical to enamel and dentin formation, and the observed tooth defects may be related more to a resulting alveolar bone phenotype. Micro-CT analysis also revealed abnormal features in the calvarial bones of the mutant mice. The cranial sutures in ClC-7 mutant mice remained open compared to the closed sutures seen in the control mice at 3 weeks. These data demonstrate that ClC-7 deficiency impacts the development of the dentition and calvaria, but does not significantly disrupt amelogenesis. PMID:25663454

Full Length Human Mutant Huntingtin with a Stable Polyglutamine Repeat Can Elicit Progressive and Selective Neuropathogenesis in BACHD Mice

PubMed Central

Gray, Michelle; Shirasaki, Dyna I.; Cepeda, Carlos; Andre, Veronique M.; Wilburn, Brian; Lu, Xiao-Hong; Tao, Jifang; Yamazaki, Irene; Li, Shi-Hua; Sun, Yi E.; Li, Xiao-Jiang; Levine, Michael S.; William Yang, X

2008-01-01

To elucidate the pathogenic mechanisms in Huntington’s disease (HD) elicited by expression of full-length human mutant huntingtin (fl-mhtt), a Bacterial Artificial Chromosome (BAC)-mediated transgenic mouse model (BACHD) was developed expressing fl-mhtt with 97 glutamine repeats under the control of endogenous htt regulatory machinery on the BAC. BACHD mice exhibit progressive motor deficits, neuronal synaptic dysfunction, and late-onset selective neuropathology, which includes significant cortical and striatal atrophy and striatal dark neuron degeneration. Power analyses reveal the robustness of the behavioral and neuropathological phenotypes, suggesting BACHD as a suitable fl-mhtt mouse model for preclinical studies. Further analyses of BACHD mice provide additional insights into how mhtt may elicit neuropathogenesis. First, unlike prior fl-mhtt mouse models, BACHD mice reveal that the slowly progressive and selective pathogenic process in HD mouse brains can occur without early and diffuse nuclear accumulation of aggregated mhtt (i.e. as detected by immunostaining with the EM48 antibody). Instead, a relatively steady-state level of predominantly full-length mhtt and a small amount of mhtt N-terminal fragments are sufficient to elicit the disease process. Second, the polyglutamine repeat within fl-mhtt in BACHD mice is encoded by a mixed CAA-CAG repeat, which is stable in both the germline and somatic tissues including the cortex and striatum at the onset of neuropathology. Therefore, our results suggest that somatic repeat instability does not play a necessary role in selective neuropathogenesis in BACHD mice. In summary, the BACHD model constitutes a novel and robust in vivo paradigm for the investigation of HD pathogenesis and treatment. PMID:18550760

Altered Body Weight Regulation in CK1ε Null and tau Mutant Mice on Regular Chow and High Fat Diets

PubMed Central

Zhou, Lili; Summa, Keith C.; Olker, Christopher; Vitaterna, Martha H.; Turek, Fred W.

2016-01-01

Disruption of circadian rhythms results in metabolic dysfunction. Casein kinase 1 epsilon (CK1ε) is a canonical circadian clock gene. Null and tau mutations in CK1ε show distinct effects on circadian period. To investigate the role of CK1ε in body weight regulation under both regular chow (RC) and high fat (HF) diet conditions, we examined body weight on both RC and HF diets in CK1ε −/− and CK1ε tau/tau mice on a standard 24 hr light-dark (LD) cycle. Given the abnormal entrainment of CK1ε tau/tau mice on a 24 hr LD cycle, a separate set of CK1ε tau/tau mice were tested under both diet conditions on a 20 hr LD cycle, which more closely matches their endogenous period length. On the RC diet, both CK1ε −/− and CK1ε tau/tau mutants on a 24 hr LD cycle and CK1ε tau/tau mice on a 20 hr LD cycle exhibited significantly lower body weights, despite similar overall food intake and activity levels. On the HF diet, CK1ε tau/tau mice on a 20 hr LD cycle were protected against the development of HF diet-induced excess weight gain. These results provide additional evidence supporting a link between circadian rhythms and energy regulation at the genetic level, particularly highlighting CK1ε involved in the integration of circadian biology and metabolic physiology. PMID:27144030

Ross River virus mutant with a deletion in the E2 gene: properties of the virion, virus-specific macromolecule synthesis, and attenuation of virulence for mice.

PubMed

Vrati, S; Faragher, S G; Weir, R C; Dalgarno, L

1986-06-01

A mutant of RRV T48 the prototype strain of Ross River virus has been isolated with a 21-nucleotide deletion in the gene coding for the envelope glycoprotein E2. Direct sequencing of the 26 S subgenomic RNA, together with HaeIII and TaqI restriction digest analysis of cDNA to RNAs from cells infected with the mutant virus (RRV dE2) and with RRV T48, were consistent with the deletion being the only major alteration in the mutant genome. The E2 protein of RRV dE2 virions had a higher electrophoretic mobility than that of RRV T48 E2 protein. Neither RRV dE2 nor RRV T48 virions contained more than trace amounts of E3, the small envelope glycoprotein found in Semliki Forest virus. RRV dE2 generated small plaques on Vero cell monolayers; plaque formation was not temperature-sensitive between 32 and 41 degrees. By comparison with RRV T48 the infectivity of RRV dE2 virions was thermolabile at 50 degrees. In BHK cells RRV dE2 grew with similar kinetics to RRV T48. Rates of synthesis of 26 S RNA and 49 S RNA were higher in cells infected with RRV dE2 than in cells infected with RRV T48. Virus-specific protein synthesis and shut-down of host protein synthesis occurred 2-3 hr earlier in RRV dE2-infected cells than in cells infected with RRV T48. Minor differences between the two viruses were observed in the profiles of virus-specific proteins generated in infected cells. In day-old mice RRV dE2 induced less severe symptoms of hind leg paralysis than did RRV T48. A small increase in LD50 and average survival time was observed in RRV dE2-infected mice by comparison with RRV T48 infected mice. Peak titers reached by RRV dE2 in the hind leg muscle, brain, and blood of day-old mice were 3-4 log units less than the titers reached during infection with RRV T48. In week-old mice the differences in virulence between the two strains were magnified: RRV dE2 induced no detectable symptoms even when injected at high doses (8 X 10(6) PFU) whereas the LD50 and average survival time for RRV T

Inhibition of Shp2 suppresses mutant EGFR-induced lung tumors in transgenic mouse model of lung adenocarcinoma

PubMed Central

Schneeberger, Valentina E.; Ren, Yuan; Luetteke, Noreen; Huang, Qingling; Chen, Liwei; Lawrence, Harshani R.; Lawrence, Nicholas J.; Haura, Eric B.; Koomen, John M.; Coppola, Domenico; Wu, Jie

2015-01-01

Epidermal growth factor receptor (EGFR) mutants drive lung tumorigenesis and are targeted for therapy. However, resistance to EGFR inhibitors has been observed, in which the mutant EGFR remains active. Thus, it is important to uncover mediators of EGFR mutant-driven lung tumors to develop new treatment strategies. The protein tyrosine phosphatase (PTP) Shp2 mediates EGF signaling. Nevertheless, it is unclear if Shp2 is activated by oncogenic EGFR mutants in lung carcinoma or if inhibiting the Shp2 PTP activity can suppress EGFR mutant-induced lung adenocarcinoma. Here, we generated transgenic mice containing a doxycycline (Dox)-inducible PTP-defective Shp2 mutant (tetO-Shp2CSDA). Using the rat Clara cell secretory protein (CCSP)-rtTA-directed transgene expression in the type II lung pneumocytes of transgenic mice, we found that the Gab1-Shp2 pathway was activated by EGFRL858R in the lungs of transgenic mice. Consistently, the Gab1-Shp2 pathway was activated in human lung adenocarcinoma cells containing mutant EGFR. Importantly, Shp2CSDA inhibited EGFRL858R-induced lung adenocarcinoma in transgenic animals. Analysis of lung tissues showed that Shp2CSDA suppressed Gab1 tyrosine phosphorylation and Gab1-Shp2 association, suggesting that Shp2 modulates a positive feedback loop to regulate its own activity. These results show that inhibition of the Shp2 PTP activity impairs mutant EGFR signaling and suppresses EGFRL858R-driven lung adenocarcinoma. PMID:25730908

Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes

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

Wang, Bo; Hikosaka, Keisuke; Sultana, Nishat

2012-01-06

Highlights: Black-Right-Pointing-Pointer Fifty percent of the mutant Rb transgenic mice produced liver tumors. Black-Right-Pointing-Pointer In the tumor, Foxm1, Skp2, Bmi1 and AP-1 mRNAs were up-regulated. Black-Right-Pointing-Pointer No increase in expression of the Myc-target genes was observed in the non-tumorous liver. Black-Right-Pointing-Pointer Tumor formation depends on up-regulation of the Myc-target genes. -- Abstract: The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, wemore » generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with {approx}50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.« less

In vivo cell-autonomous transcriptional abnormalities revealed in mice expressing mutant huntingtin in striatal but not cortical neurons.

PubMed

Thomas, Elizabeth A; Coppola, Giovanni; Tang, Bin; Kuhn, Alexandre; Kim, SoongHo; Geschwind, Daniel H; Brown, Timothy B; Luthi-Carter, Ruth; Ehrlich, Michelle E

2011-03-15

Huntington's disease (HD), caused by a CAG repeat expansion in the huntingtin (HTT) gene, is characterized by abnormal protein aggregates and motor and cognitive dysfunction. Htt protein is ubiquitously expressed, but the striatal medium spiny neuron (MSN) is most susceptible to dysfunction and death. Abnormal gene expression represents a core pathogenic feature of HD, but the relative roles of cell-autonomous and non-cell-autonomous effects on transcription remain unclear. To determine the extent of cell-autonomous dysregulation in the striatum in vivo, we examined genome-wide RNA expression in symptomatic D9-N171-98Q (a.k.a. DE5) transgenic mice in which the forebrain expression of the first 171 amino acids of human Htt with a 98Q repeat expansion is limited to MSNs. Microarray data generated from these mice were compared with those generated on the identical array platform from a pan-neuronal HD mouse model, R6/2, carrying two different CAG repeat lengths, and a relatively high degree of overlap of changes in gene expression was revealed. We further focused on known canonical pathways associated with excitotoxicity, oxidative stress, mitochondrial dysfunction, dopamine signaling and trophic support. While genes related to excitotoxicity, dopamine signaling and trophic support were altered in both DE5 and R6/2 mice, which may be either cell autonomous or non-cell autonomous, genes related to mitochondrial dysfunction, oxidative stress and the peroxisome proliferator-activated receptor are primarily affected in DE5 transgenic mice, indicating cell-autonomous mechanisms. Overall, HD-induced dysregulation of the striatal transcriptome can be largely attributed to intrinsic effects of mutant Htt, in the absence of expression in cortical neurons.

Bidirectional modulation of fear extinction by mediodorsal thalamic firing in mice.

PubMed

Lee, Sukchan; Ahmed, Touqeer; Lee, Soojung; Kim, Huisu; Choi, Sukwoo; Kim, Duk-Soo; Kim, Sang Jeong; Cho, Jeiwon; Shin, Hee-Sup

2011-12-25

The mediodorsal thalamic nucleus has been implicated in the control of memory processes. However, the underlying neural mechanism remains unclear. Here we provide evidence for bidirectional modulation of fear extinction by the mediodorsal thalamic nucleus. Mice with a knockout or mediodorsal thalamic nucleus-specific knockdown of phospholipase C β4 exhibited impaired fear extinction. Mutant mediodorsal thalamic nucleus neurons in slices showed enhanced burst firing accompanied by increased T-type Ca(2+) currents; blocking of T channels in vivo rescued the fear extinction. Tetrode recordings in freely moving mice revealed that, during extinction, the single-spike (tonic) frequency of mediodorsal thalamic nucleus neurons increased in wild-type mice, but was static in mutant mice. Furthermore, tonic-evoking microstimulations of the mediodorsal thalamic nucleus, contemporaneous with the extinction tones, rescued fear extinction in mutant mice and facilitated it in wild-type mice. In contrast, burst-evoking microstimulation suppressed extinction in wild-type mice, mimicking the mutation. These results suggest that the firing mode of the mediodorsal thalamic nucleus is critical for the modulation of fear extinction.

Plasmodium yoelii: induction of attenuated mutants by irradiation

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

Waki, S.; Yonome, I.; Suzuki, M.

When erythrocytic forms of Plasmodium yoelii nigeriensis, which is invariably fatal in mice, were exposed to X rays, the dose to reduce surviving parasites to one millionth was 100 gray (10 Krad). A suspension of 5 X 10(6) per ml of parasitized erythrocyte was irradiated at 100 gray, and 0.2 ml aliquots were inoculated into 22 mice. Eleven mice showed patent parasitemia, and in these the growth curves were less steep than that found in nonirradiated parasites. The infections of 8 mice of the 11 were self-resolving, and the attenuated feature of the parasites maintained following a limited number ofmore » blood passages. The parasites were slowly growing even in nude mice and cause self-resolving infections in intact mice. BALB/c mice immunized with the attenuated parasites were protected against subsequent challenge infections with the original virulent erythrocytic and sporogonic forms. These findings indicate that attenuated mutants of malaria parasites can be readily induced by this method.« less

Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice.

PubMed

Varela, Mariana; Pinto, Rute Maria; Caporale, Marco; Piras, Ilaria M; Taggart, Aislynn; Seehusen, Frauke; Hahn, Kerstin; Janowicz, Anna; de Souza, William Marciel; Baumgärtner, Wolfgang; Shi, Xiaohong; Palmarini, Massimo

2016-06-01

Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc

Immunization with a novel Clostridium perfringens epsilon toxin mutant rETX(Y196E)-C confers strong protection in mice.

PubMed

Yao, Wenwu; Kang, Jingjing; Kang, Lin; Gao, Shan; Yang, Hao; Ji, Bin; Li, Ping; Liu, Jing; Xin, Wenwen; Wang, Jinglin

2016-04-06

Epsilon toxin (ETX) is produced by toxinotypes B and D of Clostridium perfringens. It can induce lethal enterotoxemia in domestic animals, mainly in sheep, goats and cattle, causing serious economic losses to global animal husbandry. In this study, a novel and stable epsilon toxin mutant rETX(Y196E)-C, obtained by substituting the 196th tyrosine (Y196) with glutamic acid (E) and introducing of 23 amino acids long C-terminal peptide, was determined as a promising recombinant vaccine candidate against enterotoxemia. After the third vaccination, the antibody titers against recombinant wild type (rETX) could reach 1:10(5) in each immunized group, and the mice were completely protected from 100 × LD50 (50% lethal dose) of rETX challenge. The mice in 15 μg subcutaneously immunized group fully survived at the dose of 500 × LD50 of rETX challenge and 80% of mice survived at 180 μg (1000 × LD50) of rETX administration. In vitro, immune sera from 15 μg subcutaneously immunized group could completely protect MDCK cells from 16 × CT50 (50% lethal dose of cells) of rETX challenge and protect against 10 × LD50 dose (1.8 μg) of rETX challenge in mice. These data suggest that recombinant protein rETX(Y196E)-C is a potential vaccine candidate for future applied researches.

Altered striatal function in a mutant mouse lacking D1A dopamine receptors.

PubMed Central

Drago, J; Gerfen, C R; Lachowicz, J E; Steiner, H; Hollon, T R; Love, P E; Ooi, G T; Grinberg, A; Lee, E J; Huang, S P

1994-01-01

Of the five known dopamine receptors, D1A and D2 represent the major subtypes expressed in the striatum of the adult brain. Within the striatum, these two subtypes are differentially distributed in the two main neuronal populations that provide direct and indirect pathways between the striatum and the output nuclei of the basal ganglia. Movement disorders, including Parkinson disease and various dystonias, are thought to result from imbalanced activity in these pathways. Dopamine regulates movement through its differential effects on D1A receptors expressed by direct output neurons and D2 receptors expressed by indirect output neurons. To further examine the interaction of D1A and D2 neuronal pathways in the striatum, we used homologous recombination to generate mutant mice lacking functional D1A receptors (D1A-/-). D1A-/- mutants are growth retarded and die shortly after weaning age unless their diet is supplemented with hydrated food. With such treatment the mice gain weight and survive to adulthood. Neurologically, D1A-/- mice exhibit normal coordination and locomotion, although they display a significant decrease in rearing behavior. Examination of the striatum revealed changes associated with the altered phenotype of these mutants. D1A receptor binding was absent in striatal sections from D1A-/- mice. Striatal neurons normally expressing functional D1A receptors are formed and persist in adult homozygous mutants. Moreover, substance P mRNA, which is colocalized specifically in striatal neurons with D1A receptors, is expressed at a reduced level. In contrast, levels of enkephalin mRNA, which is expressed in striatal neurons with D2 receptors, are unaffected. These findings show that D1A-/- mice exhibit selective functional alterations in the striatal neurons giving rise to the direct striatal output pathway. Images Fig. 2 Fig. 4 PMID:7809078

Pathogenesis of coxsackievirus A9 in mice: role of the viral arginine-glycine-aspartic acid motif.

PubMed

Harvala, Heli; Kalimo, Hannu; Stanway, Glyn; Hyypiä, Timo

2003-09-01

Coxsackievirus A9 (CAV9) contains an arginine-glycine-aspartic acid (RGD) motif which participates in cell entry. Mutants with alterations in the RGD-containing region were utilized to explore the importance of the tripeptide in the pathogenesis of CAV9 in mice. Using in situ hybridization, the parental CAV9 strain was observed to infect skeletal muscle (intercostal, platysma, lingual and thigh muscles) of newborn mice, whereas the RGD-less mutants were detectable only in platysma and lingual muscles. In addition, newborn mice infected with the mutants survived longer than CAV9-infected mice. In adult mice, the parental strain of CAV9, but not the mutants, achieved moderately high titres in the pancreas. These results suggest that the RGD motif has a significant role in the pathogenesis of CAV9 in mice but also that RGD-independent entry routes can be utilized in the infection of murine tissue.

Salmonella DNA Adenine Methylase Mutants Confer Cross-Protective Immunity

PubMed Central

Heithoff, Douglas M.; Enioutina, Elena Y.; Daynes, Raymond A.; Sinsheimer, Robert L.; Low, David A.; Mahan, Michael J.

2001-01-01

Salmonella isolates that lack or overproduce DNA adenine methylase (Dam) elicited a cross-protective immune response to different Salmonella serovars. The protection afforded by the Salmonella enterica serovar Typhimurium Dam vaccine was greater than that elicited in mice that survived a virulent infection. S. enterica serovar Typhimurium Dam mutant strains exhibited enhanced sensitivity to mediators of innate immunity such as antimicrobial peptides, bile salts, and hydrogen peroxide. Also, S. enterica serovar Typhimurium Dam− vaccines were not immunosuppressive; unlike wild-type vaccines, they failed to induce increased nitric oxide levels and permitted a subsequent robust humoral response to diptheria toxoid antigen in infected mice. Dam mutant strains exhibited a low-grade persistence which, coupled with the nonimmunosuppression and the ectopic protein expression caused by altered levels of Dam, may provide an expanded source of potential antigens in vaccinated hosts. PMID:11598044

Immunization with mutant HPV16 E7 protein inhibits the growth of TC-1 cells in tumor-bearing mice.

PubMed

Li, Yan-Li; Ma, Zhong-Liang; Zhao, Yue; Zhang, Jing

2015-04-01

Two human papillomavirus (HPV) 16 oncogenic proteins, E6 and E7, are co-expressed in the majority of HPV16-induced cervical cancer cells. Thus, the E6 and E7 proteins are good targets for developing therapeutic vaccines for cervical cancer. In the present study, immunization with the mutant non-transforming HPV16 E7 (mE7) protein was demonstrated to inhibit the growth of TC-1 cells in the TC-1 mouse model. The HPV16 mE7 gene was amplified by splicing overlap extension polymerase chain reaction using pET-28a(+)-E7 as a template, and the gene was cloned into pET-28a(+) to form pET-28a(+)-mE7. Compared with the E7 protein, mE7 lacks amino acid residues 94-98, and at residue 24, there is a Cys to Gly substitution. pET-28a(+)-mE7 was then introduced into Escherichia coli Rosetta. The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside. The mE7 protein was purified using Ni-NTA agarose and detected by SDS-PAGE and western blot analysis. In the tumor prevention model, no tumor was detected in the mice vaccinated with the mE7 protein. After 40 days, the tumor-free mice and control mice were challenged with 2×10 5 TC-1 cells. All control mice developed tumors six days later, but mE7 immunized mice were tumor free until 90 days. In the tumor therapy model, the TC-1 cells were initially injected subcutaneously, and the mice were subsequently vaccinated. Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control. These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

Mutant TDP-43 within motor neurons drives disease onset but not progression in amyotrophic lateral sclerosis.

PubMed

Ditsworth, Dara; Maldonado, Marcus; McAlonis-Downes, Melissa; Sun, Shuying; Seelman, Amanda; Drenner, Kevin; Arnold, Eveline; Ling, Shuo-Chien; Pizzo, Donald; Ravits, John; Cleveland, Don W; Da Cruz, Sandrine

2017-06-01

Mutations in TDP-43 cause amyotrophic lateral sclerosis (ALS), a fatal paralytic disease characterized by degeneration and premature death of motor neurons. The contribution of mutant TDP-43-mediated damage within motor neurons was evaluated using mice expressing a conditional allele of an ALS-causing TDP-43 mutant (Q331K) whose broad expression throughout the central nervous system mimics endogenous TDP-43. TDP-43 Q331K mice develop age- and mutant-dependent motor deficits from degeneration and death of motor neurons. Cre-recombinase-mediated excision of the TDP-43 Q331K gene from motor neurons is shown to delay onset of motor symptoms and appearance of TDP-43-mediated aberrant nuclear morphology, and abrogate subsequent death of motor neurons. However, reduction of mutant TDP-43 selectively in motor neurons did not prevent age-dependent degeneration of axons and neuromuscular junction loss, nor did it attenuate astrogliosis or microgliosis. Thus, disease mechanism is non-cell autonomous with mutant TDP-43 expressed in motor neurons determining disease onset but progression defined by mutant acting within other cell types.

Impaired ventilatory acclimatization to hypoxia in mice lacking the immediate early gene fos B.

PubMed

Malik, Mohammad T; Peng, Ying-Jie; Kline, David D; Adhikary, Gautam; Prabhakar, Nanduri R

2005-01-15

Earlier studies on cell culture models suggested that immediate early genes (IEGs) play an important role in cellular adaptations to hypoxia. Whether IEGs are also necessary for hypoxic adaptations in intact animals is not known. In the present study we examined the potential importance of fos B, an IEG in ventilatory acclimatization to hypoxia. Experiments were performed on wild type and mutant mice lacking the fos B gene. Ventilation was monitored by whole body plethysmography in awake animals. Baseline ventilation under normoxia, and ventilatory response to acute hypoxia and hypercapnia were comparable between wild type and mutant mice. Hypobaric hypoxia (0.4 atm; 3 days) resulted in a significant elevation of baseline ventilation in wild type but not in mutant mice. Wild type mice exposed to hypobaric hypoxia manifested an enhanced hypoxic ventilatory response compared to pre-hypobaric hypoxia. In contrast, hypobaric hypoxia had no effect on the hypoxic ventilatory response in mutant mice. Hypercapnic ventilatory responses, however, were unaffected by hypobaric hypoxia in both groups of mice. These results suggest that the fos B, an immediate early gene, plays an important role in ventilatory acclimatization to hypoxia in mice.

Copper/zinc superoxide dismutase insufficiency impairs progesterone secretion and fertility in female mice.

PubMed

Noda, Yoshihiro; Ota, Kuniaki; Shirasawa, Takuji; Shimizu, Takahiko

2012-01-01

Copper/zinc superoxide dismutase (CuZn-SOD, SOD1) is one of the major antioxidant enzymes, and is localized in the cytoplasm to scavenge superoxide. To investigate the physiological role of SOD1 in the ovaries, we analyzed the fertility of Sod1-deficient female mice. To evaluate their hormonal metabolism, we measured pituitary and ovarian hormone levels in the plasma of the mutant mice. Plasma follicle-stimulating hormone, luteinizing hormone, and estradiol were not altered in the mutant compared to the wild-type females, while the plasma progesterone level was significantly reduced in the mutant females. Furthermore, the mutant mice showed decreased progesterone secretion under the condition of superovulation. In a histochemical analysis, we observed a remarkable reduction in the corpus luteum area in the mutant ovaries without atrophic changes. The mutant mice also displayed enhanced superoxide generation in the region surrounding the corpora lutea, which was associated with increased apoptotic cells and suppressed vasculature. These results suggested that SOD1 deficiency dysregulated luteal formation because of increased superoxide generation in the ovary. In vitro fertilization experiments showed no abnormal fertilization of Sod1-deficient oocytes. In addition, when Sod1-deficient embryos were transferred into the oviducts of wild-type females, mutant embryos developed at a normal rate, indicating that SOD1 deficiency in embryos did not cause miscarriage in the uterus of wild-type females. These results indicated that increased intracellular ROS impaired luteal formation and progesterone production in the mutant females, thus suggesting that SOD1 plays a crucial role in both the luteal function and the maintenance of fertility in female mice.

A Mutant Receptor Tyrosine Phosphatase, CD148, Causes Defects in Vascular Development

PubMed Central

Takahashi, Takamune; Takahashi, Keiko; St. John, Patricia L.; Fleming, Paul A.; Tomemori, Takuya; Watanabe, Toshio; Abrahamson, Dale R.; Drake, Christopher J.; Shirasawa, Takuji; Daniel, Thomas O.

2003-01-01

Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPη), participates in developmental vascularization. A mutant allele, CD148ΔCyGFP, was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions. PMID:12588999

Effect of CCS on the Accumulation of FALS SOD1 Mutant-containing Aggregates and on Mitochondrial Translocation of SOD1 Mutants: Implication of a Free Radical Hypothesis

PubMed Central

Kim, Ha Kun; Chung, Youn Wook; Chock, P. Boon; Yim, Moon B.

2011-01-01

Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H2O2, mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation. PMID:21354101

Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

PubMed

Kim, Ha Kun; Chung, Youn Wook; Chock, P Boon; Yim, Moon B

2011-05-15

Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H(2)O(2), mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation. Published by Elsevier Inc.

Ultrasonic vocalizations of adult male Foxp2-mutant mice: behavioral contexts of arousal and emotion.

PubMed

Gaub, S; Fisher, S E; Ehret, G

2016-02-01

Adult mouse ultrasonic vocalizations (USVs) occur in multiple behavioral and stimulus contexts associated with various levels of arousal, emotion and social interaction. Here, in three experiments of increasing stimulus intensity (water; female urine; male interacting with adult female), we tested the hypothesis that USVs of adult males express the strength of arousal and emotion via different USV parameters (18 parameters analyzed). Furthermore, we analyzed two mouse lines with heterozygous Foxp2 mutations (R552H missense, S321X nonsense), known to produce severe speech and language disorders in humans. These experiments allowed us to test whether intact Foxp2 function is necessary for developing full adult USV repertoires, and whether mutations of this gene influence instinctive vocal expressions based on arousal and emotion. The results suggest that USV calling rate characterizes the arousal level, while sound pressure and spectrotemporal call complexity (overtones/harmonics, type of frequency jumps) may provide indices of levels of positive emotion. The presence of Foxp2 mutations did not qualitatively affect the USVs; all USV types that were found in wild-type animals also occurred in heterozygous mutants. However, mice with Foxp2 mutations displayed quantitative differences in USVs as compared to wild-types, and these changes were context dependent. Compared to wild-type animals, heterozygous mutants emitted mainly longer and louder USVs at higher minimum frequencies with a higher occurrence rate of overtones/harmonics and complex frequency jump types. We discuss possible hypotheses about Foxp2 influence on emotional vocal expressions, which can be investigated in future experiments using selective knockdown of Foxp2 in specific brain circuits. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

DNase I hypersensitivity and epsilon-globin transcriptional enhancement are separable in locus control region (LCR) HS1 mutant human beta-globin YAC transgenic mice.

PubMed

Shimotsuma, Motoshi; Okamura, Eiichi; Matsuzaki, Hitomi; Fukamizu, Akiyoshi; Tanimoto, Keiji

2010-05-07

Expression of the five beta-like globin genes (epsilon, Ggamma, Agamma, delta, beta) in the human beta-globin locus depends on enhancement by the locus control region, which consists of five DNase I hypersensitive sites (5'HS1 through 5'HS5). We report here a novel enhancer activity in 5'HS1 that appears to be potent in transfected K562 cells. Deletion analyses identified a core activating element that bound to GATA-1, and a two-nucleotide mutation that disrupted GATA-1 binding in vitro abrogated 5'HS1 enhancer activity in transfection experiments. To determine the in vivo role of this GATA site, we generated multiple lines of human beta-globin YAC transgenic mice bearing the same two-nucleotide mutation. In the mutant mice, epsilon-, but not gamma-globin, gene expression in primitive erythroid cells was severely attenuated, while adult beta-globin gene expression in definitive erythroid cells was unaffected. Interestingly, DNaseI hypersensitivity near the 5'HS1 mutant sequence was eliminated in definitive erythroid cells, whereas it was only mildly affected in primitive erythroid cells. We therefore conclude that, although the GATA site in 5'HS1 is critical for efficient epsilon-globin gene expression, hypersensitive site formation per se is independent of 5'HS1 function, if any, in definitive erythroid cells.

Mecp2 truncation in male mice promotes affiliative social behavior

PubMed Central

Pearson, B.L.; Defensor, E.B.; Pobbe, R.L.H.; Yamamoto, L.H.L.; Bolivar, V.J.; Blanchard, D.C.; Blanchard, R.J.

2018-01-01

Mouse models of Rett syndrome, with targeted mutations in the Mecp2 gene, show a high degree of phenotypic consistency with the clinical syndrome. In addition to severe and age-specific regression in motor and cognitive abilities, a variety of studies have demonstrated that Mecp2 mutant mice display impaired social behavior. Conversely, other studies indicate complex enhancements of social behavior in Mecp2 mutant mice. Since social behavior is a complicated accumulation of constructs, we performed a series of classic and refined social behavior tasks and revealed a relatively consistent pattern of enhanced pro-social behavior in hypomorphic Mecp2308/Y mutant mice. Analyses of repetitive motor acts, and cognitive stereotypy did not reveal any profound differences due to genotype. Taken together, these results suggest that the mutations associated with Rett syndrome are not necessarily associated with autism-relevant social impairment in mice. However, this gene may be a valuable candidate for revealing basic mechanisms of affiliative behavior. PMID:21909962

Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation.

PubMed

Mostafa, Heba H; Thompson, Thornton W; Konen, Adam J; Haenchen, Steve D; Hilliard, Joshua G; Macdonald, Stuart J; Morrison, Lynda A; Davido, David J

2018-04-01

In the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in UL39 , which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypes in vivo , we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of UL39 in KOS-NA's decreased pathogenicity, the UL39 mutations were recombined into HSV-1 (generating UL39 mut ), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection. IMPORTANCE HSV-1 is a major human pathogen that infects ∼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in

Subthreshold membrane potential oscillations in inferior olive neurons are dynamically regulated by P/Q- and T-type calcium channels: a study in mutant mice.

PubMed

Choi, Soonwook; Yu, Eunah; Kim, Daesoo; Urbano, Francisco J; Makarenko, Vladimir; Shin, Hee-Sup; Llinás, Rodolfo R

2010-08-15

The role of P/Q- and T-type calcium channels in the rhythmic oscillatory behaviour of inferior olive (IO) neurons was investigated in mutant mice. Mice lacking either the CaV2.1 gene of the pore-forming alpha1A subunit for P/Q-type calcium channel, or the CaV3.1 gene of the pore-forming alpha1G subunit for T-type calcium channel were used. In vitro intracellular recording from IO neurons reveals that the amplitude and frequency of sinusoidal subthreshold oscillations (SSTOs) were reduced in the CaV2.1-/- mice. In the CaV3.1-/- mice, IO neurons also showed altered patterns of SSTOs and the probability of SSTO generation was significantly lower (15%, 5 of 34 neurons) than that of wild-type (78%, 31 of 40 neurons) or CaV2.1-/- mice (73%, 22 of 30 neurons). In addition, the low-threshold calcium spike and the sustained endogenous oscillation following rebound potentials were absent in IO neurons from CaV3.1-/- mice. Moreover, the phase-reset dynamics of oscillatory properties of single neurons and neuronal clusters in IO were remarkably altered in both CaV2.1-/- and CaV3.1-/- mice. These results suggest that both alpha1A P/Q- and alpha1G T-type calcium channels are required for the dynamic control of neuronal oscillations in the IO. These findings were supported by results from a mathematical IO neuronal model that incorporated T and P/Q channel kinetics.

Progressive Motor Deficit is Mediated by the Denervation of Neuromuscular Junctions and Axonal Degeneration in Transgenic Mice Expressing Mutant (P301S) Tau Protein.

PubMed

Yin, Zhuoran; Valkenburg, Femke; Hornix, Betty E; Mantingh-Otter, Ietje; Zhou, Xingdong; Mari, Muriel; Reggiori, Fulvio; Van Dam, Debby; Eggen, Bart J L; De Deyn, Peter P; Boddeke, Erik

2017-01-01

Tauopathies include a variety of neurodegenerative diseases associated with the pathological aggregation of hyperphosphorylated tau, resulting in progressive cognitive decline and motor impairment. The underlying mechanism for motor deficits related to tauopathy is not yet fully understood. Here, we use a novel transgenic tau mouse line, Tau 58/4, with enhanced neuron-specific expression of P301S mutant tau to investigate the motor abnormalities in association with the peripheral nervous system. Using stationary beam, gait, and rotarod tests, motor deficits were found in Tau 58/4 mice already 3 months after birth, which deteriorated during aging. Hyperphosphorylated tau was detected in the cell bodies and axons of motor neurons. At the age of 9 and 12 months, significant denervation of the neuromuscular junction in the extensor digitorum longus muscle was observed in Tau 58/4 mice, compared to wild-type mice. Muscle hypotrophy was observed in Tau 58/4 mice at 9 and 12 months. Using electron microscopy, we observed ultrastructural changes in the sciatic nerve of 12-month-old Tau 58/4 mice indicative of the loss of large axonal fibers and hypomyelination (assessed by g-ratio). We conclude that the accumulated hyperphosphorylated tau in the axon terminals may induce dying-back axonal degeneration, myelin abnormalities, neuromuscular junction denervation, and muscular atrophy, which may be the mechanisms responsible for the deterioration of the motor function in Tau 58/4 mice. Tau 58/4 mice represent an interesting neuromuscular degeneration model, and the pathological mechanisms might be responsible for motor signs observed in some human tauopathies.

Mutant Huntingtin Causes a Selective Decrease in the Expression of Synaptic Vesicle Protein 2C.

PubMed

Peng, Chaohua; Zhu, Gaochun; Liu, Xiangqian; Li, He

2018-04-30

Huntington's disease (HD) is a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin (Htt) protein. Mutant Htt causes synaptic transmission dysfunctions by interfering in the expression of synaptic proteins, leading to early HD symptoms. Synaptic vesicle proteins 2 (SV2s), a family of synaptic vesicle proteins including 3 members, SV2A, SV2B, and SV2C, plays important roles in synaptic physiology. Here, we investigated whether the expression of SV2s is affected by mutant Htt in the brains of HD transgenic (TG) mice and Neuro2a mouse neuroblastoma cells (N2a cells) expressing mutant Htt. Western blot analysis showed that the protein levels of SV2A and SV2B were not significantly changed in the brains of HD TG mice expressing mutant Htt with 82 glutamine repeats. However, in the TG mouse brain there was a dramatic decrease in the protein level of SV2C, which has a restricted distribution pattern in regions particularly vulnerable in HD. Immunostaining revealed that the immunoreactivity of SV2C was progressively weakened in the basal ganglia and hippocampus of TG mice. RT-PCR demonstrated that the mRNA level of SV2C progressively declined in the TG mouse brain without detectable changes in the mRNA levels of SV2A and SV2B, indicating that mutant Htt selectively inhibits the transcriptional expression of SV2C. Furthermore, we found that only SV2C expression was progressively inhibited in N2a cells expressing a mutant Htt containing 120 glutamine repeats. These findings suggest that the synaptic dysfunction in HD results from the mutant Htt-mediated inhibition of SV2C transcriptional expression. These data also imply that the restricted distribution and decreased expression of SV2C contribute to the brain region-selective pathology of HD.

Impaired locomotor activity and exploratory behavior in mice lacking histamine H1 receptors

PubMed Central

Inoue, Isao; Yanai, Kazuhiko; Kitamura, Daisuke; Taniuchi, Ichiro; Kobayashi, Takashi; Niimura, Kaku; Watanabe, Takehiko; Watanabe, Takeshi

1996-01-01

From pharmacological studies using histamine antagonists and agonists, it has been demonstrated that histamine modulates many physiological functions of the hypothalamus, such as arousal state, locomotor activity, feeding, and drinking. Three kinds of receptors (H1, H2, and H3) mediate these actions. To define the contribution of the histamine H1 receptors (H1R) to behavior, mutant mice lacking the H1R were generated by homologous recombination. In brains of homozygous mutant mice, no specific binding of [3H]pyrilamine was seen. [3H]Doxepin has two saturable binding sites with higher and lower affinities in brains of wild-type mice, but H1R-deficient mice showed only the weak labeling of [3H]doxepin that corresponds to lower-affinity binding sites. Mutant mice develop normally, but absence of H1R significantly increased the ratio of ambulation during the light period to the total ambulation for 24 hr in an accustomed environment. In addition, mutant mice significantly reduced exploratory behavior of ambulation and rearings in a new environment. These results indicate that through H1R, histamine is involved in circadian rhythm of locomotor activity and exploratory behavior as a neurotransmitter. PMID:8917588

Regulation of GABAA and Glutamate Receptor Expression, Synaptic Facilitation and Long-Term Potentiation in the Hippocampus of Prion Mutant Mice

PubMed Central

Rangel, Alejandra; Madroñal, Noelia; Massó, Agnès Gruart i.; Gavín, Rosalina; Llorens, Franc; Sumoy, Lauro; Torres, Juan María; Delgado-García, José María; Río, José Antonio Del

2009-01-01

Background Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrPsc) of the natural cellular prion protein (PrPc) encoded by the Prnp gene. Although several roles have been attributed to PrPc, its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrPc studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Methodology/Principal Findings Here we explore the role of PrPc expression in neurotransmission and neural excitability using wild-type, Prnp −/− and PrPc-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp −/− mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina™ microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp −/− and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABAA and AMPA-kainate receptors are co-regulated in both Prnp −/− and Tg20 mice. Conclusions/Significance Present results demonstrate that PrPc is necessary for the proper

Active site mutant transgene confers tolerance to human β-glucuronidase without affecting the phenotype of MPS VII mice

PubMed Central

Sly, William S.; Vogler, Carole; Grubb, Jeffrey H.; Zhou, Mi; Jiang, Jinxing; Zhou, Xiao Yan; Tomatsu, Shunji; Bi, Yanhua; Snella, Elizabeth M.

2001-01-01

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is an autosomal recessive lysosomal storage disorder due to an inherited deficiency of β-glucuronidase. A naturally occurring mouse model for this disease was discovered at The Jackson Laboratory and shown to be due to homozygosity for a 1-bp deletion in exon 10 of the gus gene. The murine model MPS VII (gusmps/mps) has been very well characterized and used extensively to evaluate experimental strategies for lysosomal storage diseases, including bone marrow transplantation, enzyme replacement therapy, and gene therapy. To enhance the value of this model for enzyme and gene therapy, we produced a transgenic mouse expressing the human β-glucuronidase cDNA with an amino acid substitution at the active site nucleophile (E540A) and bred it onto the MPS VII (gusmps/mps) background. We demonstrate here that the mutant mice bearing the active site mutant human transgene retain the clinical, morphological, biochemical, and histopathological characteristics of the original MPS VII (gusmps/mps) mouse. However, they are now tolerant to immune challenge with human β-glucuronidase. This “tolerant MPS VII mouse model” should be useful for preclinical trials evaluating the effectiveness of enzyme and/or gene therapy with the human gene products likely to be administered to human patients with MPS VII. PMID:11226217

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.

Deletion of vanilloid receptor (TRPV1) in mice alters behavioral effects of ethanol

PubMed Central

Blednov, Y.A.; Harris, R.A.

2009-01-01

The vanilloid receptor TRPV1 is activated by ethanol and this may be important for some of the central and peripheral actions of ethanol. To determine if this receptor has a role in ethanol-mediated behaviors, we studied null mutant mice in which the Trpv1 gene was deleted. Mice lacking this gene showed significantly higher preference for ethanol and consumed more ethanol in a two-bottle choice test as compared with wild type littermates. Null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol (2 g/kg). However, there were no differences between null mutant and wild type mice in severity of ethanol-induced acute withdrawal (4 g/kg) or conditioned taste aversion to ethanol (2.5 g/kg). Two behavioral phenotypes (decreased sensitivity to ethanol-induced sedation and faster recovery from ethanol-induced motor incoordination) seen in null mutant mice were reproduced in wild type mice by injection of a TRPV1 antagonist, capsazepine (10 mg/kg). These two ethanol behaviors were changed in the opposite direction after injection of capsaicin, a selective TRPV1 agonist, in wild type mice. The studies provide the first evidence that TRPV1 is important for specific behavioral actions of ethanol. PMID:19705551

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/Wv mutant mouse colon

PubMed Central

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice. PMID:26727725

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/Wv mutant mouse colon.

PubMed

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon.The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers,but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice.

PAC1- and VPAC2 receptors in light regulated behavior and physiology: Studies in single and double mutant mice

PubMed Central

Georg, Birgitte; Fahrenkrug, Jan

2017-01-01

The two sister peptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) and their receptors, the PAC1 –and the VPAC2 receptors, are involved in regulation of the circadian timing system. PACAP as a neurotransmitter in the retinohypothalamic tract (RHT) and VIP as a neurotransmitter, involved in synchronization of SCN neurons. Behavior and physiology in VPAC2 deficient mice are strongly regulated by light most likely as a result of masking. Consequently, we used VPAC2 and PAC1/VPAC2 double mutant mice in comparison with PAC1 receptor deficient mice to further elucidate the role of PACAP in the light mediated regulation of behavior and physiology of the circadian system. We compared circadian rhythms in mice equipped with running wheels or implanted radio-transmitter measuring core body temperature kept in a full photoperiod ((FPP)(12:12 h light dark-cycles (LD)) and skeleton photo periods (SPP) at high and low light intensity. Furthermore, we examined the expression of PAC1- and VPAC2 receptors in the SCN of the different genotypes in combination with visualization of PACAP and VIP and determined whether compensatory changes in peptide and/or receptor expression in the reciprocal knockouts (KO) (PAC1 and VPAC2) had occurred. Our data demonstrate that in although being closely related at both ligand and receptor structure/sequence, PACAP/PAC1 receptor signaling are independent of VIP/VPAC2 receptor signaling and vice versa. Furthermore, lack of either of the receptors does not result in compensatory changes at neither the physiological or anatomical level. PACAP/PAC1 signaling is important for light regulated behavior, VIP/VPAC2signaling for stable clock function and both signaling pathways may play a role in shaping diurnality versus nocturnality. PMID:29155851

PAC1- and VPAC2 receptors in light regulated behavior and physiology: Studies in single and double mutant mice.

PubMed

Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

2017-01-01

The two sister peptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) and their receptors, the PAC1 -and the VPAC2 receptors, are involved in regulation of the circadian timing system. PACAP as a neurotransmitter in the retinohypothalamic tract (RHT) and VIP as a neurotransmitter, involved in synchronization of SCN neurons. Behavior and physiology in VPAC2 deficient mice are strongly regulated by light most likely as a result of masking. Consequently, we used VPAC2 and PAC1/VPAC2 double mutant mice in comparison with PAC1 receptor deficient mice to further elucidate the role of PACAP in the light mediated regulation of behavior and physiology of the circadian system. We compared circadian rhythms in mice equipped with running wheels or implanted radio-transmitter measuring core body temperature kept in a full photoperiod ((FPP)(12:12 h light dark-cycles (LD)) and skeleton photo periods (SPP) at high and low light intensity. Furthermore, we examined the expression of PAC1- and VPAC2 receptors in the SCN of the different genotypes in combination with visualization of PACAP and VIP and determined whether compensatory changes in peptide and/or receptor expression in the reciprocal knockouts (KO) (PAC1 and VPAC2) had occurred. Our data demonstrate that in although being closely related at both ligand and receptor structure/sequence, PACAP/PAC1 receptor signaling are independent of VIP/VPAC2 receptor signaling and vice versa. Furthermore, lack of either of the receptors does not result in compensatory changes at neither the physiological or anatomical level. PACAP/PAC1 signaling is important for light regulated behavior, VIP/VPAC2signaling for stable clock function and both signaling pathways may play a role in shaping diurnality versus nocturnality.

Rescue of volume-regulated anion current by bestrophin mutants with altered charge selectivity.

PubMed

Chien, Li-Ting; Hartzell, H Criss

2008-11-01

Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca(2+)-activated Cl(-) channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca(2+)-activated Cl(-) currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) (P(Cs)/P(Cl) = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-). The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1(-/-)/best2(-/-)). VRACs were identical in wild-type and best1(-/-)/best2(-/-) mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.

Variations of L- and D-amino acid levels in the brain of wild-type and mutant mice lacking D-amino acid oxidase activity.

PubMed

Du, Siqi; Wang, Yadi; Weatherly, Choyce A; Holden, Kylie; Armstrong, Daniel W

2018-05-01

D-amino acids are now recognized to be widely present in organisms and play essential roles in biological processes. Some D-amino acids are metabolized by D-amino acid oxidase (DAO), while D-Asp and D-Glu are metabolized by D-aspartate oxidase (DDO). In this study, levels of 22 amino acids and the enantiomeric compositions of the 19 chiral proteogenic entities have been determined in the whole brain of wild-type ddY mice (ddY/DAO +/+ ), mutant mice lacking DAO activity (ddY/DAO -/- ), and the heterozygous mice (ddY/DAO +/- ) using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). No significant differences were observed for L-amino acid levels among the three strains except for L-Trp which was markedly elevated in the DAO +/- and DAO -/- mice. The question arises as to whether this is an unknown effect of DAO inactivity. The three highest levels of L-amino acids were L-Glu, L-Asp, and L-Gln in all the three strains. The lowest L-amino acid level was L-Cys in ddY/DAO +/- and ddY/DAO -/- mice, while L-Trp showed the lowest level in ddY/DAO +/+ mice. The highest concentration of D-amino acid was found to be D-Ser, which also had the highest % D value (~ 25%). D-Glu had the lowest % D value (~ 0.01%) in all the three strains. Significant differences of D-Leu, D-Ala, D-Ser, D-Arg, and D-Ile were observed in ddY/DAO +/- and ddY/DAO -/- mice compared to ddY/DAO +/+ mice. This work provides the most complete baseline analysis of L- and D-amino acids in the brains of ddY/DAO +/+ , ddY/DAO +/- , and ddY/DAO -/- mice yet reported. It also provides the most effective and efficient analytical approach for measuring these analytes in biological samples. This study provides fundamental information on the role of DAO in the brain and may be relevant for future development involving novel drugs for DAO regulation.

Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation

PubMed Central

2011-01-01

Background Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. Results In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II), to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus), infected with mutant parasites did not develop any sign of pathology. Conclusions The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis. PMID:21794145

Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation.

PubMed

Carrión, Javier; Folgueira, Cristina; Soto, Manuel; Fresno, Manuel; Requena, Jose M

2011-07-27

Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II), to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus), infected with mutant parasites did not develop any sign of pathology. The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

Juvenile spermatogonial depletion (jsd): a genetic defect of germ cell proliferation of male mice.

PubMed

Beamer, W G; Cunliffe-Beamer, T L; Shultz, K L; Langley, S H; Roderick, T H

1988-05-01

Adult C57BL/6J male mice homozygous for the mutant gene, juvenile spermatogonial depletion (jsd/jsd), show azoosper4ia and testes reduced to one-third normal size, but are otherwise phenotypically normal. In contrast, adult jsd/jsd females are fully fertile. This feature facilitated mapping the jsd gene to the centromeric end of chromosome 1; the gene order is jsd-Isocitrate dehydrogenase-1 (Idh-1)-Peptidase-3 (Pep-3). Analysis of testicular histology from jsd/jsd mice aged 3-10 wk revealed that these mutant mice experience one wave of spermatogenesis, but fail to continue mitotic proliferation of type A spermatogonial cells at the basement membrane. As a consequence, histological sections of testes from mutant mice aged 8-52 wk showed tubules populated by modest numbers of Sertoli cells, with only an occasional spermatogonial cell. Some sperm with normal morphology and motility were observed in epididymides of 6.5- but not in 8-wk or older mutants. Treatment with retinol failed to alter the loss of spermatogenesis in jsd/jsd mice. Analyses of serum hormones of jsd/jsd males showed that testosterone levels were normal at all ages--a finding corroborated by normal seminal vesicle and vas deferens weights, whereas serum follicle-stimulating hormone levels were significantly elevated in mutant mice from 4 to 20 wk of age. We hypothesize the jsd/jsd male may be deficient in proliferative signals from Sertoli cells that are needed for spermatogenesis.

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/W(v) mutant mouse colon.

PubMed

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/W(v) mice carrying W and W(v) mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/W(v) mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/W(v) mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/W(v) mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/W(v) mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/W(v) mutant mice.

Voronoi-based spatial analysis reveals selective interneuron changes in the cortex of FALS mice.

PubMed

Minciacchi, Diego; Kassa, Roman M; Del Tongo, Claudia; Mariotti, Raffaella; Bentivoglio, Marina

2009-01-01

The neurodegenerative disease amyotrophic lateral sclerosis affects lower motoneurons and corticospinal cells. Mice expressing human mutant superoxide dismutase (SOD)1 provide widely investigated models of the familial form of disease, but information on cortical changes in these mice is still limited. We here analyzed the spatial organization of interneurons characterized by parvalbumin immunoreactivity in the motor, somatosensory, and visual cortical areas of SOD1(G93A) mice. Cell number and sociological spatial behavior were assessed by digital charts of cell location in cortical samples, cell counts, and generation of two-dimensional Voronoi diagrams. In end-stage SOD1-mutant mice, an increase of parvalbumin-containing cortical interneurons was found in the motor and somatosensory areas (about 35% and 20%, respectively) with respect to wild-type littermates. Changes in cell spatial distribution, as documented by Voronoi-derived coefficients of variation, indicated increased tendency of parvalbumin cells to aggregate into clusters in the same areas of the SOD1-mutant cortex. Counts and coefficients of variation of parvalbumin cells in the visual cortex gave instead similar results in SOD1-mutant and wild-type mice. Analyses of motor and somatosensory areas in presymptomatic SOD1-mutant mice provided findings very similar to those obtained at end-stage, indicating early changes of interneurons in these cortical areas during the pathology. Altogether the data reveal in the SOD1-mutant mouse cortex an altered architectonic pattern of interneurons, which selectively affects areas involved in motor control. The findings, which can be interpreted as pathogenic factors or early disease-related adaptations, point to changes in the cortical regulation and modulation of the motor circuit during motoneuron disease.

Vaccination with a Leishmania infantum HSP70-II null mutant confers long-term protective immunity against Leishmania major infection in two mice models

PubMed Central

Solana, José Carlos; Ramírez, Laura; Corvo, Laura; de Oliveira, Camila Indiani; Barral-Netto, Manoel; Requena, José María

2017-01-01

Background The immunization with genetically attenuated Leishmania cell lines has been associated to the induction of memory and effector T cell responses against Leishmania able to control subsequent challenges. A Leishmania infantum null mutant for the HSP70-II genes has been described, possessing a non-virulent phenotype. Methodology/Principal findings The L. infantum attenuated parasites (LiΔHSP70-II) were inoculated in BALB/c (intravenously and subcutaneously) and C57BL/6 (subcutaneously) mice. An asymptomatic infection was generated and parasites diminished progressively to become undetectable in most of the analyzed organs. However, inoculation resulted in the long-term induction of parasite specific IFN-γ responses able to control the disease caused by a challenge of L. major infective promastigotes. BALB/c susceptible mice showed very low lesion development and a drastic decrease in parasite burdens in the lymph nodes draining the site of infection and internal organs. C57BL/6 mice did not show clinical manifestation of disease, correlated to the rapid migration of Leishmania specific IFN-γ producing T cells to the site of infection. Conclusion/Significance Inoculation of the LiΔHSP70-II attenuated line activates mammalian immune system for inducing moderate pro-inflammatory responses. These responses are able to confer long-term protection in mice against the infection of L. major virulent parasites. PMID:28558043

Subthreshold membrane potential oscillations in inferior olive neurons are dynamically regulated by P/Q- and T-type calcium channels: a study in mutant mice

PubMed Central

Choi, Soonwook; Yu, Eunah; Kim, Daesoo; Urbano, Francisco J; Makarenko, Vladimir; Shin, Hee-Sup; Llinás, Rodolfo R

2010-01-01

The role of P/Q- and T-type calcium channels in the rhythmic oscillatory behaviour of inferior olive (IO) neurons was investigated in mutant mice. Mice lacking either the CaV2.1 gene of the pore-forming α1A subunit for P/Q-type calcium channel, or the CaV3.1 gene of the pore-forming α1G subunit for T-type calcium channel were used. In vitro intracellular recording from IO neurons reveals that the amplitude and frequency of sinusoidal subthreshold oscillations (SSTOs) were reduced in the CaV2.1−/− mice. In the CaV3.1−/− mice, IO neurons also showed altered patterns of SSTOs and the probability of SSTO generation was significantly lower (15%, 5 of 34 neurons) than that of wild-type (78%, 31 of 40 neurons) or CaV2.1−/− mice (73%, 22 of 30 neurons). In addition, the low-threshold calcium spike and the sustained endogenous oscillation following rebound potentials were absent in IO neurons from CaV3.1−/− mice. Moreover, the phase-reset dynamics of oscillatory properties of single neurons and neuronal clusters in IO were remarkably altered in both CaV2.1−/− and CaV3.1−/− mice. These results suggest that both α1A P/Q- and α1G T-type calcium channels are required for the dynamic control of neuronal oscillations in the IO. These findings were supported by results from a mathematical IO neuronal model that incorporated T and P/Q channel kinetics. PMID:20547676

Learning-Induced Suboptimal Compensation for PKCι/λ Function in Mutant Mice.

PubMed

Sheng, Tao; Wang, Shaoli; Qian, Dandan; Gao, Jun; Ohno, Shigeo; Lu, Wei

2017-06-01

PKCι/λ has been proposed to be crucial in the early expression of long-term potentiation (LTP). Here, we further investigate the potential role of PKCι/λ in learning and memory by generating PKCι/λ conditional knockout mice specifically lacking PKCι/λ in the hippocampal CA1 pyramidal cells. Surprisingly, PKCι/λ cKO mice show normal hippocampal LTP and memory. Further close-up observation reveals compensation for PKCι/λ expression by PKMζ in PKCι/λ cKO mice. This compensation was not observed under basal conditions, but was detected either after LTP induction or learning-associated behavioral training. Accordingly, in the early stage of LTP expression, a switch from PKCι/λ- to PKMζ-dependent molecular mechanisms was detected in PKCι/λ cKO mice. Notably, when cKO mice were challenged with more difficult hippocampus-dependent learning tasks, moderate learning deficits were detected, suggesting a suboptimal compensation for PKCι/λ's function in PKCι/λ cKO mice. Thus, under physiological conditions, PKCι/λ is essential for hippocampal early-LTP and long-term memory (LTM). © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy.

PubMed

Ong, Han B; Sienkiewicz, Natasha; Wyllie, Susan; Patterson, Stephen; Fairlamb, Alan H

2013-10-01

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy

PubMed Central

Ong, Han B; Sienkiewicz, Natasha; Wyllie, Susan; Patterson, Stephen; Fairlamb, Alan H

2013-01-01

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) – a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite. PMID:23980694

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

The effect of mild traumatic brain injury on peripheral nervous system pathology in wild-type mice and the G93A mutant mouse model of motor neuron disease.

PubMed

Evans, T M; Jaramillo, C A; Sataranatarajan, K; Watts, L; Sabia, M; Qi, W; Van Remmen, H

2015-07-09

Traumatic brain injury (TBI) is associated with a risk of neurodegenerative disease. Some suggest a link between TBI and motor neuron disease (MND), including amyotrophic lateral sclerosis (ALS). To investigate the potential mechanisms linking TBI to MND, we measured motor function and neuropathology following mild-TBI in wild-type and a transgenic model of ALS, G93A mutant mice. Mild-TBI did not alter the lifespan of G93A mice or age of onset; however, rotarod performance was impaired in G93A verses wild-type mice. Grip strength was reduced only in G93A mice after mild-TBI. Increased electromyography (EMG) abnormalities and markers of denervation (AchR, Runx1) indicate that mild-TBI may result in peripheral effects that are exaggerated in G93A mice. Markers of inflammation (cell edema, astrogliosis and microgliosis) were detected at 24 and 72h in the brain and spinal cord in wild-type and G93A mice. Levels of F2-isoprostanes, a marker of oxidative stress, were increased in the spinal cord 24h post mild-TBI in wild-type mice but were not affected by TBI in G93A mice. In summary, our data demonstrate that mild-TBI induces inflammation and oxidative stress and negatively impacts muscle denervation and motor performance, suggesting mild-TBI can potentiate motor neuron pathology and influence the development of MND in mice. Published by Elsevier Ltd.

Evaluation of Nitrofurantoin Combination Therapy of Metronidazole-Sensitive and -Resistant Helicobacter pylori Infections in Mice

PubMed Central

Jenks, Peter J.; Ferrero, Richard L.; Tankovic, Jacques; Thiberge, Jean-Michel; Labigne, Agnès

2000-01-01

The main objectives of this study were to determine whether the nitroreductase enzyme encoded by the rdxA gene of Helicobacter pylori was responsible for reductive activation of nitrofurantoin and whether a triple-therapy regimen with nitrofurantoin was able to eradicate metronidazole-sensitive and -resistant H. pylori infections from mice. The susceptibilities to nitrofurantoin of parent and isogenic rdxA mutant strains (three pairs), as well as a series of matched metronidazole-sensitive and -resistant strains isolated from mice (30) and patients (20), were assessed by agar dilution determination of the MIC. Groups of mice colonized with the metronidazole-sensitive H. pylori SS1 strain or a metronidazole-resistant rdxA SS1 mutant were treated with either metronidazole or nitrofurantoin as part of a triple-therapy regimen. One month after the completion of treatment the mice were sacrificed and their stomachs were cultured for H. pylori. The nitrofurantoin MICs for all strains tested were between 0.5 and 4.0 μg/ml. There was no significant difference between the susceptibility to nitrofurantoin of the parental strains and those of respective rdxA mutants or between those of matched metronidazole-sensitive and -resistant H. pylori isolates. The regimen with metronidazole eradicated infection from all eight SS1-infected mice and from one of eight mice inoculated with the rdxA mutant (P ≤ 0.001). The regimen with nitrofurantoin failed to eradicate infection from any of the six SS1-infected mice (P ≤ 0.001) and cleared infection from one of seven mice inoculated with the rdxA mutant. These results demonstrate that, despite the good in vitro activity of nitrofurantoin against H. pylori and the lack of cross-resistance between metronidazole and nitrofurantoin, eradication regimens involving nitrofurantoin are unable to eradicate either metronidazole-sensitive or -resistant H. pylori infections from mice. PMID:10991835

ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair

PubMed Central

Chen, Chun-Chin; Kass, Elizabeth M.; Yen, Wei-Feng; Ludwig, Thomas; Moynahan, Mary Ellen; Chaudhuri, Jayanta; Jasin, Maria

2017-01-01

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1–53BP1 antagonism and that its HDR function can become critical in certain contexts. PMID:28659469

ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair.

PubMed

Chen, Chun-Chin; Kass, Elizabeth M; Yen, Wei-Feng; Ludwig, Thomas; Moynahan, Mary Ellen; Chaudhuri, Jayanta; Jasin, Maria

2017-07-18

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1 S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1 S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1 S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1-53BP1 antagonism and that its HDR function can become critical in certain contexts.

Iron metabolism mutant hbd mice have a deletion in Sec15l1, which has homology to a yeast gene for vesicle docking.

PubMed

White, Robert A; Boydston, Leigh A; Brookshier, Terri R; McNulty, Steven G; Nsumu, Ndona N; Brewer, Brandon P; Blackmore, Krista

2005-12-01

Defects in iron absorption and utilization lead to iron deficiency and anemia. While iron transport by transferrin receptor-mediated endocytosis is well understood, it is not completely clear how iron is transported from the endosome to the mitochondria where heme is synthesized. We undertook a positional cloning project to identify the causative mutation for the hemoglobin-deficit (hbd) mouse mutant, which suffers from a microcytic, hypochromic anemia apparently due to defective iron transport in the endocytosis cycle. As shown by previous studies, reticulocyte iron accumulation in homozygous hbd/hbd mice is deficient despite normal binding of transferrin to its receptor and normal transferrin uptake in the cell. We have identified a strong candidate gene for hbd, Sec15l1, a homologue to yeast SEC15, which encodes a key protein in vesicle docking. The hbd mice have an exon deletion in Sec15l1, which is the first known mutation of a SEC gene homologue in mammals.

Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging.

PubMed

Hall, Mary P; Woodroofe, Carolyn C; Wood, Monika G; Que, Ivo; Van't Root, Moniek; Ridwan, Yanto; Shi, Ce; Kirkland, Thomas A; Encell, Lance P; Wood, Keith V; Löwik, Clemens; Mezzanotte, Laura

2018-01-09

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice.

Cerebral Amyloid Angiopathy and Parenchymal Amyloid Deposition in Transgenic Mice Expressing the Danish Mutant Form of Human BRI2

PubMed Central

Vidal, Ruben; Barbeito, Ana G; Miravalle, Leticia; Ghetti, Bernardino

2009-01-01

Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease clinically characterized by the presence of cataracts, hearing impairment, cerebellar ataxia and dementia. Neuropathologically, FDD is characterized by the presence of widespread cerebral amyloid angiopathy (CAA), parenchymal amyloid deposition and neurofibrillary tangles. FDD is caused by a 10-nucleotide duplication-insertion in the BRI2 gene that generates a larger-than-normal precursor protein, of which the Danish amyloid subunit (ADan) comprises the last 34 amino acids. Here, we describe a transgenic mouse model for FDD (Tg-FDD) in which the mouse Prnp (prion protein) promoter drives the expression of the Danish mutant form of human BRI2. The main neuropathological findings in Tg-FDD mice are the presence of widespread CAA and parenchymal deposition of ADan. In addition, we observe the presence of amyloid-associated gliosis, an inflammatory response and deposition of oligomeric ADan. As the animals aged, they showed abnormal grooming behavior, an arched back, and walked with a wide-based gait and shorter steps. This mouse model may give insights on the pathogenesis of FDD and will prove useful for the development of therapeutics. Moreover, the study of Tg-FDD mice may offer new insights into the role of amyloid in neurodegeneration in other disorders, including Alzheimer disease. PMID:18410407

Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2.

PubMed

Vidal, Ruben; Barbeito, Ana G; Miravalle, Leticia; Ghetti, Bernardino

2009-01-01

Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease clinically characterized by the presence of cataracts, hearing impairment, cerebellar ataxia and dementia. Neuropathologically, FDD is characterized by the presence of widespread cerebral amyloid angiopathy (CAA), parenchymal amyloid deposition and neurofibrillary tangles. FDD is caused by a 10-nucleotide duplication-insertion in the BRI(2) gene that generates a larger-than-normal precursor protein, of which the Danish amyloid subunit (ADan) comprises the last 34 amino acids. Here, we describe a transgenic mouse model for FDD (Tg-FDD) in which the mouse Prnp (prion protein) promoter drives the expression of the Danish mutant form of human BRI(2). The main neuropathological findings in Tg-FDD mice are the presence of widespread CAA and parenchymal deposition of ADan. In addition, we observe the presence of amyloid-associated gliosis, an inflammatory response and deposition of oligomeric ADan. As the animals aged, they showed abnormal grooming behavior, an arched back, and walked with a wide-based gait and shorter steps. This mouse model may give insights on the pathogenesis of FDD and will prove useful for the development of therapeutics. Moreover, the study of Tg-FDD mice may offer new insights into the role of amyloid in neurodegeneration in other disorders, including Alzheimer disease.

Efficacy of BET bromodomain inhibition in Kras-mutant non-small cell lung cancer

PubMed Central

Shimamura, Takeshi; Chen, Zhao; Soucheray, Margaret; Carretero, Julian; Kikuchi, Eiki; Tchaicha, Jeremy H.; Gao, Yandi; Cheng, Katherine A.; Cohoon, Travis J.; Qi, Jun; Akbay, Esra; Kimmelman, Alec C.; Kung, Andrew L.; Bradner, James E.; Wong, Kwok-Kin

2013-01-01

Purpose Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that BET bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven NSCLC with BET inhibition. Experimental Design We performed functional assays to evaluate the effects of JQ1 in genetically defined NSCLC cells lines harboring KRAS and/or LKB1 mutations. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. Effects of bromodomain inhibition on transcriptional pathways were explored and validated by expression analysis. Results While JQ1 is broadly active in NSCLC cells, activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knock-down of LKB1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1. Conclusion Bromodomain inhibition comprises a promising therapeutic strategy for KRAS mutant NSCLC with wild-type LKB1, via inhibition of MYC function. Clinical studies of BET bromodomain inhibitors in aggressive NSCLC will be actively pursued. PMID:24045185

Lgl1 Is Required for Olfaction and Development of Olfactory Bulb in Mice.

PubMed

Li, Zhenzu; Zhang, Tingting; Lin, Zhuchun; Hou, Congzhe; Zhang, Jian; Men, Yuqin; Li, Huashun; Gao, Jiangang

2016-01-01

Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and functioned as a key regulator of epithelial polarity and asymmetric cell division. In this study, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in olfactory bulb (OB). Next, we examined the effects of Lgl1 loss in the OB. First, we determined the expression patterns of Lgl1 in the neurogenic regions of the embryonic dorsal region of the LGE (dLGE) and postnatal OB. Furthermore, the Lgl1 conditional mutants exhibited abnormal morphological characteristics of the OB. Our behavioral analysis exhibited greatly impaired olfaction in Lgl1 mutant mice. To elucidate the possible mechanisms of impaired olfaction in Lgl1 mutant mice, we investigated the development of the OB. Interestingly, reduced thickness of the MCL and decreased density of mitral cells (MCs) were observed in Lgl1 mutant mice. Additionally, we observed a dramatic loss in SP8+ interneurons (e.g. calretinin and GABAergic/non-dopaminergic interneurons) in the GL of the OB. Our results demonstrate that Lgl1 is required for the development of the OB and the deletion of Lgl1 results in impaired olfaction in mice.

Lgl1 Is Required for Olfaction and Development of Olfactory Bulb in Mice

PubMed Central

Li, Zhenzu; Zhang, Tingting; Lin, Zhuchun; Hou, Congzhe; Zhang, Jian; Men, Yuqin; Li, Huashun

2016-01-01

Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and functioned as a key regulator of epithelial polarity and asymmetric cell division. In this study, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in olfactory bulb (OB). Next, we examined the effects of Lgl1 loss in the OB. First, we determined the expression patterns of Lgl1 in the neurogenic regions of the embryonic dorsal region of the LGE (dLGE) and postnatal OB. Furthermore, the Lgl1 conditional mutants exhibited abnormal morphological characteristics of the OB. Our behavioral analysis exhibited greatly impaired olfaction in Lgl1 mutant mice. To elucidate the possible mechanisms of impaired olfaction in Lgl1 mutant mice, we investigated the development of the OB. Interestingly, reduced thickness of the MCL and decreased density of mitral cells (MCs) were observed in Lgl1 mutant mice. Additionally, we observed a dramatic loss in SP8+ interneurons (e.g. calretinin and GABAergic/non-dopaminergic interneurons) in the GL of the OB. Our results demonstrate that Lgl1 is required for the development of the OB and the deletion of Lgl1 results in impaired olfaction in mice. PMID:27603780

The effect of nitric oxide synthase inhibitors nitro-L-arginine and 7-nitroindazole on spatial learning and motor functions in Lurcher mutant and wild type mice.

PubMed

Markvartová, V; Vozeh, F

2008-01-01

Nitric oxide (NO) is an intercellular messenger that, among other things, plays an important role in the nervous system as a gaseous neurotransmitter, modulating long-term potentiation (LTP) induction of synaptic transmission. LTP has been suggested to be the basis of memory formation. On the other hand NO also participates in excitotoxic processes which play an important role in many neuropathological states. The aim of this work was to observe the effect of two NO synthase (NOS) inhibitors (N omega-Nitro-L-arginine, NA; 7-nitroindazole, NI) on spontaneous behaviour, spatial learning and motor functions in Lurcher (+/Lc) and wild type (+/+) mice, derived from the B6CBA strain. Heterozygous Lurcher mutant mice represent a natural model of the olivocerebellar degeneration. They suffer from postnatal, practically total, extinction of cerebellar Purkinje cells (due to the excitotoxic apoptosis) and a partial decrease of granule cells and inferior olive neurons (ION) because of the lost target of their axons. +/+ animals are healthy littermates of +/Lc. NA is a nonselective NOS inhibitor which influences, except neuronal (n), also endothelial (e) NOS with an impact on blood pressure, NI is a selective nNOS inhibitor without any circulatory effect. The adult animals of both types (+/Lc; +/+) were influenced by acute administration of both inhibitors (25 mg/kg i.p. 30 min. before experiments) and newborns only by both acute and long-term administration of NI (1 month, starting from postnatal day 2, P2). Control solutions - saline or solvents of both NA and NI inhibitors--diluted 1M HCl and dimethyl sulfoxide (DMSO) respectively, were given at a relevant volume in the same way. The effect of both inhibitors and control solutions on motor functions was tested using four standard procedures (horizontal wire, slanting ladder, rotating cylinder, foot-bridge); in newborns at the age of 14 days. Spatial learning ability was examined in five-day long procedure in the Morris

Noonan syndrome-causing SHP2 mutants impair ERK-dependent chondrocyte differentiation during endochondral bone growth.

PubMed

Tajan, Mylène; Pernin-Grandjean, Julie; Beton, Nicolas; Gennero, Isabelle; Capilla, Florence; Neel, Benjamin G; Araki, Toshiyuki; Valet, Philippe; Tauber, Maithé; Salles, Jean-Pierre; Yart, Armelle; Edouard, Thomas

2018-04-12

Growth retardation is a constant feature of Noonan syndrome (NS) but its physiopathology remains poorly understood. We previously reported that hyperactive NS-causing SHP2 mutants impair the systemic production of insulin-like growth factor 1 (IGF1) through hyperactivation of the RAS/extracellular signal-regulated kinases (ERK) signalling pathway. Besides endocrine defects, a direct effect of these mutants on growth plate has not been explored, although recent studies have revealed an important physiological role for SHP2 in endochondral bone growth. We demonstrated that growth plate length was reduced in NS mice, mostly due to a shortening of the hypertrophic zone and to a lesser extent of the proliferating zone. These histological features were correlated with decreased expression of early chondrocyte differentiation markers, and with reduced alkaline phosphatase staining and activity, in NS murine primary chondrocytes. Although IGF1 treatment improved growth of NS mice, it did not fully reverse growth plate abnormalities, notably the decreased hypertrophic zone. In contrast, we documented a role of RAS/ERK hyperactivation at the growth plate level since 1) NS-causing SHP2 mutants enhance RAS/ERK activation in chondrocytes in vivo (NS mice) and in vitro (ATDC5 cells) and 2) inhibition of RAS/ERK hyperactivation by U0126 treatment alleviated growth plate abnormalities and enhanced chondrocyte differentiation. Similar effects were obtained by chronic treatment of NS mice with statins.In conclusion, we demonstrated that hyperactive NS-causing SHP2 mutants impair chondrocyte differentiation during endochondral bone growth through a local hyperactivation of the RAS/ERK signalling pathway, and that statin treatment may be a possible therapeutic approach in NS.

Vectored Intracerebral Immunization with the Anti-Tau Monoclonal Antibody PHF1 Markedly Reduces Tau Pathology in Mutant Tau Transgenic Mice.

PubMed

Liu, Wencheng; Zhao, Lingzhi; Blackman, Brittany; Parmar, Mayur; Wong, Man Ying; Woo, Thomas; Yu, Fangmin; Chiuchiolo, Maria J; Sondhi, Dolan; Kaminsky, Stephen M; Crystal, Ronald G; Paul, Steven M

2016-12-07

Passive immunization with anti-tau monoclonal antibodies has been shown by several laboratories to reduce age-dependent tau pathology and neurodegeneration in mutant tau transgenic mice. These studies have used repeated high weekly doses of various tau antibodies administered systemically for several months and have reported reduced tau pathology of ∼40-50% in various brain regions. Here we show that direct intrahippocampal administration of the adeno-associated virus (AAV)-vectored anti-phospho-tau antibody PHF1 to P301S tau transgenic mice results in high and durable antibody expression, primarily in neurons. Hippocampal antibody levels achieved after AAV delivery were ∼50-fold more than those reported following repeated systemic administration. In contrast to systemic passive immunization, we observed markedly reduced (≥80-90%) hippocampal insoluble pathological tau species and neurofibrillary tangles following a single dose of AAV-vectored PHF1 compared with mice treated with an AAV-IgG control vector. Moreover, the hippocampal atrophy observed in untreated P301S mice was fully rescued by treatment with the AAV-vectored PHF1 antibody. Vectored passive immunotherapy with an anti-tau monoclonal antibody may represent a viable therapeutic strategy for treating or preventing such tauopathies as frontotemporal dementia, progressive supranuclear palsy, or Alzheimer's disease. We have used an adeno-associated viral (AAV) vector to deliver the genes encoding an anti-phospho-tau monoclonal antibody, PHF1, directly to the brain of mice that develop neurodegeneration due to a tau mutation that causes frontotemporal dementia (FTD). When administered systemically, PHF1 has been shown to modestly reduce tau pathology and neurodegeneration. Since such antibodies do not readily cross the blood-brain barrier, we used an AAV vector to deliver antibody directly to the hippocampus and observed much higher antibody levels and a much greater reduction in tau pathology. Using

β-Myosin heavy chain variant Val606Met causes very mild hypertrophic cardiomyopathy in mice, but exacerbates HCM phenotypes in mice carrying other HCM mutations.

PubMed

Blankenburg, Robert; Hackert, Katarzyna; Wurster, Sebastian; Deenen, René; Seidman, J G; Seidman, Christine E; Lohse, Martin J; Schmitt, Joachim P

2014-07-07

Approximately 40% of hypertrophic cardiomyopathy (HCM) is caused by heterozygous missense mutations in β-cardiac myosin heavy chain (β-MHC). Associating disease phenotype with mutation is confounded by extensive background genetic and lifestyle/environmental differences between subjects even from the same family. To characterize disease caused by β-cardiac myosin heavy chain Val606Met substitution (VM) that has been identified in several HCM families with wide variation of clinical outcomes, in mice. Unlike 2 mouse lines bearing the malignant myosin mutations Arg453Cys (RC/+) or Arg719Trp (RW/+), VM/+ mice with an identical inbred genetic background lacked hallmarks of HCM such as left ventricular hypertrophy, disarray of myofibers, and interstitial fibrosis. Even homozygous VM/VM mice were indistinguishable from wild-type animals, whereas RC/RC- and RW/RW-mutant mice died within 9 days after birth. However, hypertrophic effects of the VM mutation were observed both in mice treated with cyclosporine, a known stimulator of the HCM response, and compound VM/RC heterozygous mice, which developed a severe HCM phenotype. In contrast to all heterozygous mutants, both systolic and diastolic function of VM/RC hearts was severely impaired already before the onset of cardiac remodeling. The VM mutation per se causes mild HCM-related phenotypes; however, in combination with other HCM activators it exacerbates the HCM phenotype. Double-mutant mice are suitable for assessing the severity of benign mutations. © 2014 American Heart Association, Inc.

Csf3r mutations in mice confer a strong clonal HSC advantage via activation of Stat5

PubMed Central

Liu, Fulu; Kunter, Ghada; Krem, Maxwell M.; Eades, William C.; Cain, Jennifer A.; Tomasson, Michael H.; Hennighausen, Lothar; Link, Daniel C.

2008-01-01

A fundamental property of leukemic stem cells is clonal dominance of the bone marrow microenvironment. Truncation mutations of CSF3R, which encodes the G-CSF receptor (G-CSFR), are implicated in leukemic progression in patients with severe congenital neutropenia. Here we show that expression of a truncated mutant Csf3r in mice confers a strong clonal advantage at the HSC level that is dependent upon exogenous G-CSF. G-CSF–induced proliferation, phosphorylation of Stat5, and transcription of Stat5 target genes were increased in HSCs isolated from mice expressing the mutant Csf3r. Conversely, the proliferative advantage conferred by the mutant Csf3r was abrogated in myeloid progenitors lacking both Stat5A and Stat5B, and HSC function was reduced in mice expressing a truncated mutant Csf3r engineered to have impaired Stat5 activation. These data indicate that in mice, inappropriate Stat5 activation plays a key role in establishing clonal dominance by stem cells expressing mutant Csf3r. PMID:18292815

l-tyrosine induces melanocyte differentiation in novel pink-eyed dilution castaneus mouse mutant showing age-related pigmentation.

PubMed

Hirobe, Tomohisa; Ishikawa, Akira

2015-12-01

The mouse pink-eyed dilution (oculocutaneous albinism II; p/Oca2(p)) locus is known to control tyrosinase activity, melanin content, and melanosome development in melanocytes. Pink-eyed dilution castaneus (p(cas)/Oca2(p-cas)) is a novel mutant allele on mouse chromosome 7 that arose spontaneously in Indonesian wild mice, Mus musculus castaneus. Mice homozygous for Oca2(p-cas) usually exhibit pink eyes and beige-colored coat on nonagouti C57BL/6 (B6) background. Recently, a novel spontaneous mutation occurred in the progeny between this mutant and B6 mice. The eyes of this novel mutant progressively become black from pink and the coat becomes dark gray from beige with aging. The aim of this study is to clarify whatever differences exist in melanocyte proliferation and differentiation between the ordinary (pink-eyed) and novel (black-eyed) mutant using serum-free primary culture system. The characteristics of melanocyte proliferation and differentiation were investigated by serum-free primary culture system using melanocyte-proliferation medium (MDMD). The proliferation of melanoblasts in MDMD did not differ between the two mice. However, when the epidermal cell suspensions were cultured with MDMD supplemented with l-tyrosine (Tyr), the differentiation of black-eyed melanocytes was greatly induced in a concentration-dependent manner compared with pink-eyed melanocytes. Immunocytochemistry demonstrated that the expression of tyrosinase and tyrosinase-related protein-1 (Tyrp1) was greatly induced or stimulated both in pink-eyed and black-eyed melanocytes, whereas the expression of microphthalmia-associated transcription factor (Mitf) was stimulated only in black-eyed melanocytes. These results suggest that the age-related coat darkening in black-eyed mutant may be caused by the increased ability of melanocyte differentiation dependent on l-Tyr through the upregulation of tyrosinase, Tyrp1, and Mitf. This mutant mouse may be useful for animal model to clarify the

Mutations in Prickle Orthologs Cause Seizures in Flies, Mice, and Humans

PubMed Central

Tao, Hirotaka; Manak, J. Robert; Sowers, Levi; Mei, Xue; Kiyonari, Hiroshi; Abe, Takaya; Dahdaleh, Nader S.; Yang, Tian; Wu, Shu; Chen, Shan; Fox, Mark H.; Gurnett, Christina; Montine, Thomas; Bird, Thomas; Shaffer, Lisa G.; Rosenfeld, Jill A.; McConnell, Juliann; Madan-Khetarpal, Suneeta; Berry-Kravis, Elizabeth; Griesbach, Hilary; Saneto, Russell P.; Scott, Matthew P.; Antic, Dragana; Reed, Jordan; Boland, Riley; Ehaideb, Salleh N.; El-Shanti, Hatem; Mahajan, Vinit B.; Ferguson, Polly J.; Axelrod, Jeffrey D.; Lehesjoki, Anna-Elina; Fritzsch, Bernd; Slusarski, Diane C.; Wemmie, John; Ueno, Naoto; Bassuk, Alexander G.

2011-01-01

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution. PMID:21276947

Impact of retinoic acid exposure on midfacial shape variation and manifestation of holoprosencephaly in Twsg1 mutant mice

PubMed Central

Billington, Charles J.; Schmidt, Brian; Marcucio, Ralph S.; Hallgrimsson, Benedikt; Gopalakrishnan, Rajaram; Petryk, Anna

2015-01-01

Holoprosencephaly (HPE) is a developmental anomaly characterized by inadequate or absent midline division of the embryonic forebrain and midline facial defects. It is believed that interactions between genes and the environment play a role in the widely variable penetrance and expressivity of HPE, although direct investigation of such effects has been limited. The goal of this study was to examine whether mice carrying a mutation in a gene encoding the bone morphogenetic protein (BMP) antagonist twisted gastrulation (Twsg1), which is associated with a low penetrance of HPE, are sensitized to retinoic acid (RA) teratogenesis. Pregnant Twsg1+/− dams were treated by gavage with a low dose of all-trans RA (3.75 mg/kg of body weight). Embryos were analyzed between embryonic day (E)9.5 and E11.5 by microscopy and geometric morphometric analysis by micro-computed tomography. P19 embryonal carcinoma cells were used to examine potential mechanisms mediating the combined effects of increased BMP and retinoid signaling. Although only 7% of wild-type embryos exposed to RA showed overt HPE or neural tube defects (NTDs), 100% of Twsg1−/− mutants exposed to RA manifested severe HPE compared to 17% without RA. Remarkably, up to 30% of Twsg1+/− mutants also showed HPE (23%) or NTDs (7%). The majority of shape variation among Twsg1+/− mutants was associated with narrowing of the midface. In P19 cells, RA induced the expression of Bmp2, acted in concert with BMP2 to increase p53 expression, caspase activation and oxidative stress. This study provides direct evidence for modifying effects of the environment in a genetic mouse model carrying a predisposing mutation for HPE in the Twsg1 gene. Further study of the mechanisms underlying these gene-environment interactions in vivo will contribute to better understanding of the pathogenesis of birth defects and present an opportunity to explore potential preventive interventions. PMID:25468951

Airways in smooth muscle α-actin null mice experience a compensatory mechanism that modulates their contractile response.

PubMed

Shardonofsky, Felix R; Moore, Joan; Schwartz, Robert J; Boriek, Aladin M

2012-03-01

We hypothesized that ablation of smooth muscle α-actin (SM α-A), a contractile-cytoskeletal protein expressed in airway smooth muscle (ASM) cells, abolishes ASM shortening capacity and decreases lung stiffness. In both SM α-A knockout and wild-type (WT) mice, airway resistance (Raw) determined by the forced oscillation technique rose in response to intravenous methacholine (Mch). However, the slope of Raw (cmH(2)O·ml(-1)·s) vs. log(2) Mch dose (μg·kg(-1)·min(-1)) was lower (P = 0.007) in mutant (0.54 ± 0.14) than in WT mice (1.23 ± 0.19). RT-PCR analysis performed on lung tissues confirmed that mutant mice lacked SM α-A mRNA and showed that these mice had robust expressions of both SM γ-A mRNA and skeletal muscle (SKM) α-A mRNA, which were not expressed in WT mice, and an enhanced SM22 mRNA expression relative to that in WT mice. Compared with corresponding spontaneously breathing mice, mechanical ventilation-induced lung mechanical strain increased the expression of SM α-A mRNA in WT lungs; in mutant mice, it augmented the expressions of SM γ-A mRNA and SM22 mRNA and did not alter that of SKM α-A mRNA. In mutant mice, the expression of SM γ-A mRNA in the lung during spontaneous breathing and its enhanced expression following mechanical ventilation are consistent with the likely possibility that in the absence of SM α-A, SM γ-A underwent polymerization and interacted with smooth muscle myosin to produce ASM shortening during cholinergic stimulation. Thus our data are consistent with ASM in mutant mice experiencing compensatory mechanisms that modulated its contractile muscle capacity.

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.

Overactivation of hedgehog signaling alters development of the ovarian vasculature in mice.

PubMed

Ren, Yi; Cowan, Robert G; Migone, Fernando F; Quirk, Susan M

2012-06-01

The hedgehog (HH) signaling pathway is critical for ovarian function in Drosophila, but its role in the mammalian ovary has not been defined. Previously, expression of a dominant active allele of the HH signal transducer protein smoothened (SMO) in Amhr2(cre/+)SmoM2 mice caused anovulation in association with a lack of smooth muscle in the theca of developing follicles. The current study examined events during the first 2 wk of life in Amhr2(cre/+)SmoM2 mice to gain insight into the cause of anovulation. Expression of transcriptional targets of HH signaling, Gli1, Ptch1, and Hhip, which are used as measures of pathway activity, were elevated during the first several days of life in Amhr2(cre/+)SmoM2 mice compared to controls but were similar to controls in older mice. Microarray analysis showed that genes with increased expression in 2-day-old mutants compared to controls were enriched for the processes of vascular and tube development and steroidogenesis. The density of platelet endothelial cell adhesion molecule (PECAM)-labeled endothelial tubes was increased in the cortex of newborn ovaries of mutant mice. Costaining of preovulatory follicles for PECAM and smooth muscle actin showed that muscle-type vascular support cells are deficient in theca of mutant mice. Expression of genes for steroidogenic enzymes that are normally expressed in the fetal adrenal gland were elevated in newborn ovaries of mutant mice. In summary, overactivation of HH signaling during early life alters gene expression and vascular development and this is associated with the lifelong development of anovulatory follicles in which the thecal vasculature fails to mature appropriately.

THE REELIN RECEPTORS VLDLR AND ApoER2 REGULATE SENSORIMOTOR GATING IN MICE

PubMed Central

Barr, Alasdair M.; Fish, Kenneth N.; Markou, Athina

2007-01-01

Summary Postmortem brain loss of reelin is noted in schizophrenia patients. Accordingly, heterozygous reeler mutant mice have been proposed as a putative model of this disorder. Little is known, however, about the involvement of the two receptors for reelin, Very-Low-Density Lipoprotein Receptor (VLDLR) and Apolipoprotein E Receptor 2 (ApoER2), on pre-cognitive processes of relevance to deficits seen in schizophrenia. Thus, we evaluated sensorimotor gating in mutant mice heterozygous or homozygous for the two reelin receptors. Mutant mice lacking one of these reelin receptors were tested for prepulse inhibition (PPI) of the acoustic startle reflex prior to and following puberty, and on a crossmodal PPI task, involving the presentation of acoustic and tactile stimuli. Furthermore, because schizophrenia patients show increased sensitivity to N-methyl-D-aspartate (NMDA) receptor blockade, we assessed the sensitivity of these mice to the PPI-disruptive effects of the NMDA receptor antagonist phencyclidine. The results demonstrated that acoustic PPI did not differ between mutant and wildtype mice. However, VLDLR homozygous mice displayed significant deficits in crossmodal PPI, while ApoER2 heterozygous and homozygous mice displayed significantly increased crossmodal PPI. Both ApoER2 and VLDLR heterozygous and homozygous mice exhibited greater sensitivity to the PPI-disruptive effects of phencyclidine than wildtype mice. These results indicate that partial or complete loss of either one of the reelin receptors results in a complex pattern of alterations in PPI function that include alterations in crossmodal, but not acoustic, PPI and increased sensitivity to NMDA receptor blockade. Thus, reelin receptor function appears to be critically involved in crossmodal PPI and the modulation of the PPI response by NMDA receptors. These findings have relevance to a range of neuropsychiatric disorders that involve sensorimotor gating deficits, including schizophrenia.. PMID:17261317

The effects of a skeletal muscle titin mutation on walking in mice.

PubMed

Pace, Cinnamon M; Mortimer, Sarah; Monroy, Jenna A; Nishikawa, Kiisa C

2017-01-01

Titin contributes to sarcomere assembly, muscle signaling, and mechanical properties of muscle. The mdm mouse exhibits a small deletion in the titin gene resulting in dystrophic mutants and phenotypically normal heterozygotes. We examined the effects of this mutation on locomotion to assess how, and if, changes to muscle phenotype explain observed locomotor differences. Mutant mice are much smaller in size than their siblings and gait abnormalities may be driven by differences in limb proportions and/or by changes to muscle phenotype caused by the titin mutation. We quantified differences in walking gait among mdm genotypes and also determined whether genotypes vary in limb morphometrics. Mice were filmed walking, and kinematic and morphological variables were measured. Mutant mice had a smaller range of motion at the ankle, shorter stride lengths, and shorter stance duration, but walked at the same relative speeds as the other genotypes. Although phenotypically similar to wildtype mice, heterozygous mice frequently exhibited intermediate gait mechanics. Morphological differences among genotypes in hindlimb proportions were small and do not explain the locomotor differences. We suggest that differences in locomotion among mdm genotypes are due to changes in muscle phenotype caused by the titin mutation.

Lack of tau proteins rescues neuronal cell death and decreases amyloidogenic processing of APP in APP/PS1 mice.

PubMed

Leroy, Karelle; Ando, Kunie; Laporte, Vincent; Dedecker, Robert; Suain, Valérie; Authelet, Michèle; Héraud, Céline; Pierrot, Nathalie; Yilmaz, Zehra; Octave, Jean-Noël; Brion, Jean-Pierre

2012-12-01

Lack of tau expression has been reported to protect against excitotoxicity and to prevent memory deficits in mice expressing mutant amyloid precursor protein (APP) identified in familial Alzheimer disease. In APP mice, mutant presenilin 1 (PS1) enhances generation of Aβ42 and inhibits cell survival pathways. It is unknown whether the deficient phenotype induced by concomitant expression of mutant PS1 is rescued by absence of tau. In this study, we have analyzed the effect of tau deletion in mice expressing mutant APP and PS1. Although APP/PS1/tau(+/+) mice had a reduced survival, developed spatial memory deficits at 6 months and motor impairments at 12 months, these deficits were rescued in APP/PS1/tau(-/-) mice. Neuronal loss and synaptic loss in APP/PS1/tau(+/+) mice were rescued in the APP/PS1/tau(-/-) mice. The amyloid plaque burden was decreased by roughly 50% in the cortex and the spinal cord of the APP/PS1/tau(-/-) mice. The levels of soluble and insoluble Aβ40 and Aβ42, and the Aβ42/Aβ40 ratio were reduced in APP/PS1/tau(-/-) mice. Levels of phosphorylated APP, of β-C-terminal fragments (CTFs), and of β-secretase 1 (BACE1) were also reduced, suggesting that β-secretase cleavage of APP was reduced in APP/PS1/tau(-/-) mice. Our results indicate that tau deletion had a protective effect against amyloid induced toxicity even in the presence of mutant PS1 and reduced the production of Aβ. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

PubMed Central

Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

2014-01-01

The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

Forebrain-Specific Loss of BMPRII in Mice Reduces Anxiety and Increases Object Exploration.

PubMed

McBrayer, Zofeyah L; Dimova, Jiva; Pisansky, Marc T; Sun, Mu; Beppu, Hideyuki; Gewirtz, Jonathan C; O'Connor, Michael B

2015-01-01

To investigate the role of Bone Morphogenic Protein Receptor Type II (BMPRII) in learning, memory, and exploratory behavior in mice, a tissue-specific knockout of BMPRII in the post-natal hippocampus and forebrain was generated. We found that BMPRII mutant mice had normal spatial learning and memory in the Morris water maze, but showed significantly reduced swimming speeds with increased floating behavior. Further analysis using the Porsolt Swim Test to investigate behavioral despair did not reveal any differences in immobility between mutants and controls. In the Elevated Plus Maze, BMPRII mutants and Smad4 mutants showed reduced anxiety, while in exploratory tests, BMPRII mutants showed more interest in object exploration. These results suggest that loss of BMPRII in the mouse hippocampus and forebrain does not disrupt spatial learning and memory encoding, but instead impacts exploratory and anxiety-related behaviors.

Forebrain-Specific Loss of BMPRII in Mice Reduces Anxiety and Increases Object Exploration

PubMed Central

McBrayer, Zofeyah L.; Dimova, Jiva; Pisansky, Marc T.; Sun, Mu; Beppu, Hideyuki; Gewirtz, Jonathan C.; O’Connor, Michael B.

2015-01-01

To investigate the role of Bone Morphogenic Protein Receptor Type II (BMPRII) in learning, memory, and exploratory behavior in mice, a tissue-specific knockout of BMPRII in the post-natal hippocampus and forebrain was generated. We found that BMPRII mutant mice had normal spatial learning and memory in the Morris water maze, but showed significantly reduced swimming speeds with increased floating behavior. Further analysis using the Porsolt Swim Test to investigate behavioral despair did not reveal any differences in immobility between mutants and controls. In the Elevated Plus Maze, BMPRII mutants and Smad4 mutants showed reduced anxiety, while in exploratory tests, BMPRII mutants showed more interest in object exploration. These results suggest that loss of BMPRII in the mouse hippocampus and forebrain does not disrupt spatial learning and memory encoding, but instead impacts exploratory and anxiety-related behaviors. PMID:26444546

Fibroblast growth factor receptor two (FGFR2) regulates uterine epithelial integrity and fertility in mice.

PubMed

Filant, Justyna; DeMayo, Franco J; Pru, James K; Lydon, John P; Spencer, Thomas E

2014-01-01

Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate luminal epithelial (LE) cell proliferation in the adult mouse uterus. This study tested the hypothesis that FGFR2 has a biological role in postnatal development and function of the uterus by conditionally deleting Fgfr2 after birth using progesterone receptor (Pgr)-Cre mice. Adult Fgfr2 mutant female mice were initially subfertile and became infertile with increasing parity. No defects in uterine gland development were observed in conditional Fgfr2 mutant mice. In the adult, Fgfr2 mutant mice possessed a histologically normal reproductive tract with the exception of the uterus. The LE of the Fgfr2 mutant uterus was stratified, but no obvious histological differences were observed in the glandular epithelium, stroma, or myometrium. Within the stratified LE, cuboidal basal cells were present and positive for basal cell markers (KRT14 and TRP63). Nulliparous bred Fgfr2 mutants contained normal numbers of blastocysts on Day 3.5 postmating, but the number of embryo implantation sites was substantially reduced on Day 5.5 postmating. These results support the idea that loss of FGFR2 in the uterus after birth alters its development, resulting in LE stratification and peri-implantation pregnancy loss.

Impairment of autophagosome-lysosome fusion in the buff mutant mice with the VPS33AD251E mutation

PubMed Central

Zhen, Yuanli; Li, Wei

2015-01-01

The HOPS (homotypic fusion and protein sorting) complex functions in endocytic and autophagic pathways in both lower eukaryotes and mammalian cells through its involvement in fusion events between endosomes and lysosomes or autophagosomes and lysosomes. However, the differential molecular mechanisms underlying these fusion processes are largely unknown. Buff (bf) is a mouse mutant that carries an Asp251-to-Glu point mutation (D251E) in the VPS33A protein, a tethering protein and a core subunit of the HOPS complex. Bf mice showed impaired spontaneous locomotor activity, motor learning, and autophagic activity. Although the gross anatomy of the brain was apparently normal, the number of Purkinje cells was significantly reduced. Furthermore, we found that fusion between autophagosomes and lysosomes was defective in bf cells without compromising the endocytic pathway. The direct association of mutant VPS33AD251E with the autophagic SNARE complex, STX17 (syntaxin 17)-VAMP8-SNAP29, was enhanced. In addition, the VPS33AD251E mutation enhanced interactions with other HOPS subunits, namely VPS41, VPS39, VPS18, and VPS11, except for VPS16. Reduction of the interactions between VPS33AY440D and several other HOPS subunits led to decreased association with STX17. These results suggest that the VPS33AD251E mutation plays dual roles by increasing the HOPS complex assembly and its association with the autophagic SNARE complex, which selectively affects the autophagosome-lysosome fusion that impairs basal autophagic activity and induces Purkinje cell loss. PMID:26259518

A 1-bp deletion in the gammaC-crystallin leads to dominant cataracts in mice.

PubMed

Zhao, Liya; Li, Kai; Bao, Shimin; Zhou, Yuxun; Liang, Yinming; Zhao, Guoji; Chen, Ye; Xiao, Junhua

2010-08-01

To date around 140 genetic alleles have been identified as being responsible for mouse cataract pathology, including Crya, Cryb, Cryg, Maf, Pax6, Pitx3, Sox, Connexins, MIP, and Lim-2. We obtained a dominant cataract mouse model from a spontaneous mutation in the F1 hybrids of outbred strain ICR mice crossed to the inbred strain BALB/cJ mice. Heterozygous and homozygous mutants expressed a nuclear cataract in both eyes. In 8-day-old mice, histological analysis showed that polygon epithelial cells were in the equatorial region and cortex underneath, and vacuole and sponge-like degeneration were in the cortical area underneath the posterior lens capsule. The nucleus of the lens was a deeply stained pink, with the shorter fibers losing their normal arrangement. For the entire eye, there was a blank zone in the equatorial region in 8-day-old mice; however, there was a certain degree of atrophy in cornea tension and retina in the lens in 3-month-old mice. The lens had been serious damaged in the homozygous mutants. For mutation mapping, heterozygous carriers were mated to wild-type C3H/HeJ mice, and offspring (F1 generation) with cataracts were backcrossed to the wild-type C3H/HeJ mice again. N2 mice with cataracts were used for genotyping. Using genome-wide linkage analysis, the mutation was mapped to chromosome 1 and the Cryg gene cluster between two markers was confirmed as the candidate gene. After direct sequencing the cDNA of the Cryg gene cluster, a 1-bp deletion was found in exon 3 of the Crygc gene, leading to a stop codon at the 76th amino acid of exon 3 which results in production of a truncated protein in mutant mice (Leu160Stop). Bioinformatic analysis of the mutant gammaC-crystallin reveals that the COOH-terminal of the mutant protein deletes a beta-sheet, which affects the function of the lens proteins and leads to the development of cataracts.

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

Overactivation of Hedgehog Signaling Alters Development of the Ovarian Vasculature in Mice1

PubMed Central

Ren, Yi; Cowan, Robert G.; Migone, Fernando F.; Quirk, Susan M.

2012-01-01

ABSTRACT The hedgehog (HH) signaling pathway is critical for ovarian function in Drosophila, but its role in the mammalian ovary has not been defined. Previously, expression of a dominant active allele of the HH signal transducer protein smoothened (SMO) in Amhr2cre/+SmoM2 mice caused anovulation in association with a lack of smooth muscle in the theca of developing follicles. The current study examined events during the first 2 wk of life in Amhr2cre/+SmoM2 mice to gain insight into the cause of anovulation. Expression of transcriptional targets of HH signaling, Gli1, Ptch1, and Hhip, which are used as measures of pathway activity, were elevated during the first several days of life in Amhr2cre/+SmoM2 mice compared to controls but were similar to controls in older mice. Microarray analysis showed that genes with increased expression in 2-day-old mutants compared to controls were enriched for the processes of vascular and tube development and steroidogenesis. The density of platelet endothelial cell adhesion molecule (PECAM)-labeled endothelial tubes was increased in the cortex of newborn ovaries of mutant mice. Costaining of preovulatory follicles for PECAM and smooth muscle actin showed that muscle-type vascular support cells are deficient in theca of mutant mice. Expression of genes for steroidogenic enzymes that are normally expressed in the fetal adrenal gland were elevated in newborn ovaries of mutant mice. In summary, overactivation of HH signaling during early life alters gene expression and vascular development and this is associated with the lifelong development of anovulatory follicles in which the thecal vasculature fails to mature appropriately. PMID:22402963

Persistent increase of D-aspartate in D-aspartate oxidase mutant mice induces a precocious hippocampal age-dependent synaptic plasticity and spatial memory decay.

PubMed

Errico, Francesco; Nisticò, Robert; Napolitano, Francesco; Oliva, Alessandra Bonito; Romano, Rosaria; Barbieri, Federica; Florio, Tullio; Russo, Claudio; Mercuri, Nicola B; Usiello, Alessandro

2011-11-01

The atypical amino acid d-aspartate (d-Asp) occurs at considerable amounts in the developing brain of mammals. However, during postnatal life, d-Asp levels diminish following the expression of d-aspartate oxidase (DDO) enzyme. The strict control of DDO over its substrate d-Asp is particularly evident in the hippocampus, a brain region crucially involved in memory, and highly vulnerable to age-related deterioration processes. Herein, we explored the influence of deregulated higher d-Asp brain content on hippocampus-related functions during aging of mice lacking DDO (Ddo(-/-)). Strikingly, we demonstrated that the enhancement of hippocampal synaptic plasticity and cognition in 4/5-month-old Ddo(-/-) mice is followed by an accelerated decay of basal glutamatergic transmission, NMDAR-dependent LTP and hippocampus-related reference memory at 13/14 months of age. Therefore, the precocious deterioration of hippocampal functions observed in mutants highlights for the first time a role for DDO enzyme in controlling the rate of brain aging process in mammals. Copyright © 2009 Elsevier Inc. All rights reserved.

Postnatal Changes in K+/Cl- Cotransporter-2 Expression in the Forebrain of Mice Bearing a Mutant Nicotinic Subunit Linked to Sleep-Related Epilepsy.

PubMed

Amadeo, Alida; Coatti, Aurora; Aracri, Patrizia; Ascagni, Miriam; Iannantuoni, Davide; Modena, Debora; Carraresi, Laura; Brusco, Simone; Meneghini, Simone; Arcangeli, Annarosa; Pasini, Maria Enrica; Becchetti, Andrea

2018-06-24

The Na + /K + /Cl - cotransporter-1 (NKCC1) and the K + /Cl - cotransporter-2 (KCC2) set the transmembrane Cl - gradient in the brain, and are implicated in epileptogenesis. We studied the postnatal distribution of NKCC1 and KCC2 in wild-type (WT) mice, and in a mouse model of sleep-related epilepsy, carrying the mutant β2-V287L subunit of the nicotinic acetylcholine receptor (nAChR). In WT neocortex, immunohistochemistry showed a wide distribution of NKCC1 in neurons and astrocytes. At birth, KCC2 was localized in neuronal somata, whereas at subsequent stages it was mainly found in the somatodendritic compartment. The cotransporters' expression was quantified by densitometry in the transgenic strain. KCC2 expression increased during the first postnatal weeks, while the NKCC1 amount remained stable, after birth. In mice expressing β2-V287L, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8), with no concomitant change in NKCC1. Consistently, the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying β2-V287L. At P60, the amount of KCC2 was instead higher in mice bearing the transgene. Irrespective of genotype, NKCC1 and KCC2 were abundantly expressed in the neuropil of most thalamic nuclei since birth. However, KCC2 expression decreased by P60 in the reticular nucleus, and more so in mice expressing β2-V287L. Therefore, a complex regulatory interplay occurs between heteromeric nAChRs and KCC2 in postnatal forebrain. The pathogenetic effect of β2-V287L may depend on altered KCC2 amounts in PFC during synaptogenesis, as well as in mature thalamocortical circuits. Copyright © 2018. Published by Elsevier Ltd.

Milk composition and lactation of beta-casein-deficient mice.

PubMed Central

Kumar, S; Clarke, A R; Hooper, M L; Horne, D S; Law, A J; Leaver, J; Springbett, A; Stevenson, E; Simons, J P

1994-01-01

beta-Casein is a major protein component of milk and, in conjunction with the other caseins, it is assembled into micelles. The casein micelles determine many of the physical characteristics of milk, which are important for stability during storage and for milk-processing properties. There is evidence that suggests that beta-casein may also possess other, nonnutritional functions. To address the function of beta-casein, the mouse beta-casein gene was disrupted by gene targeting in embryonic stem cells. Homozygous beta-casein mutant mice are viable and fertile; females can lactate and successfully rear young. beta-Casein was expressed at a reduced level in heterozygotes and was completely absent from the milk of homozygous mutant mice. Despite the deficiency of beta-casein, casein micelles were assembled in heterozygous and homozygous mutants, albeit with reduced diameters. The absence of beta-casein expression was reflected in a reduced total protein concentration in milk, although this was partially compensated for by an increased concentration of other proteins. The growth of pups feeding on the milk of homozygous mutants was reduced relative to those feeding on the milk of wild-type mice. Various genetic manipulations of caseins have been proposed for the qualitative improvement of cow's milk composition. The results presented here demonstrate that beta-casein has no essential function and that the casein micelle is remarkably tolerant of changes in composition. Images PMID:8016126

Mice expressing a “hyper-sensitive” form of the CB1 cannabinoid receptor (CB1) show modestly enhanced alcohol preference and consumption

PubMed Central

Gonek, Maciej; Zee, Michael L.; Farnsworth, Jill C.; Amin, Randa A.; Andrews, Mary-Jeanette; Davis, Brian J.; Mackie, Ken; Morgan, Daniel J.

2017-01-01

We recently characterized S426A/S430A mutant mice expressing a desensitization-resistant form of the CB1 receptor. These mice display an enhanced response to endocannabinoids and ∆9-THC. In this study, S426A/S430A mutants were used as a novel model to test whether ethanol consumption, morphine dependence, and reward for these drugs are potentiated in mice with a “hyper-sensitive” form of CB1. Using an unlimited-access, two-bottle choice, voluntary drinking paradigm, S426A/S430A mutants exhibit modestly increased intake and preference for low (6%) but not higher concentrations of ethanol. S426A/S430A mutants and wild-type mice show similar taste preference for sucrose and quinine, exhibit normal sensitivity to the hypothermic and ataxic effects of ethanol, and have normal blood ethanol concentrations following administration of ethanol. S426A/S430A mutants develop robust conditioned place preference for ethanol (2 g/kg), morphine (10 mg/kg), and cocaine (10 mg/kg), demonstrating that drug reward is not changed in S426A/S430A mutants. Precipitated morphine withdrawal is also unchanged in opioid-dependent S426A/S430A mutant mice. Although ethanol consumption is modestly changed by enhanced CB1 signaling, reward, tolerance, and acute sensitivity to ethanol and morphine are normal in this model. PMID:28426670

Pathogenicty and immune prophylaxis of cag pathogenicity island gene knockout homogenic mutants

PubMed Central

Lin, Huan-Jian; Xue, Jing; Bai, Yang; Wang, Ji-De; Zhang, Ya-Li; Zhou, Dian-Yuan

2004-01-01

AIM: To clarify the role of cag pathogenicity island (cagPAI) of Helicobacter pylori (H pylori) in the pathogenicity and immune prophylaxis of H pylori infection. METHODS: Three pairs of H pylori including 3 strains of cagPAI positive wildtype bacteria and their cagPAI knockout homogenic mutants were utilized. H pylori binding to the gastric epithelial cells was analyzed by flow cytometry assays. Apoptosis of gastric epithelial cells induced by H pylori was determined by ELISA assay. Prophylaxis effect of the wildtype and mutant strains was compared by immunization with the sonicate of the bacteria into mice model. RESULTS: No difference was found in the apoptasis between cagPAI positive and knockout H pylori strains in respective of the ability in the binding to gastric epithelial cells as well as the induction of apoptosis. Both types of the bacteria were able to protect the mice from the infection of H pylori after immunization, with no difference between them regarding to the protection rate as well as the stimulation of the proliferation of splenocytes of the mice. CONCLUSION: The role of cagPAI in the pathogenicity and prophylaxis of H pylori infection remains to be cleared. PMID:15484302

Myostatin gene mutated mice induced with tale nucleases.

PubMed

Zhou, Fangfang; Sun, Ruilin; Chen, Hongyan; Fei, Jian; Lu, Daru

2015-01-01

Myostain gene (MSTN) is expressed primarily in skeletal muscle, and negatively regulates skeletal muscle mass; it has been suggested that mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. Therefore, it is important to establish a fast and effective gene editing method. In this report, we established the myostatin mutated-mouse model by microinjection of Transcription Activator-Like Effector Nucleases (TALENs) mRNA within the mouse fertilized oocytes and achieved high rates of mutagenesis of the mouse MSTN in C57BL/6J. Six of 45 born mice carried target mutations and we appointed one as the parental mating with wild mouse to produce the F1 and backcross to produce the F2 generation. All the mutations of the mice were examined quickly and efficiently by high-resolution melting curve analysis (HRMA) and then verified by direct sequencing. We obtained the homozygous of the F2 generation which transmitted the mutant alleles to the progeny with 100% efficiency. Mutant mice exhibited increases in muscle mass comparable to those observed in wild-type mice. Therefore, combining TALEN-mediated gene targeting with HRMA technology is a superior method of constructing genetically modified mice through microinjection in the mouse fertilized oocytes with high efficiency and short time of selection.

Multiple Renal Cyst Development but Not Situs Abnormalities in Transgenic RNAi Mice against Inv::GFP Rescue Gene

PubMed Central

Kamijho, Yuki; Shiozaki, Yayoi; Sakurai, Eiki; Hanaoka, Kazunori; Watanabe, Daisuke

2014-01-01

In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments. PMID:24586938

The Ovary Is an Alternative Site of Origin for High-Grade Serous Ovarian Cancer in Mice

PubMed Central

Coffey, Donna M.; Ma, Lang; Matzuk, Martin M.

2015-01-01

Although named “ovarian cancer,” it has been unclear whether the cancer actually arises from the ovary, especially for high-grade serous carcinoma (HGSC), also known as high-grade serous ovarian cancer, the most common and deadliest ovarian cancer. In addition, the tumor suppressor p53 is the most frequently mutated gene in HGSC. However, whether mutated p53 can cause HGSC remains unknown. In this study, we bred a p53 mutation, p53R172H, into conditional Dicer-Pten double-knockout (DKO) mice, a mouse model duplicating human HGSC, to generate triple-mutant (TKO) mice. Like DKO mice, these TKO mice develop metastatic HGSCs originating from the fallopian tube. Unlike DKO mice, however, even after fallopian tubes are removed in TKO mice, ovaries alone can develop metastatic HGSCs, indicating that a p53 mutation can drive HGSC arising from the ovary. To confirm this, we generated p53R172H-Pten double-mutant mice, one of the genetic control lines for TKO mice. As anticipated, these double-mutant mice also develop metastatic HGSCs from the ovary, verifying the HGSC-forming ability of ovaries with a p53 mutation. Our study therefore shows that ovaries harboring a p53 mutation, as well as fallopian tubes, can be a distinct tissue source of high-grade serous ovarian cancer in mice. PMID:25815421

The ovary is an alternative site of origin for high-grade serous ovarian cancer in mice.

PubMed

Kim, Jaeyeon; Coffey, Donna M; Ma, Lang; Matzuk, Martin M

2015-06-01

Although named "ovarian cancer," it has been unclear whether the cancer actually arises from the ovary, especially for high-grade serous carcinoma (HGSC), also known as high-grade serous ovarian cancer, the most common and deadliest ovarian cancer. In addition, the tumor suppressor p53 is the most frequently mutated gene in HGSC. However, whether mutated p53 can cause HGSC remains unknown. In this study, we bred a p53 mutation, p53(R172H), into conditional Dicer-Pten double-knockout (DKO) mice, a mouse model duplicating human HGSC, to generate triple-mutant (TKO) mice. Like DKO mice, these TKO mice develop metastatic HGSCs originating from the fallopian tube. Unlike DKO mice, however, even after fallopian tubes are removed in TKO mice, ovaries alone can develop metastatic HGSCs, indicating that a p53 mutation can drive HGSC arising from the ovary. To confirm this, we generated p53(R172H)-Pten double-mutant mice, one of the genetic control lines for TKO mice. As anticipated, these double-mutant mice also develop metastatic HGSCs from the ovary, verifying the HGSC-forming ability of ovaries with a p53 mutation. Our study therefore shows that ovaries harboring a p53 mutation, as well as fallopian tubes, can be a distinct tissue source of high-grade serous ovarian cancer in mice.

A Dominant Loss-of-Function GJA1 (Cx43) Mutant Impairs Parturition in the Mouse1

PubMed Central

Tong, Dan; Lu, Xuerong; Wang, Hong-Xing; Plante, Isabelle; Lui, Ed; Laird, Dale W.; Bai, Donglin; Kidder, Gerald M.

2009-01-01

Expression of GJA1 (commonly known as connexin43 or Cx43), a major myometrial gap junction protein, is upregulated before the onset of delivery, suggesting an essential role for Cx43-mediated gap junctional intercellular communication (GJIC) in normal uterine contraction during parturition. To determine how a disease-linked Cx43 mutation affects myometrial function, we studied a mutant mouse model carrying an autosomal dominant mutation (Gja1Jrt) in the gene encoding Cx43 that displays features of the human genetic disease oculodentodigital dysplasia. We found that Cx43 level, specifically the phosphorylated species of the protein, is significantly reduced in the myometrium of the mutant mice (Gja1Jrt/+), as revealed by Western blotting and immunostaining. Patch-clamp electrophysiological measurements demonstrated that coupling between myometrial smooth muscle cells is reduced to <15% of wild-type, indicating that the mutant protein acts dominantly on its wild-type counterpart. The phosphorylated species of Cx43 in the mutant myometrium failed to increase prior to parturition as well as in response to exogenous estrogen. Correspondingly, in vitro experiments with uterine strips revealed weaker contraction of the mutant myometrium and reduced responsiveness to oxytocin, providing an explanation for the prolonged gestation and presence of suffocated fetuses in the uteri that were observed in some of the mutant mice. We conclude that the Gja1Jrt mutation has a dominant-negative effect on Cx43 function in the myometrium, severely reducing GJIC, leading to impaired parturition. PMID:19176884

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.

Salmonella enterica serovar Typhimurium mutants unable to convert malate to pyruvate and oxaloacetate are avirulent and immunogenic in BALB/c mice.

PubMed

Mercado-Lubo, Regino; Leatham, Mary P; Conway, Tyrrell; Cohen, Paul S

2009-04-01

Previously, we showed that the Salmonella enterica serovar Typhimurium SR-11 tricarboxylic acid (TCA) cycle must operate as a complete cycle for full virulence after oral infection of BALB/c mice (M. Tchawa Yimga, M. P. Leatham, J. H. Allen, D. C. Laux, T. Conway, and P. S. Cohen, Infect. Immun. 74:1130-1140, 2006). In the same study, we showed that for full virulence, malate must be converted to both oxaloacetate and pyruvate. Moreover, it was recently demonstrated that blocking conversion of succinyl-coenzyme A to succinate attenuates serovar Typhimurium SR-11 but does not make it avirulent; however, blocking conversion of succinate to fumarate renders it completely avirulent and protective against subsequent oral infection with the virulent serovar Typhimurium SR-11 wild-type strain (R. Mercado-Lubo, E. J. Gauger, M. P. Leatham, T. Conway, and P. S. Cohen, Infect. Immun. 76:1128-1134, 2008). Furthermore, the ability to convert succinate to fumarate appeared to be required only after serovar Typhimurium SR-11 became systemic. In the present study, evidence is presented that serovar Typhimurium SR-11 mutants that cannot convert fumarate to malate or that cannot convert malate to both oxaloacetate and pyruvate are also avirulent and protective in BALB/c mice. These results suggest that in BALB/c mice, the malate that is removed from the TCA cycle in serovar Typhimurium SR-11 for conversion to pyruvate must be replenished by succinate or one of its precursors, e.g., arginine or ornithine, which might be available in mouse phagocytes.

Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice.

PubMed

Tsai, Peter T; Hull, Court; Chu, YunXiang; Greene-Colozzi, Emily; Sadowski, Abbey R; Leech, Jarrett M; Steinberg, Jason; Crawley, Jacqueline N; Regehr, Wade G; Sahin, Mustafa

2012-08-30

Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders, but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. However, the extent of cerebellar contribution to the pathogenesis of ASDs remains unclear. Tuberous sclerosis complex (TSC) is a genetic disorder with high rates of comorbid ASDs that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. TSC is an intriguing model to investigate the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies in TSC patients demonstrate cerebellar pathology and correlate cerebellar pathology with increased ASD symptomatology. Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs. However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.

Electroacupuncture remediates glial dysfunction and ameliorates neurodegeneration in the astrocytic α-synuclein mutant mouse model.

PubMed

Deng, Jiahui; Lv, E; Yang, Jian; Gong, Xiaoli; Zhang, Wenzhong; Liang, Xibin; Wang, Jiazeng; Jia, Jun; Wang, Xiaomin

2015-05-28

The acupuncture or electroacupuncture (EA) shows the therapeutic effect on various neurodegenerative diseases. This effect was thought to be partially achieved by its ability to alleviate existing neuroinflammation and glial dysfunction. In this study, we systematically investigated the effect of EA on abnormal neurochemical changes and motor symptoms in a mouse neurodegenerative disease model. The transgenic mouse which expresses a mutant α-synuclein (α-syn) protein, A53T α-syn, in brain astrocytic cells was used. These mice exhibit extensive neuroinflammatory and motor phenotypes of neurodegenerative disorders. In this study, the effects of EA on these phenotypic changes were examined in these mice. EA improved the movement detected in multiple motor tests in A53T mutant mice. At the cellular level, EA significantly reduced the activation of microglia and prevented the loss of dopaminergic neurons in the midbrain and motor neurons in the spinal cord. At the molecular level, EA suppressed the abnormal elevation of proinflammatory factors (tumor necrosis factor-α and interleukin-1β) in the striatum and midbrain of A53T mice. In contrast, EA increased striatal and midbrain expression of a transcription factor, nuclear factor E2-related factor 2, and its downstream antioxidants (heme oxygenase-1 and glutamate-cysteine ligase modifier subunits). These results suggest that EA possesses the ability to ameliorate mutant α-syn-induced motor abnormalities. This ability may be due to that EA enhances both anti-inflammatory and antioxidant activities and suppresses aberrant glial activation in the diseased sites of brains.

Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity

PubMed Central

Sun, Xin; Marque, Leonard O.; Cordner, Zachary; Pruitt, Jennifer L.; Bhat, Manik; Li, Pan P.; Kannan, Geetha; Ladenheim, Ellen E.; Moran, Timothy H.; Margolis, Russell L.; Rudnicki, Dobrila D.

2014-01-01

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT. Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been of particular interest due to their potential capacity to discriminate between normal and mutant HTT transcripts. Here, we focus on phosphorodiamidate morpholino oligomers (PMOs), ASOs that are especially stable, highly soluble and non-toxic. We designed three PMOs to selectively target expanded CAG repeat tracts (CTG22, CTG25 and CTG28), and two PMOs to selectively target sequences flanking the HTT CAG repeat (HTTex1a and HTTex1b). In HD patient–derived fibroblasts with expanded alleles containing 44, 77 or 109 CAG repeats, HTTex1a and HTTex1b were effective in suppressing the expression of mutant and non-mutant transcripts. CTGn PMOs also suppressed HTT expression, with the extent of suppression and the specificity for mutant transcripts dependent on the length of the targeted CAG repeat and on the CTG repeat length and concentration of the PMO. PMO CTG25 reduced HTT-induced cytotoxicity in vitro and suppressed mutant HTT expression in vivo in the N171-82Q transgenic mouse model. Finally, CTG28 reduced mutant HTT expression and improved the phenotype of HdhQ7/Q150 knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT. PMID:25035419

Immunological Development and Cardiovascular Function Are Normal in Annexin VI Null Mutant Mice

PubMed Central

Hawkins, Tim E.; Roes, Jürgen; Rees, Daryl; Monkhouse, Jayne; Moss, Stephen E.

1999-01-01

Annexins are calcium-binding proteins of unknown function but which are implicated in important cellular processes, including anticoagulation, ion flux regulation, calcium homeostasis, and endocytosis. To gain insight into the function of annexin VI, we performed targeted disruption of its gene in mice. Matings between heterozygous mice produced offspring with a normal Mendelian pattern of inheritance, indicating that the loss of annexin VI did not interfere with viability in utero. Mice lacking annexin VI reached sexual maturity at the same age as their normal littermates, and both males and females were fertile. Because of interest in the role of annexin VI in cardiovascular function, we examined heart rate and blood pressure in knockout and wild-type mice and found these to be identical in the two groups. Similarly, the cardiovascular responses of both sets of mice to septic shock were indistinguishable. We also examined components of the immune system and found no differences in thymic, splenic, or bone marrow lymphocyte levels between knockout and wild-type mice. This is the first study of annexin knockout mice, and the lack of a clear phenotype has broad implications for current views of annexin function. PMID:10567528

Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant

PubMed Central

Song, Xiufeng; Seo, Jungwon; Baameur, Faiza; Vishnivetskiy, Sergey A.; Chen, Qiuyan; Kook, Seunghyi; Kim, Miyeon; Brooks, Evan K.; Altenbach, Christian; Hong, Yuan; Hanson, Susan M.; Palazzo, Maria C.; Chen, Jeannie; Hubbell, Wayne L.; Gurevich, Eugenia V.; Gurevich, Vsevolod V.

2013-01-01

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization. PMID:24012956

Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant.

PubMed

Song, Xiufeng; Seo, Jungwon; Baameur, Faiza; Vishnivetskiy, Sergey A; Chen, Qiuyan; Kook, Seunghyi; Kim, Miyeon; Brooks, Evan K; Altenbach, Christian; Hong, Yuan; Hanson, Susan M; Palazzo, Maria C; Chen, Jeannie; Hubbell, Wayne L; Gurevich, Eugenia V; Gurevich, Vsevolod V

2013-12-01

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization. © 2013.

K-RasV14I recapitulates Noonan syndrome in mice

PubMed Central

Hernández-Porras, Isabel; Fabbiano, Salvatore; Schuhmacher, Alberto J.; Aicher, Alexandra; Cañamero, Marta; Cámara, Juan Antonio; Cussó, Lorena; Desco, Manuel; Heeschen, Christopher; Mulero, Francisca; Bustelo, Xosé R.; Guerra, Carmen; Barbacid, Mariano

2014-01-01

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-RasV14I, a recurrent KRAS mutation in NS patients. K-RasV14I–mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-RasV14I–mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome. PMID:25359213

The targeted overexpression of a Claudin mutant in the epidermis of transgenic mice elicits striking epidermal and hair follicle abnormalities.

PubMed

Troy, Tammy-Claire; Turksen, Kursad

2007-06-01

Skin is one of the largest organs of the body, and is formed during development through a highly orchestrated process involving mesenchymal-epithelial interactions, cell commitment, and terminal differentiation. It protects against microorganism invasion and UV irradiation, inhibits water loss, regulates body temperature, and is an important part of the immune system. Using transgenic mouse technology, we have demonstrated that Claudin (Cldn)-containing tight junctions (TJs) are intricately involved in cell signaling during epidermal differentiation and that an epidermal suprabasal overexpression of Cldn6 results in a perturbed epidermal terminal differentiation program with distinct phenotypic abnormalities. To delineate the role of the Cldn cytoplasmic tail domain in epidermal differentiation, we engineered transgenic mice targeting the overexpression of a Cldn6 cytoplasmic tail-truncation mutant in the epidermis. Transgenic mice were characterized by a lethal barrier dysfunction in addition to the existence of hyperproliferative squamous invaginations/cysts replacing hair follicles. Immunohistochemical analysis revealed an epidermal cytoplasmic accumulation of Cldn6, Cldn11, Cldn12, and Cldn18, downregulation of Cldn1 and aberrant expression of various classical markers of epidermal differentiation; namely the basal keratins as well as K1, involucrin, loricrin, and filaggrin. Collectively these studies suggest an important role for Cldns in epidermal/hair follicle differentiation programs likely involving cross talk to signaling pathways (e.g., Notch) directing cell fate selection and differentiation.

Defeat mutant KRAS with synthetic lethality

PubMed Central

Pang, Xiufeng; Liu, Mingyao

2017-01-01

ABSTRACT Ras proteins are considered as the founding members of a large superfamily of small GTPases that control fundamental cellular functions. Mutationally activated RAS genes were discovered in human cancer cells more than 3 decades ago, but intensive efforts on Ras structure, biochemistry, function and signaling continue even now. Because mutant Ras proteins are inherently difficult to inhibit and have yet been therapeutically conquered, it was designated as “the Everest of oncogenes” in the cancer genome landscape, further promoting a “renaissance” in RAS research. Different paths to directly or indirectly targeting mutant Ras signaling are currently under investigation in the hope of finding an efficacious regimen. Inhibitors directly binding to KRASG12C to block its downstream signaling have been revealed, supporting the notion of Ras' druggability. An alternative indirect approach by targeting synthetic lethal interactors of mutant RAS is underway. We recently employed a synthetic lethal drug screen plus a combinatorial strategy using a panel of clinical agents and discovered that KRAS-mutant cancers were fragile to the combined inhibition of polo-like kinase 1 (Plk1) and RhoA/Rho kinase (ROCK). The combined regimen of BI-2536 (a Plk1 inhibitor) and fasudil (a ROCK inhibitor) promoted a significant inhibition of patient-derived lung cancer xenografts and prolonged the survival of LSL-KRASG12D mice. In this commentary, we will summarize the state-of-the art for the direction of synthetic lethality, and also speculate on the future development of this approach. PMID:27463838

A noninhibitory mutant of the caveolin-1 scaffolding domain enhances eNOS-derived NO synthesis and vasodilation in mice

PubMed Central

Bernatchez, Pascal; Sharma, Arpeeta; Bauer, Philip M.; Marin, Ethan; Sessa, William C.

2011-01-01

Aberrant regulation of eNOS and associated NO release are directly linked with various vascular diseases. Caveolin-1 (Cav-1), the main coat protein of caveolae, is highly expressed in endothelial cells. Its scaffolding domain serves as an endogenous negative regulator of eNOS function. Structure-function analysis of Cav-1 has shown that phenylalanine 92 (F92) is critical for the inhibitory actions of Cav-1 toward eNOS. Herein, we show that F92A–Cav-1 and a mutant cell–permeable scaffolding domain peptide called Cavnoxin can increase basal NO release in eNOS-expressing cells. Cavnoxin reduced vascular tone ex vivo and lowered blood pressure in normal mice. In contrast, similar experiments performed with eNOS- or Cav-1–deficient mice showed that the vasodilatory effect of Cavnoxin is abolished in the absence of these gene products, which indicates a high level of eNOS/Cav-1 specificity. Mechanistically, biochemical assays indicated that noninhibitory F92A–Cav-1 and Cavnoxin specifically disrupted the inhibitory actions of endogenous Cav-1 toward eNOS and thereby enhanced basal NO release. Collectively, these data raise the possibility of studying the inhibitory influence of Cav-1 on eNOS without interfering with the other actions of endogenous Cav-1. They also suggest a therapeutic application for regulating the eNOS/Cav-1 interaction in diseases characterized by decreased NO release. PMID:21804187

Enhanced radiosensitization of p53 mutant cells by oleamide.

PubMed

Lee, Yoon-Jin; Chung, Da Yeon; Lee, Su-Jae; Ja Jhon, Gil; Lee, Yun-Sil

2006-04-01

Effect of oleamide, an endogenous fatty-acid primary amide, on tumor cells exposed to ionizing radiation (IR) has never before been explored. NCI H460, human lung cancer cells, and human astrocytoma cell lines, U87 and U251, were used. The cytotoxicity of oleamide alone or in combination with IR was determined by clonogenic survival assay, and induction of apoptosis was estimated by FACS analysis. Protein expressions were confirmed by Western blotting, and immunofluorescence analysis of Bax by use of confocal microscopy was also performed. The combined effect of IR and oleamide to suppress tumor growth was studied by use of xenografts in the thighs of nude mice. Oleamide in combination with IR had a synergistic effect that decreased clonogenic survival of lung-carcinoma cell lines and also sensitized xenografts in nude mice. Enhanced induction of apoptosis of the cells by the combined treatment was mediated by loss of mitochondrial membrane potential, which resulted in the activation of caspase-8, caspase-9, and caspase-3 accompanied by cytochrome c release and Bid cleavage. The synergistic effects of the combined treatment were more enhanced in p53 mutant cells than in p53 wild-type cells. In p53 wild-type cells, both oleamide and radiation induced Bax translocation to mitochondria. On the other hand, in p53 mutant cells, radiation alone slightly induced Bax translocation to mitochondria, whereas oleamide induced a larger translocation. Oleamide may exhibit synergistic radiosensitization in p53 mutant cells through p53-independent Bax translocation to mitochondria.

A targeted deletion/insertion in the mouse Pcsk1 locus is associated with homozygous embryo preimplantation lethality, mutant allele preferential transmission and heterozygous female susceptibility to dietary fat.

PubMed

Mbikay, Majambu; Croissandeau, Gilles; Sirois, Francine; Anini, Younes; Mayne, Janice; Seidah, Nabil G; Chrétien, Michel

2007-06-15

Proprotein convertase 1 (PC1) is a neuroendocrine proteinase involved in the proteolytic activation of precursors to hormones and neuropeptides. To determine the physiological importance of PC1, we produced a mutant mouse from embryonic stem cells in which its locus (Pcsk1) had been inactivated by homologous recombination. The inactivating mutation consisted of a 32.7-kb internal deletion and a 1.8 kb insertion of the bacterial neomycin resistance gene (neo) under the mouse phosphoglycerate kinase 1 protein (PGKneo). Intercross of Pcsk1(+/-) mice produced no Pcsk1(-/-) offspring or blastocysts; in addition, more than 80% of the offspring were Pcsk1(+/-). These observations suggested that the mutation caused preimplantation lethality of homozygous embryos and preferential transmission of the mutant allele. Interestingly, RT-PCR analysis on RNA from endocrine tissues from Pcsk1(+/-) mice revealed the presence of aberrant transcripts specifying the N-terminal half of the PC1 propeptide fused to neo gene product. Mass spectrometric profiles of proopiomelanocortin-derived peptides in the anterior pituitary were similar between Pcsk1(+/-) and Pcsk1(+/+) mice, but significantly different between male and female mice of the same genotype. Relative to their wild-type counterparts, female mutant mice exhibited stunted growth under a low fat diet, and catch-up growth under a high-fat diet. The complex phenotype exhibited by this Pcsk1 mutant mouse model may be due to PC1 deficiency aggravated by expression of aberrant gene products from the mutant allele.

Dopamine-Dependent Compensation Maintains Motor Behavior in Mice with Developmental Ablation of Dopaminergic Neurons

PubMed Central

DeMaro, Joseph A.; Knoten, Amanda; Hoshi, Masato; Pehek, Elizabeth; Johnson, Eugene M.; Gereau, Robert W.

2013-01-01

The loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and consequent depletion of striatal dopamine are known to underlie the motor deficits observed in Parkinson's disease (PD). Adaptive changes in dopaminergic terminals and in postsynaptic striatal neurons can compensate for significant losses of striatal dopamine, resulting in preservation of motor behavior. In addition, compensatory changes independent of striatal dopamine have been proposed based on PD therapies that modulate nondopaminergic circuits within the basal ganglia. We used a genetic strategy to selectively destroy dopaminergic neurons in mice during development to determine the necessity of these neurons for the maintenance of normal motor behavior in adult and aged mice. We find that loss of 90% of SNc dopaminergic neurons and consequent depletion of >95% of striatal dopamine does not result in changes in motor behavior in young-adult or aged mice as evaluated by an extensive array of motor behavior tests. Treatment of aged mutant mice with the dopamine receptor antagonist haloperidol precipitated motor behavior deficits in aged mutant mice, indicating that <5% of striatal dopamine is sufficient to maintain motor function in these mice. We also found that mutant mice exhibit an exaggerated response to l-DOPA compared with control mice, suggesting that preservation of motor function involves sensitization of striatal dopamine receptors. Our results indicate that congenital loss of dopaminergic neurons induces remarkable adaptions in the nigrostriatal system where limited amounts of dopamine in the dorsal striatum can maintain normal motor function. PMID:24155314

Suppression of gain-of-function mutant p53 with metabolic inhibitors reduces tumor growth in vivo

PubMed Central

Jung, Chae Lim; Mun, Hyemin; Jo, Se-Young; Oh, Ju-Hee; Lee, ChuHee; Choi, Eun-Kyung; Jang, Se Jin; Suh, Young-Ah

2016-01-01

Mutation of p53 occasionally results in a gain of function, which promotes tumor growth. We asked whether destabilizing the gain-of-function protein would kill tumor cells. Downregulation of the gene reduced cell proliferation in p53-mutant cells, but not in p53-null cells, indicating that the former depended on the mutant protein for survival. Moreover, phenformin and 2-deoxyglucose suppressed cell growth and simultaneously destabilized mutant p53. The AMPK pathway, MAPK pathway, chaperone proteins and ubiquitination all contributed to this process. Interestingly, phenformin and 2-deoxyglucose also reduced tumor growth in syngeneic mice harboring the p53 mutation. Thus, destabilizing mutant p53 protein in order to kill cells exhibiting “oncogene addiction” could be a promising strategy for combatting p53 mutant tumors. PMID:27765910

Suppression of gain-of-function mutant p53 with metabolic inhibitors reduces tumor growth in vivo.

PubMed

Jung, Chae Lim; Mun, Hyemin; Jo, Se-Young; Oh, Ju-Hee; Lee, ChuHee; Choi, Eun-Kyung; Jang, Se Jin; Suh, Young-Ah

2016-11-22

Mutation of p53 occasionally results in a gain of function, which promotes tumor growth. We asked whether destabilizing the gain-of-function protein would kill tumor cells. Downregulation of the gene reduced cell proliferation in p53-mutant cells, but not in p53-null cells, indicating that the former depended on the mutant protein for survival. Moreover, phenformin and 2-deoxyglucose suppressed cell growth and simultaneously destabilized mutant p53. The AMPK pathway, MAPK pathway, chaperone proteins and ubiquitination all contributed to this process. Interestingly, phenformin and 2-deoxyglucose also reduced tumor growth in syngeneic mice harboring the p53 mutation. Thus, destabilizing mutant p53 protein in order to kill cells exhibiting "oncogene addiction" could be a promising strategy for combatting p53 mutant tumors.

Impaired eye-blink conditioning in waggler, a mutant mouse with cerebellar BDNF deficiency.

PubMed

Bao, S; Chen, L; Qiao, X; Knusel, B; Thompson, R F

1998-01-01

In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expression of brain-derived neurotrophic factor (BDNF) was selectively absent in the cerebellar granule cells. The cytoarchitecture of the waggler cerebellum appeared to be normal at the light microscope level. The mutant mice exhibited no sensory deficits to auditory stimuli or heat-induced pain. However, they were massively impaired in classic eye-blink conditioning. These results suggest that BDNF may have a role in normal cerebellar neuronal function, which, in turn, is essential for classic eye-blink conditioning.

Parent-of-origin effects on schizophrenia-relevant behaviours of type III neuregulin 1 mutant mice.

PubMed

Shang, Kani; Talmage, David A; Karl, Tim

2017-08-14

A robust, disease-relevant phenotype is paramount to the validity of genetic mouse models, which are an important tool in understanding complex diseases. Recent evidence from genome-wide association studies suggests the genetic contribution of parents to offspring is not equivalent. Despite this, few studies to date have examined the potential impact of parent genotype (i.e. origin of mutation) on the offspring of disease-relevant genetic mouse models. To elucidate the potential impact of the sex of the mutant parent on offspring phenotype, we characterized male and female offspring of an established schizophrenia mouse model, which had been generated using two different breeding schemes, in a range of disease-relevant behaviours. We compared heterozygous type III neuregulin 1 mutant (type III Nrg1 +/- ) and wild type-like control (WT) offspring from mutant father x WT mother pairings with offspring from mutant mother x WT father pairings. Offspring were tested in schizophrenia-relevant paradigms including the elevated plus maze (EPM), fear conditioning (FC), prepulse inhibition (PPI), social interaction (SI), and open field (OF). We found type III Nrg1 +/- males from mutant fathers, but not mutant mothers, showed deficits in contextual fear-associated memory and exhibited increased social interaction, compared to their WT littermates. Type III Nrg1 +/- females across breeding colonies only exhibited a subtle change to their acoustic startle response and sensorimotor gating. These results suggest a paternal-dependent transmission of genetically induced behavioural characteristics. Though the mechanisms governing this phenomenon are unclear, our results show that parental origin of mutation can alter the behavioural phenotype of genetic mouse models. Thus, researchers should carefully consider their breeding scheme when dealing with genetic mouse models of diseases such as schizophrenia. Copyright © 2017. Published by Elsevier B.V.

Test systems for measuring ocular parameters and visual function in mice.

PubMed

Schaeffel, Frank

2008-05-01

New techniques are described to measure refractive state, pupil responses, corneal curvature, ocular dimensions and spatial vision in mice. These variables are important for studies on myopia development in mice, but they are also valuable for phenotyping mouse mutants and for pharmacological studies.

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.

Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy

PubMed Central

Freidl, Raphaela; Gstoettner, Antonia; Baranyi, Ulrike; Swoboda, Ines; Stolz, Frank; Focke-Tejkl, Margarete; Wekerle, Thomas; van Ree, Ronald; Valenta, Rudolf; Linhart, Birgit

2017-01-01

Background Fish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed. Objectives This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunization with a hypoallergenic mutant of the major carp allergen protect against allergic symptoms in sensitized mice. Methods C3H/HeJ mice were sensitized with recombinant wildtype Cyp c 1 or carp extract by intragastric gavage. Antibody, cellular immune responses, and epitope specificity in sensitized mice were investigated by ELISA, rat basophil leukemia assay, T-cell proliferation experiments using recombinant wildtype Cyp c 1, and overlapping peptides spanning the Cyp c 1 sequence. Anti-hypoallergenic Cyp c 1 mutant mouse and rabbit sera were tested for their ability to inhibit IgE recognition of Cyp c 1, Cyp c 1–specific basophil degranulation, and Cyp c 1–induced allergic symptoms in the mouse model. Results A mouse model of fish allergy mimicking human disease regarding IgE epitope recognition and symptoms as close as possible was established. Administration of antisera generated in mice and rabbits by immunization with a hypoallergenic Cyp c 1 mutant inhibited IgE binding to Cyp c 1, Cyp c 1–induced basophil degranulation, and allergic symptoms caused by allergen challenge in sensitized mice. Conclusions Antibodies induced by immunization with a hypoallergenic Cyp c 1 mutant protect against allergic reactions in a murine model of fish allergy. PMID:27876628

Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy.

PubMed

Freidl, Raphaela; Gstoettner, Antonia; Baranyi, Ulrike; Swoboda, Ines; Stolz, Frank; Focke-Tejkl, Margarete; Wekerle, Thomas; van Ree, Ronald; Valenta, Rudolf; Linhart, Birgit

2017-06-01

Fish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed. This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunization with a hypoallergenic mutant of the major carp allergen protect against allergic symptoms in sensitized mice. C3H/HeJ mice were sensitized with recombinant wildtype Cyp c 1 or carp extract by intragastric gavage. Antibody, cellular immune responses, and epitope specificity in sensitized mice were investigated by ELISA, rat basophil leukemia assay, T-cell proliferation experiments using recombinant wildtype Cyp c 1, and overlapping peptides spanning the Cyp c 1 sequence. Anti-hypoallergenic Cyp c 1 mutant mouse and rabbit sera were tested for their ability to inhibit IgE recognition of Cyp c 1, Cyp c 1-specific basophil degranulation, and Cyp c 1-induced allergic symptoms in the mouse model. A mouse model of fish allergy mimicking human disease regarding IgE epitope recognition and symptoms as close as possible was established. Administration of antisera generated in mice and rabbits by immunization with a hypoallergenic Cyp c 1 mutant inhibited IgE binding to Cyp c 1, Cyp c 1-induced basophil degranulation, and allergic symptoms caused by allergen challenge in sensitized mice. Antibodies induced by immunization with a hypoallergenic Cyp c 1 mutant protect against allergic reactions in a murine model of fish allergy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation.

PubMed

Kawabe, T; Naka, T; Yoshida, K; Tanaka, T; Fujiwara, H; Suematsu, S; Yoshida, N; Kishimoto, T; Kikutani, H

1994-06-01

An engagement of CD40 with CD40 ligand (CD40L) expressed on activated T cells is known to provide an essential costimulatory signal to B cells in vitro. To investigate the role of CD40 in in vivo immune responses, CD40-deficient mice were generated by gene targeting. The significant reduction of CD23 expression on mature B cells and relatively decreased number of IgM bright and IgD dull B cells were observed in the mutant mice. The mutant mice mounted IgM responses but no IgG, IgA, and IgE responses to thymus-dependent (TD) antigens. However, IgG as well as IgM responses to thymus-independent (TI) antigens were normal. Furthermore, the germinal center formation was defective in the mutant mice. These results suggest that CD40 is essential for T cell-dependent immunoglobulin class switching and germinal center formation, but not for in vivo T cell-dependent IgM responses and T cell-independent antibody responses.

Suppressed cellular oscillations in after-hours mutant mice are associated with enhanced circadian phase-resetting

PubMed Central

Guilding, Clare; Scott, Fiona; Bechtold, David A; Brown, Timothy M; Wegner, Sven; Piggins, Hugh D

2013-01-01

Within the core molecular clock, protein phosphorylation and degradation play a vital role in determining circadian period. The ‘after-hours’ (Afh) mutation in mouse slows the degradation of the core clock protein Cryptochrome, lengthening the period of the molecular clock in the suprachiasmatic nuclei (SCN) and behavioural wheel-running rhythms. However, we do not yet know how the Afh mutation affects other aspects of physiology or the activity of circadian oscillators in other brain regions. Here we report that daily rhythms of metabolism and ingestive behaviours are altered in these animals, as are PERIOD2::LUCIFERASE (PER2::LUC) rhythms in mediobasal hypothalamic nuclei, which influence these behaviours. Overall there is a trend towards period lengthening and a decrease in amplitude of PER2::LUC rhythms throughout the brain. Imaging of single cells from the arcuate and dorsomedial hypothalamic nuclei revealed this reduction in tissue oscillator amplitude to be due to a decrease in the amplitude, rather than a desynchrony, of single cells. Consistent with existing models of oscillator function, this cellular phenotype was associated with a greater susceptibility to phase-shifting stimuli in vivo and in vitro, with light evoking high-amplitude Type 0 resetting in Afh mutant mice. Together, these findings reveal unexpected consequences of the Afh mutation on the amplitude and synchrony of individual cellular oscillators in the SCN. PMID:23207594

Neuronal-specific overexpression of a mutant valosin-containing protein associated with IBMPFD promotes aberrant ubiquitin and TDP-43 accumulation and cognitive dysfunction in transgenic mice.

PubMed

Rodriguez-Ortiz, Carlos J; Hoshino, Hitomi; Cheng, David; Liu-Yescevitz, Liqun; Blurton-Jones, Mathew; Wolozin, Benjamin; LaFerla, Frank M; Kitazawa, Masashi

2013-08-01

Mutations in valosin-containing protein (VCP) cause a rare, autosomal dominant disease called inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD). One-third of patients with IBMPFD develop frontotemporal dementia, characterized by an extensive neurodegeneration in the frontal and temporal lobes. Neuropathologic hallmarks include nuclear and cytosolic inclusions positive to ubiquitin and transactive response DNA-binding protein 43 (TDP-43) in neurons and glial activation in affected regions. However, the pathogenic mechanisms by which mutant VCP triggers neurodegeneration remain unknown. Herein, we generated a mouse model selectively overexpressing a human mutant VCP in neurons to study pathogenic mechanisms of mutant VCP-mediated neurodegeneration and cognitive impairment. The overexpression of VCPA232E mutation in forebrain regions produced significant progressive impairments of cognitive function, including deficits in spatial memory, object recognition, and fear conditioning. Although overexpressed or endogenous VCP did not seem to focally aggregate inside neurons, TDP-43 and ubiquitin accumulated with age in transgenic mouse brains. TDP-43 was also found to co-localize with stress granules in the cytosolic compartment. Together with the appearance of high-molecular-weight TDP-43 in cytosolic fractions, these findings demonstrate the mislocalization and accumulation of abnormal TDP-43 in the cytosol of transgenic mice, which likely lead to an increase in cellular stress and cognitive impairment. Taken together, these results highlight an important pathologic link between VCP and cognition. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Functional Motor Recovery from Motoneuron Axotomy Is Compromised in Mice with Defective Corticospinal Projections

PubMed Central

Ding, Yuetong; Qu, Yibo; Feng, Jia; Wang, Meizhi; Han, Qi; So, Kwok-Fai; Wu, Wutian; Zhou, Libing

2014-01-01

Brachial plexus injury (BPI) and experimental spinal root avulsion result in loss of motor function in the affected segments. After root avulsion, significant motoneuron function is restored by re-implantation of the avulsed root. How much this functional recovery depends on corticospinal inputs is not known. Here, we studied that question using Celsr3|Emx1 mice, in which the corticospinal tract (CST) is genetically absent. In adult mice, we tore off right C5–C7 motor and sensory roots and re-implanted the right C6 roots. Behavioral studies showed impaired recovery of elbow flexion in Celsr3|Emx1 mice compared to controls. Five months after surgery, a reduced number of small axons, and higher G-ratio of inner to outer diameter of myelin sheaths were observed in mutant versus control mice. At early stages post-surgery, mutant mice displayed lower expression of GAP-43 in spinal cord and of myelin basic protein (MBP) in peripheral nerves than control animals. After five months, mutant animals had atrophy of the right biceps brachii, with less newly formed neuromuscular junctions (NMJs) and reduced peak-to-peak amplitudes in electromyogram (EMG), than controls. However, quite unexpectedly, a higher motoneuron survival rate was found in mutant than in control mice. Thus, following root avulsion/re-implantation, the absence of the CST is probably an important reason to hamper axonal regeneration and remyelination, as well as target re-innervation and formation of new NMJ, resulting in lower functional recovery, while fostering motoneuron survival. These results indicate that manipulation of corticospinal transmission may help improve functional recovery following BPI. PMID:25003601

Localization of efferent neurotransmitters in the inner ear of the homozygous Bronx waltzer mutant mouse.

PubMed

Kong, W J; Scholtz, A W; Hussl, B; Kammen-Jolly, K; Schrott-Fischer, A

2002-05-01

Naturally occurring mutant mice provide an excellent model for the study of genetic malformations of the inner ear. Mice homozygous for the Bronx waltzer (bv/bv) mutation are severely hearing impaired or deaf and exhibit a 'waltzing' gait. Functional aspects of cochlear and vestibular efferents in the bv/bv mutant mouse are not well known. The present study was designed to evaluate several candidates of efferent neurotransmitters or neuromodulators including choline acetyltransferase (ChAT), gamma-aminobutyric acid (GABA), and calcitonin gene-related peptide (CGRP) in the inner ear of the bv/bv mutant mouse. Ultrastructural investigations at both light and electron microscopic level were performed. Ultrastructural morphologic evaluations of the cochlea and the vestibular end-organs were also undertaken. It is demonstrated that ChAT, GABA and CGRP immunoreactivities are present in the cochlea and in vestibular end-organs of bv/bv mutant mice. In the organ of Corti, immunoreactivity of ChAT, GABA and CGRP is confined to the inner spiral fibers, tunnel-crossing fibers, and the vesiculated nerve endings synapsing with outer hair cells. Interestingly, immunoreactivity was detectable even where inner hair cells appeared missing. Results also revealed malformations of the outer hair cells with synaptic contacts to efferent nerve endings consistently intact. In the neurosensory epithelia of the vestibular end-organs, the presence of ChAT, GABA, and CGRP immunoreactivity was localized at the vestibular efferents, with the exception of the macula of saccule. In one 8-month-old macula of utricle where the depletion of hair cells appeared highest, ChAT immunostaining was still discernible. Ultrastructural investigation demonstrated that vesiculated efferent nerve endings make synaptic contact with the outer hair cells in the organ of Corti and with type II hair cells in the vestibular end-organs. The present study provides further support that the efferent system in the bv

Mutant WDR36 directly affects axon growth of retinal ganglion cells leading to progressive retinal degeneration in mice

PubMed Central

Chi, Zai-Long; Yasumoto, Fumie; Sergeev, Yuri; Minami, Masayoshi; Obazawa, Minoru; Kimura, Itaru; Takada, Yuichiro; Iwata, Takeshi

2010-01-01

Primary open-angle glaucoma (POAG) is one of the three principal subtypes of glaucoma and among the leading cause of blindness worldwide. POAG is defined by cell death of the retinal ganglion cells (RGCs) and surrounding neuronal cells at higher or normal intraocular pressure (IOP). Coded by one of the three genes responsible for POAG, WD repeat-containing protein 36 (WDR36) has two domains with a similar folding. To address whether WDR36 is functionally important in the retina, we developed four transgenic mice strains overexpressing a wild-type (Wt) and three mutant variants of D606G, deletion of amino acids at positions 605–607 (Del605–607) and at 601–640 (Del601–640) equivalent to the location of the D658G mutation observed in POAG patients. A triple amino acid deletion of mouse Wdr36 at positions 605–607 corresponding to the deletion at positions 657–659 in humans developed progressive retinal degeneration at the peripheral retina with normal IOP. RGCs and connecting amacrine cell synapses were affected at the peripheral retina. Axon outgrowth rate of cultured RGC directly isolated from transgenic animal was significantly reduced by the Wdr36 mutation compared with Wt. Molecular modeling of wild and mutant mouse Wdr36 revealed that deletion at positions 605–607 removed three residues and a hydrogen bond, required to stabilize anti-parallel β-sheet of the 6th β-propeller in the second domain. We concluded that WDR36 plays an important functional role in the retina homeostasis and mutation to this gene can cause devastating retinal damage. These data will improve understanding of the functional property of WDR36 in the retina and provide a new animal model for glaucoma therapeutics. PMID:20631153

Thymidine Kinase-Negative Herpes Simplex Virus 1 Can Efficiently Establish Persistent Infection in Neural Tissues of Nude Mice.

PubMed

Huang, Chih-Yu; Yao, Hui-Wen; Wang, Li-Chiu; Shen, Fang-Hsiu; Hsu, Sheng-Min; Chen, Shun-Hua

2017-02-15

Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484-490, 1994, https://doi.org/10.1006/viro.1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1. Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drug-resistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of immunocompromised

Thymidine Kinase-Negative Herpes Simplex Virus 1 Can Efficiently Establish Persistent Infection in Neural Tissues of Nude Mice

PubMed Central

Huang, Chih-Yu; Yao, Hui-Wen; Wang, Li-Chiu; Shen, Fang-Hsiu

2016-01-01

ABSTRACT Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484–490, 1994, https://doi.org/10.1006/viro.1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1. IMPORTANCE Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drug-resistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of

Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes.

PubMed

Wang, Bo; Hikosaka, Keisuke; Sultana, Nishat; Sharkar, Mohammad Tofael Kabir; Noritake, Hidenao; Kimura, Wataru; Wu, Yi-Xin; Kobayashi, Yoshimasa; Uezato, Tadayoshi; Miura, Naoyuki

2012-01-06

The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with ∼50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age. Copyright © 2011 Elsevier Inc. All rights reserved.

Chir99021 and Valproic acid reduce the proliferative advantage of Apc mutant cells.

PubMed

Langlands, Alistair J; Carroll, Thomas D; Chen, Yu; Näthke, Inke

2018-02-15

More than 90% of colorectal cancers carry mutations in Apc that drive tumourigenesis. A 'just-right' signalling model proposes that Apc mutations stimulate optimal, but not excessive Wnt signalling, resulting in a growth advantage of Apc mutant over wild-type cells. Reversal of this growth advantage constitutes a potential therapeutic approach. We utilised intestinal organoids to compare the growth of Apc mutant and wild-type cells. Organoids derived from Apc Min/+ mice recapitulate stages of intestinal polyposis in culture. They eventually form spherical cysts that reflect the competitive growth advantage of cells that have undergone loss of heterozygosity (LOH). We discovered that this emergence of cysts was inhibited by Chiron99021 and Valproic acid, which potentiates Wnt signalling. Chiron99021 and Valproic acid restrict the growth advantage of Apc mutant cells while stimulating that of wild-type cells, suggesting that excessive Wnt signalling reduces the relative fitness of Apc mutant cells. As a proof of concept, we demonstrated that Chiron99021-treated Apc mutant organoids were rendered susceptible to TSA-induced apoptosis, while wild-type cells were protected.

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

PubMed

Baldwin, Thomas T; Basenko, Evelina; Harb, Omar; Brown, Neil A; Urban, Martin; Hammond-Kosack, Kim E; Bregitzer, Phil P

2018-06-01

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species. Published by Elsevier Inc.

Compensation for PKMζ in long-term potentiation and spatial long-term memory in mutant mice.

PubMed

Tsokas, Panayiotis; Hsieh, Changchi; Yao, Yudong; Lesburguères, Edith; Wallace, Emma Jane Claire; Tcherepanov, Andrew; Jothianandan, Desingarao; Hartley, Benjamin Rush; Pan, Ling; Rivard, Bruno; Farese, Robert V; Sajan, Mini P; Bergold, Peter John; Hernández, Alejandro Iván; Cottrell, James E; Shouval, Harel Z; Fenton, André Antonio; Sacktor, Todd Charlton

2016-05-17

PKMζ is a persistently active PKC isoform proposed to maintain late-LTP and long-term memory. But late-LTP and memory are maintained without PKMζ in PKMζ-null mice. Two hypotheses can account for these findings. First, PKMζ is unimportant for LTP or memory. Second, PKMζ is essential for late-LTP and long-term memory in wild-type mice, and PKMζ-null mice recruit compensatory mechanisms. We find that whereas PKMζ persistently increases in LTP maintenance in wild-type mice, PKCι/λ, a gene-product closely related to PKMζ, persistently increases in LTP maintenance in PKMζ-null mice. Using a pharmacogenetic approach, we find PKMζ-antisense in hippocampus blocks late-LTP and spatial long-term memory in wild-type mice, but not in PKMζ-null mice without the target mRNA. Conversely, a PKCι/λ-antagonist disrupts late-LTP and spatial memory in PKMζ-null mice but not in wild-type mice. Thus, whereas PKMζ is essential for wild-type LTP and long-term memory, persistent PKCι/λ activation compensates for PKMζ loss in PKMζ-null mice.

An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

PubMed Central

Qualls, David A.; Crosby, Keith; Brown, Hilda; Borchelt, David R.

2013-01-01

Background By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures. Methodology/Principal Findings In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures. Conclusions/Significance Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells. PMID:24391857

Mutant KRAS promotes malignant pleural effusion formation

PubMed Central

Αgalioti, Theodora; Giannou, Anastasios D.; Krontira, Anthi C.; Kanellakis, Nikolaos I.; Kati, Danai; Vreka, Malamati; Pepe, Mario; Spella, Μagda; Lilis, Ioannis; Zazara, Dimitra E.; Nikolouli, Eirini; Spiropoulou, Nikolitsa; Papadakis, Andreas; Papadia, Konstantina; Voulgaridis, Apostolos; Harokopos, Vaggelis; Stamou, Panagiota; Meiners, Silke; Eickelberg, Oliver; Snyder, Linda A.; Antimisiaris, Sophia G.; Kardamakis, Dimitrios; Psallidas, Ioannis; Μarazioti, Antonia; Stathopoulos, Georgios T.

2017-01-01

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition. PMID:28508873

Mouse mutants from chemically mutagenized embryonic stem cells

PubMed Central

Munroe, Robert J.; Bergstrom, Rebecca A.; Zheng, Qing Yin; Libby, Brian; Smith, Richard; John, Simon W.M.; Schimenti, Kerry J.; Browning, Victoria L.; Schimenti, John C.

2010-01-01

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain1 and interlocus2 variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chi-maeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives. PMID:10700192

Amyloid Precursor Protein Haploinsufficiency Preferentially Mediates Brain Iron Accumulation in Mice Transgenic for The Huntington's Disease Mutation.

PubMed

Berggren, Kiersten; Agrawal, Sonal; Fox, Julia A; Hildenbrand, Justin; Nelson, Ryan; Bush, Ashley I; Fox, Jonathan H

2017-01-01

Huntington's disease (HD) is an autosomal dominant disorder caused by a CAG expansion in the huntingtin gene that results in expression of mutant huntingtin protein. Iron accumulates in HD brain neurons. Amyloid precursor protein (APP) promotes neuronal iron export. However, the role of APP in brain iron accumulation in HD is unclear. To determine the effects of APP insufficiency on HD in YAC128 mice. We crossed APP hemizygous mice (APP+/-) with YAC128 mice that are transgenic (Tg) for human mutant huntingtin (hmHTT) to generate APP+/+ hmHTT-/-, APP+/- hmHTT-/-, APP+/+ hmHTT+/- and APP+/- hmHTT+/- progeny. Mice were evaluated for behavioral, biochemical and neuropathology HD outcomes at 2-12 months of age. APP heterozygosity decreased cortical APP 25% and 60% in non-Tg and Tg mice, respectively. Cerebral and striatal iron levels were increased by APP knockdown in Tg mice only. Nest-building behavior was decreased in Tg mice; APP knockdown decreased nest building in non-Tg but not Tg mice. Rota-rod endurance was decreased in Tg mice. APP+/- hHTT+/- mice demonstrated additional decreases in rota-rod endurance from 4-10 months of age. Tg mice had smaller striatal volumes and fewer striatal neurons but were not affected by APP knockdown. APP heterozygosity results in greater decreases of cortical APP in Tg versus non-Tg mice. Mutant huntingtin transgenic mice develop brain iron accumulation as a result of greater suppression of APP levels. Elevated brain iron in Tg mice was associated with a decline in motor endurance consistent with a disease promoting effect of iron in the YAC128 model of human HD.

TTSS2-deficient hha mutant of Salmonella Typhimurium exhibits significant systemic attenuation in immunocompromised hosts

PubMed Central

Vishwakarma, Vikalp; Pati, Niladri Bhusan; Ray, Shilpa; Das, Susmita; Suar, Mrutyunjay

2014-01-01

Non-typhoidal Salmonella (NTS) infections are emerging as leading problem worldwide and the variations in host immune status append to the concern of NTS. Salmonella enterica serovar Typhimurium is one of the causative agents of NTS infections and has been extensively studied. The inactivation of Salmonella pathogenicity island 2 (SPI2) encoded type-III secretion system 2 (TTSS2) has been reported rendering the strain incapable for systemic dissemination to host sites and has also been proposed as live-attenuated vaccine. However, infections from TTSS2-deficient Salmonella have also been reported. In this study, mutant strain MT15 was developed by inactivation of the hemolysin expression modulating protein (hha) in TTSS2-deficient S. Typhimurium background. The MT15 strain showed significant level of attenuation in immune-deprived murine colitis model when tested in iNos−/−, IL10−/−, and CD40L−/− mice groups in C57BL/6 background. Further, the mutation in hha does not implicate any defect in bacterial colonization to the host gut. The long-term infection of developed mutant strain conferred protective immune responses to suitably immunized streptomycin pre-treated C57BL/6 mice. The immunization enhanced the CD4+ and CD8+ cell types involved in bacterial clearance. The serum IgG and luminal secretory IgA (sIgA) was also found to be elevated after the due course of infection. Additionally, the immunized C57BL/6 mice were protected from the subsequent lethal infection of Salmonella Typhimurium. Collectively, these findings implicate the involvement of hemolysin expression modulating protein (Hha) in establishment of bacterial infection. In light of the observed attenuation of the developed mutant strain, this study proposes the possible significance of SPI2-deficient hha mutant as an alternative live-attenuated vaccine strain for use against lethal Salmonella infections. PMID:24401482

Muscarinic cholinergic receptor (M2) plays a crucial role in the development of myopia in mice

PubMed Central

Barathi, Veluchamy A.; Kwan, Jia Lin; Tan, Queenie S. W.; Weon, Sung Rhan; Seet, Li Fong; Goh, Liang Kee; Vithana, Eranga N.; Beuerman, Roger W.

2013-01-01

SUMMARY Myopia is a huge public health problem worldwide, reaching the highest incidence in Asia. Identification of susceptible genes is crucial for understanding the biological basis of myopia. In this paper, we have identified and characterized a functional myopia-associated gene using a specific mouse-knockout model. Mice lacking the muscarinic cholinergic receptor gene (M2; also known as Chrm2) were less susceptible to lens-induced myopia compared with wild-type mice, which showed significantly increased axial length and vitreous chamber depth when undergoing experimental induction of myopia. The key findings of this present study are that the sclera of M2 mutant mice has higher expression of collagen type I and lower expression of collagen type V than do wild-type mice and mice that are mutant for other muscarinic subtypes, and, therefore, M2 mutant mice were resistant to the development of experimental myopia. Pharmacological blockade of M2 muscarinic receptor proteins retarded myopia progression in the mouse. These results suggest for the first time a role of M2 in growth-related changes in extracellular matrix genes during myopia development in a mammalian model. M2 receptor antagonists might thus provide a targeted therapeutic approach to the management of this refractive error. PMID:23649821

Defective transport of the obesity mutant PC1/3 N222D contributes to loss of function.

PubMed

Prabhu, Yogikala; Blanco, Elias H; Liu, Ming; Peinado, Juan R; Wheeler, Matthew C; Gekakis, Nicholas; Arvan, Peter; Lindberg, Iris

2014-07-01

Mutations in the PCSK1 gene encoding prohormone convertase 1/3 (PC1/3) are strongly associated with obesity in humans. The PC1/3(N222D) mutant mouse thus far represents the only mouse model that mimics the PC1/3 obesity phenotype in humans. The present investigation addresses the cell biology of the N222D mutation. Metabolic labeling experiments reveal a clear defect in the kinetics of insulin biosynthesis in islets from PC1/3(N222D) mutant mice, resulting in an increase in both proinsulin and its processing intermediates, predominantly lacking cleavage at the Arg-Arg site. Although the mutant PC1/3 zymogen is correctly processed to the 87-kDa form, pulse-chase immunoprecipitation experiments, labeling, and immunohistochemical experiments using uncleavable variants all demonstrate that the PC1/3-N222D protein is largely mislocalized compared with similar wild-type (WT) constructs, being predominantly retained in the endoplasmic reticulum. The PC1/3-N222D mutant also undergoes more efficient degradation via the ubiquitin-proteasome system than the WT enzyme. Lastly, the mutant PC1/3-N222D protein coimmunoprecipitates with WT PC1/3 and exerts a modest effect on intracellular retention of the WT enzyme. These profound alterations in the cell biology of PC1/3-N222D are likely to contribute to the defective insulin biosynthetic events observed in the mutant mice and may be relevant to the dramatic contributions of polymorphisms in this gene to human obesity.

Defective Transport of the Obesity Mutant PC1/3 N222D Contributes to Loss of Function

PubMed Central

Prabhu, Yogikala; Blanco, Elias H.; Liu, Ming; Peinado, Juan R.; Wheeler, Matthew C.; Gekakis, Nicholas; Arvan, Peter

2014-01-01

Mutations in the PCSK1 gene encoding prohormone convertase 1/3 (PC1/3) are strongly associated with obesity in humans. The PC1/3N222D mutant mouse thus far represents the only mouse model that mimics the PC1/3 obesity phenotype in humans. The present investigation addresses the cell biology of the N222D mutation. Metabolic labeling experiments reveal a clear defect in the kinetics of insulin biosynthesis in islets from PC1/3N222D mutant mice, resulting in an increase in both proinsulin and its processing intermediates, predominantly lacking cleavage at the Arg-Arg site. Although the mutant PC1/3 zymogen is correctly processed to the 87-kDa form, pulse-chase immunoprecipitation experiments, labeling, and immunohistochemical experiments using uncleavable variants all demonstrate that the PC1/3-N222D protein is largely mislocalized compared with similar wild-type (WT) constructs, being predominantly retained in the endoplasmic reticulum. The PC1/3-N222D mutant also undergoes more efficient degradation via the ubiquitin-proteasome system than the WT enzyme. Lastly, the mutant PC1/3-N222D protein coimmunoprecipitates with WT PC1/3 and exerts a modest effect on intracellular retention of the WT enzyme. These profound alterations in the cell biology of PC1/3-N222D are likely to contribute to the defective insulin biosynthetic events observed in the mutant mice and may be relevant to the dramatic contributions of polymorphisms in this gene to human obesity. PMID:24828610

Aberrant Muscle Antigen Exposure in Mice Is Sufficient to Cause Myositis in a Treg Cell–Deficient Milieu

PubMed Central

Young, Nicholas A; Sharma, Rahul; Friedman, Alexandra K; Kaffenberger, Benjamin H; Bolon, Brad; Jarjour, Wael N

2013-01-01

Objective Myositis is associated with muscle-targeted inflammation and is observed in some Treg cell–deficient mouse models. Because an autoimmune pathogenesis has been strongly implicated, the aim of this study was to investigate the hypothesis that abnormal exposure to muscle antigens, as observed in muscle injury, can induce autoimmune-mediated myositis in susceptible hosts. Methods FoxP3 mutant (scurfy) mice were mated to synaptotagmin VII (Syt VII) mutant mice, which resulted in a new mouse strain that combines impaired membrane resealing with Treg cell deficiency. Lymphocyte preparations from double-mutant mice were adoptively transferred intraperitoneally, with or without purified Treg cells, into recombination-activating gene 1 (RAG-1)–null recipients. Lymph node cells from mice with the FoxP3 mutation were transferred into RAG-1–null mice either 1) intraperitoneally in conjunction with muscle homogenate or purified myosin protein or 2) intramuscularly with or without cotransfer of purified Treg cells. Results FoxP3-deficient mouse lymph node cells transferred in conjunction with myosin protein or muscle homogenate induced robust skeletal muscle inflammation. The infiltrates consisted predominantly of CD4+ and CD8+ T cells, a limited number of macrophages, and no B cells. Significant inflammation was also seen in similar experiments using lymph node cells from FoxP3/Syt VII double-mutant mice but was absent in experiments using adoptive transfer of FoxP3 mutant mouse cells alone. The cotransfer of Treg cells completely suppressed myositis. Conclusion These data, derived from a new, reproducible model, demonstrate the critical roles of Treg cell deficiency and aberrant muscle antigen exposure in the priming of autoreactive cells to induce myositis. This mouse system has multifaceted potential for examining the interplay in vivo between tissue injury and autoimmunity. PMID:24022275

Aberrant muscle antigen exposure in mice is sufficient to cause myositis in a Treg cell-deficient milieu.

PubMed

Young, Nicholas A; Sharma, Rahul; Friedman, Alexandra K; Kaffenberger, Benjamin H; Bolon, Brad; Jarjour, Wael N

2013-12-01

Myositis is associated with muscle-targeted inflammation and is observed in some Treg cell-deficient mouse models. Because an autoimmune pathogenesis has been strongly implicated, the aim of this study was to investigate the hypothesis that abnormal exposure to muscle antigens, as observed in muscle injury, can induce autoimmune-mediated myositis in susceptible hosts. FoxP3 mutant (scurfy) mice were mated to synaptotagmin VII (Syt VII) mutant mice, which resulted in a new mouse strain that combines impaired membrane resealing with Treg cell deficiency. Lymphocyte preparations from double-mutant mice were adoptively transferred intraperitoneally, with or without purified Treg cells, into recombination-activating gene 1 (RAG-1)-null recipients. Lymph node cells from mice with the FoxP3 mutation were transferred into RAG-1-null mice either 1) intraperitoneally in conjunction with muscle homogenate or purified myosin protein or 2) intramuscularly with or without cotransfer of purified Treg cells. FoxP3-deficient mouse lymph node cells transferred in conjunction with myosin protein or muscle homogenate induced robust skeletal muscle inflammation. The infiltrates consisted predominantly of CD4+ and CD8+ T cells, a limited number of macrophages, and no B cells. Significant inflammation was also seen in similar experiments using lymph node cells from FoxP3/Syt VII double-mutant mice but was absent in experiments using adoptive transfer of FoxP3 mutant mouse cells alone. The cotransfer of Treg cells completely suppressed myositis. These data, derived from a new, reproducible model, demonstrate the critical roles of Treg cell deficiency and aberrant muscle antigen exposure in the priming of autoreactive cells to induce myositis. This mouse system has multifaceted potential for examining the interplay in vivo between tissue injury and autoimmunity. © 2013 The Authors. Arthritis & Rheumatism is published by Wiley Periodicals, Inc. on behalf of the American College of

Compensation for PKMζ in long-term potentiation and spatial long-term memory in mutant mice

PubMed Central

Tsokas, Panayiotis; Hsieh, Changchi; Yao, Yudong; Lesburguères, Edith; Wallace, Emma Jane Claire; Tcherepanov, Andrew; Jothianandan, Desingarao; Hartley, Benjamin Rush; Pan, Ling; Rivard, Bruno; Farese, Robert V; Sajan, Mini P; Bergold, Peter John; Hernández, Alejandro Iván; Cottrell, James E; Shouval, Harel Z; Fenton, André Antonio; Sacktor, Todd Charlton

2016-01-01

PKMζ is a persistently active PKC isoform proposed to maintain late-LTP and long-term memory. But late-LTP and memory are maintained without PKMζ in PKMζ-null mice. Two hypotheses can account for these findings. First, PKMζ is unimportant for LTP or memory. Second, PKMζ is essential for late-LTP and long-term memory in wild-type mice, and PKMζ-null mice recruit compensatory mechanisms. We find that whereas PKMζ persistently increases in LTP maintenance in wild-type mice, PKCι/λ, a gene-product closely related to PKMζ, persistently increases in LTP maintenance in PKMζ-null mice. Using a pharmacogenetic approach, we find PKMζ-antisense in hippocampus blocks late-LTP and spatial long-term memory in wild-type mice, but not in PKMζ-null mice without the target mRNA. Conversely, a PKCι/λ-antagonist disrupts late-LTP and spatial memory in PKMζ-null mice but not in wild-type mice. Thus, whereas PKMζ is essential for wild-type LTP and long-term memory, persistent PKCι/λ activation compensates for PKMζ loss in PKMζ-null mice. DOI: http://dx.doi.org/10.7554/eLife.14846.001 PMID:27187150

Adaptive failure to high-fat diet characterizes steatohepatitis in Alms1 mutant mice.

PubMed

Arsov, Todor; Larter, Claire Z; Nolan, Christopher J; Petrovsky, Nikolai; Goodnow, Christopher C; Teoh, Narcissus C; Yeh, Matthew M; Farrell, Geoffrey C

2006-04-21

The biochemical differences between simple steatosis, a benign liver disease, and non-alcoholic steatohepatitis, which leads to cirrhosis, are unclear. Fat aussie is an obese mouse strain with a truncating mutation (foz) in the Alms1 gene. Chow-fed female foz/foz mice develop obesity, diabetes, and simple steatosis. We fed foz/foz and wildtype mice a high-fat diet. Foz/foz mice developed serum ALT elevation and severe steatohepatitis with hepatocyte ballooning, inflammation, and fibrosis; wildtype mice showed simple steatosis. Biochemical pathways favoring hepatocellular lipid accumulation (fatty acid uptake; lipogenesis) and lipid disposal (fatty acid beta-oxidation; triglyceride egress) were both induced by high-fat feeding in wildtype but not foz/foz mice. The resulting extremely high hepatic triglyceride levels were associated with induction of mitochondrial uncoupling protein-2 and adipocyte-specific fatty acid binding protein-2, but not cytochrome P4502e1 or lipid peroxidation. In this model of metabolic syndrome, transition of steatosis to steatohepatitis was associated with hypoadiponectinemia, a mediator of hepatic fatty acid disposal pathways.

Eye hyperdeviation in mouse cerebellar mutants is comparable to the gravity-dependent component of human downbeat nystagmus.

PubMed

Stahl, John S; Oommen, Brian S

2008-01-01

Humans with cerebellar degeneration commonly exhibit downbeat nystagmus (DBN). DBN has gravity-independent and -dependent components, and the latter has been proposed to reflect hyperactive tilt maculo-ocular reflexes (tilt-MOR). Mice with genetically determined cerebellar ataxia do not exhibit DBN, but they do exhibit tonic hyperdeviation of the eyes, which we have proposed to be the DBN equivalent. As such, the tilt-MOR might be predicted to be hyperactive in these mutant mice. We measured the tilt-MOR in 10 normal C57BL/6 mice and in 6 tottering, a mutant exhibiting ataxia and ocular motor abnormalities due to mutation of the P/Q calcium channel. Awake mice were placed in body orientations spanning 360 degrees about the pitch axis. The absolute, equilibrium vertical angular deviations of one eye were measured using infrared videooculography. In both strains, eye elevation varied quasi-sinusoidally with tilt angle in the range of 90 degrees nose-up to 90 degrees nose-down. Beyond this range the eye returned to a neutral position. Deviation over +/-30 degrees of tilt was an approximately linear function of the projection of the gravity vector into the animal's horizontal plane, and can thus be summarized by its slope (sensitivity). Sensitivity measured 14.9 degrees/g for C57BL/6 and 20.3 degrees/g for tottering, a statistically significant difference. Thus the pitch otolithic reflex of the ataxic mutants is hyperactive relative to controls and could explain tonic hyperdeviation of the eyes, consistent with the idea that the tonic hyperdeviation is analogous to DBN.

Serum response factor: positive and negative regulation of an epithelial gene expression network in the destrin mutant cornea

PubMed Central

Kawakami-Schulz, Sharolyn V.; Verdoni, Angela M.; Sattler, Shannon G.; Jessen, Erik; Kao, Winston W.-Y.; Ikeda, Akihiro

2014-01-01

Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstncorn1 mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstncorn1 mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstncorn1 cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstncorn1 cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstncorn1 mice. Comparison of gene expression values from WT, Dstncorn1 mutant, and Dstncorn1 rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstncorn1 mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstncorn1 mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstncorn1 mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis. PMID:24550211

Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

PubMed Central

Perera, Nirma D.; Sheean, Rebecca K.; Lau, Chew L.; Shin, Yea Seul; Beart, Philip M.; Horne, Malcolm K.; Turner, Bradley J.

2018-01-01

ABSTRACT Macroautophagy/autophagy is the main intracellular catabolic pathway in neurons that eliminates misfolded proteins, aggregates and damaged organelles associated with ageing and neurodegeneration. Autophagy is regulated by both MTOR-dependent and -independent pathways. There is increasing evidence that autophagy is compromised in neurodegenerative disorders, which may contribute to cytoplasmic sequestration of aggregation-prone and toxic proteins in neurons. Genetic or pharmacological modulation of autophagy to promote clearance of misfolded proteins may be a promising therapeutic avenue for these disorders. Here, we demonstrate robust autophagy induction in motor neuronal cells expressing SOD1 or TARDBP/TDP-43 mutants linked to amyotrophic lateral sclerosis (ALS). Treatment of these cells with rilmenidine, an anti-hypertensive agent and imidazoline-1 receptor agonist that induces autophagy, promoted autophagic clearance of mutant SOD1 and efficient mitophagy. Rilmenidine administration to mutant SOD1G93A mice upregulated autophagy and mitophagy in spinal cord, leading to reduced soluble mutant SOD1 levels. Importantly, rilmenidine increased autophagosome abundance in motor neurons of SOD1G93A mice, suggesting a direct action on target cells. Despite robust induction of autophagy in vivo, rilmenidine worsened motor neuron degeneration and symptom progression in SOD1G93A mice. These effects were associated with increased accumulation and aggregation of insoluble and misfolded SOD1 species outside the autophagy pathway, and severe mitochondrial depletion in motor neurons of rilmenidine-treated mice. These findings suggest that rilmenidine treatment may drive disease progression and neurodegeneration in this mouse model due to excessive mitophagy, implying that alternative strategies to beneficially stimulate autophagy are warranted in ALS. PMID:28980850

FUS/TLS acts as an aggregation-dependent modifier of polyglutamine disease model mice.

PubMed

Kino, Yoshihiro; Washizu, Chika; Kurosawa, Masaru; Yamada, Mizuki; Doi, Hiroshi; Takumi, Toru; Adachi, Hiroaki; Katsuno, Masahisa; Sobue, Gen; Hicks, Geoffrey G; Hattori, Nobutaka; Shimogori, Tomomi; Nukina, Nobuyuki

2016-10-14

FUS/TLS is an RNA/DNA-binding protein associated with neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Previously, we found that a prion-like domain in the N-terminus of FUS/TLS mediates co-aggregation between FUS/TLS and mutant huntingtin, the gene product of Huntington's disease (HD). Here, we show that heterozygous knockout of FUS/TLS worsened the phenotypes of model mice of (HD, but not spinal and bulbar muscular atrophy (SBMA). This difference was correlated with the degree of pathological association between disease proteins and FUS/TLS. Co-aggregation between FUS/TLS and mutant huntingtin resulted in the depletion of free FUS/TLS protein in HD mice that was detected as a monomer in SDS-PAGE analysis. Recently, we found that FUS/TLS paralogs, TAF15 and EWS, were up-regulated in homozygous FUS/TLS knockout mice. These two proteins were up-regulated in both HD and FUS/TLS heterozygote mice, and were further elevated in HD-TLS +/- double mutant mice, consistent with the functional impairment of FUS/TLS. These results suggest that FUS/TLS sequestration by co-aggregation is a rate-limiting factor of disease phenotypes of HD and that inclusions may have an adverse aspect, rather than being simply benign or protective. In addition, our results highlight inclusions as repositories of potential modifiers of neurodegeneration.

Targeted Deletion of Kynurenine 3-Monooxygenase in Mice

PubMed Central

Giorgini, Flaviano; Huang, Shao-Yi; Sathyasaikumar, Korrapati V.; Notarangelo, Francesca M.; Thomas, Marian A. R.; Tararina, Margarita; Wu, Hui-Qiu; Schwarcz, Robert; Muchowski, Paul J.

2013-01-01

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo−/− mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo−/− mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo−/− mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD+, did not differ between Kmo−/− and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo−/− mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo−/− mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease. PMID:24189070

Structure based discovery of clomifene as a potent inhibitor of cancer-associated mutant IDH1

PubMed Central

Luan, Shanshan; Li, Dan; Chen, Renqi; Zhang, Qian; Chen, Lixia; Huang, Jiangeng; Li, Hua

2017-01-01

Isocitrate dehydrogenase (IDH) plays an indispensable role in the tricarboxylic acid cycle, and IDH mutations are present in nearly 75% of glioma and 20% of acute myeloid leukemia. One IDH1R132H inhibitor (clomifene citrate) was found by virtual screening method, which can selectively suppress mutant enzyme activities in vitro and in vivo with a dose-dependent manner. The molecular docking indicated that clomifene occupied the allosteric site of the mutant IDH1. Enzymatic kinetics also demonstrated that clomifene inhibited mutant enzyme in a non-competitive manner. Moreover, knockdown of mutant IDH1 in HT1080 cells decreased the sensitivity to clomifene. In vivo studies indicated that clomifene significantly suppressed the tumor growth of HT1080-bearing CB-17/Icr-scid mice with oral administration of 100 mg/kg and 50 mg/kg per day. In short, our findings highlight clomifene may have clinical potential in tumor therapies as a safe and effective inhibitor of mutant IDH1. PMID:28498812

Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice.

PubMed

Kotajima-Murakami, Hiroko; Narumi, Sakae; Yuzaki, Michisuke; Yanagihara, Dai

2016-01-01

Lesions in the cerebellar vermis abolish acquisition of fear-conditioned bradycardia in animals and human patients. The δ2 glutamate receptor (GluD2) is predominantly expressed in cerebellar Purkinje cells. The mouse mutant ho15J carries a spontaneous mutation in GluD2 and these mice show a primary deficiency in parallel fiber-Purkinje cell synapses, multiple innervations of Purkinje cells by climbing fibers, and impairment of long-term depression. In the present study, we used ho15J mice to investigate the role of the cerebellum in fear-conditioned bradycardia. We recorded changes in heart rate of ho15J mice induced by repeated pairing of an acoustic (conditioned) stimulus (CS) with an aversive (unconditioned) stimulus (US). The mice acquired conditioned bradycardia on Day 1 of the CS-US phase, similarly to wild-type mice. However, the magnitude of the conditioned bradycardia was not stable in the mutant mice, but rather was exaggerated on Days 2-5 of the CS-US phase. We examined the effects of reversibly inactivating the cerebellum by injection of an antagonist against the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR). The antagonist abolished expression of conditioned responses in both wild-type and ho15J mice. We conclude that the GluD2 mutation in the ho15J mice affects stable retention of the acquired conditioned bradycardia.

A robust and reliable non-invasive test for stress responsivity in mice.

PubMed

Zimprich, Annemarie; Garrett, Lillian; Deussing, Jan M; Wotjak, Carsten T; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabě; Wurst, Wolfgang; Hölter, Sabine M

2014-01-01

Stress and an altered stress response have been associated with many multifactorial diseases, such as psychiatric disorders or neurodegenerative diseases. As currently mouse mutants for each single gene are generated and phenotyped in a large-scale manner, it seems advisable also to test these mutants for alterations in their stress responses. Here we present the determinants of a robust and reliable non-invasive test for stress-responsivity in mice. Stress is applied through restraining the mice in tubes and recording behavior in the Open Field 20 min after cessation of the stress. Two hours, but not 15 or 50 min of restraint lead to a robust and reproducible increase in distance traveled and number of rearings during the first 5 min in the Open Field in C57BL/6 mice. This behavioral response is blocked by the corticosterone synthesis inhibitor metyrapone, but not by RU486 treatment, indicating that it depends on corticosteroid secretion, but is not mediated via the glucocorticoid receptor type II. We assumed that with a stress duration of 15 min one could detect hyper-responsivity, and with a stress duration of 2 h hypo-responsivity in mutant mouse lines. This was validated with two mutant lines known to show opposing effects on corticosterone secretion after stress exposure, corticotropin-releasing hormone (CRH) over-expressing mice and CRH receptor 1 knockout (KO) mice. Both lines showed the expected phenotype, i.e., increased stress responsivity in the CRH over-expressing mouse line (after 15 min restraint stress) and decreased stress responsivity in the CRHR1-KO mouse line (after 2 h of restraint stress). It is possible to repeat the acute stress test several times without the stressed animal adapting to it, and the behavioral response can be robustly evoked at different ages, in both sexes and in different mouse strains. Thus, locomotor and rearing behavior in the Open Field after an acute stress challenge can be used as reliable, non-invasive indicators of

A robust and reliable non-invasive test for stress responsivity in mice

PubMed Central

Zimprich, Annemarie; Garrett, Lillian; Deussing, Jan M.; Wotjak, Carsten T.; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabě; Wurst, Wolfgang; Hölter, Sabine M.

2014-01-01

Stress and an altered stress response have been associated with many multifactorial diseases, such as psychiatric disorders or neurodegenerative diseases. As currently mouse mutants for each single gene are generated and phenotyped in a large-scale manner, it seems advisable also to test these mutants for alterations in their stress responses. Here we present the determinants of a robust and reliable non-invasive test for stress-responsivity in mice. Stress is applied through restraining the mice in tubes and recording behavior in the Open Field 20 min after cessation of the stress. Two hours, but not 15 or 50 min of restraint lead to a robust and reproducible increase in distance traveled and number of rearings during the first 5 min in the Open Field in C57BL/6 mice. This behavioral response is blocked by the corticosterone synthesis inhibitor metyrapone, but not by RU486 treatment, indicating that it depends on corticosteroid secretion, but is not mediated via the glucocorticoid receptor type II. We assumed that with a stress duration of 15 min one could detect hyper-responsivity, and with a stress duration of 2 h hypo-responsivity in mutant mouse lines. This was validated with two mutant lines known to show opposing effects on corticosterone secretion after stress exposure, corticotropin-releasing hormone (CRH) over-expressing mice and CRH receptor 1 knockout (KO) mice. Both lines showed the expected phenotype, i.e., increased stress responsivity in the CRH over-expressing mouse line (after 15 min restraint stress) and decreased stress responsivity in the CRHR1-KO mouse line (after 2 h of restraint stress). It is possible to repeat the acute stress test several times without the stressed animal adapting to it, and the behavioral response can be robustly evoked at different ages, in both sexes and in different mouse strains. Thus, locomotor and rearing behavior in the Open Field after an acute stress challenge can be used as reliable, non-invasive indicators of

ENU-mutagenesis mice with a non-synonymous mutation in Grin1 exhibit abnormal anxiety-like behaviors, impaired fear memory, and decreased acoustic startle response

PubMed Central

2013-01-01

Background The Grin1 (glutamate receptor, ionotropic, NMDA1) gene expresses a subunit of N-methyl-D-aspartate (NMDA) receptors that is considered to play an important role in excitatory neurotransmission, synaptic plasticity, and brain development. Grin1 is a candidate susceptibility gene for neuropsychiatric disorders, including schizophrenia, bipolar disorder, and attention deficit/hyperactivity disorder (ADHD). In our previous study, we examined an N-ethyl-N-nitrosourea (ENU)-generated mutant mouse strain (Grin1Rgsc174/Grin1+) that has a non-synonymous mutation in Grin1. These mutant mice showed hyperactivity, increased novelty-seeking to objects, and abnormal social interactions. Therefore, Grin1Rgsc174/Grin1+ mice may serve as a potential animal model of neuropsychiatric disorders. However, other behavioral characteristics related to these disorders, such as working memory function and sensorimotor gating, have not been fully explored in these mutant mice. In this study, to further investigate the behavioral phenotypes of Grin1Rgsc174/Grin1+ mice, we subjected them to a comprehensive battery of behavioral tests. Results There was no significant difference in nociception between Grin1Rgsc174/Grin1+ and wild-type mice. The mutants did not display any abnormalities in the Porsolt forced swim and tail suspension tests. We confirmed the previous observations that the locomotor activity of these mutant mice increased in the open field and home cage activity tests. They displayed abnormal anxiety-like behaviors in the light/dark transition and the elevated plus maze tests. Both contextual and cued fear memory were severely deficient in the fear conditioning test. The mutant mice exhibited slightly impaired working memory in the eight-arm radial maze test. The startle amplitude was markedly decreased in Grin1Rgsc174/Grin1+ mice, whereas no significant differences between genotypes were detected in the prepulse inhibition (PPI) test. The mutant mice showed no obvious

Male Fertility Defect Associated with Disrupted BRCA1-PALB2 Interaction in Mice*

PubMed Central

Simhadri, Srilatha; Peterson, Shaun; Patel, Dharm S.; Huo, Yanying; Cai, Hong; Bowman-Colin, Christian; Miller, Shoreh; Ludwig, Thomas; Ganesan, Shridar; Bhaumik, Mantu; Bunting, Samuel F.; Jasin, Maria; Xia, Bing

2014-01-01

PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis. PMID:25016020

Nootropic nefiracetam inhibits proconvulsant action of peripheral-type benzodiazepines in epileptic mutant EL mice.

PubMed

Nakamoto, Yurie; Shiotani, Tadashi; Watabe, Shigeo; Nabeshima, Toshitaka; Yoshii, Mitsunobu

2004-10-01

Piracetam and structurally related nootropics are known to potentiate the anticonvulsant effects of antiepileptic drugs. It remains to be seen, however, whether these nootropics inhibit proconvulsant actions of many toxic agents including Ro 5-4864, a specific agonist for peripheral-type benzodiazepine receptors (PBR). The present study was designed to address this issue using EL mice, an animal model of epilepsy. In behavioral pharmacological experiments, EL mice were highly susceptible to convulsions induced by Ro 5-4864 (i.p.) in comparison with nonepileptic DDY mice. Nefiracetam administered orally to EL mice inhibited spontaneous seizures. In DDY mice, convulsions induced by Ro 5-4864 were prevented by nefiracetam when administered by i.v. injection. Aniracetam (i.v.) was partially effective, but piracetam and oxiracetam were ineffective as anticonvulsants. Binding assay for brain tissues revealed a higher density of mitochondrial PBR in EL mice compared with DDY mice. Binding of the PBR ligands Ro 5-4864 to either EL or DDY mouse brain was inhibited by micromolar concentrations of these nootropic agents in the sequence of nefiracetam > aniracetam > oxiracetam, piracetam. This rank order is identical to potency as anticonvulsants. These data suggest that nefiracetam may prevent toxic effects of PBR agonists through interacting with PBR.

Accumulation of Mutant Neuroserpin Precedes Development of Clinical Symptoms in Familial Encephalopathy with Neuroserpin Inclusion Bodies

PubMed Central

Galliciotti, Giovanna; Glatzel, Markus; Kinter, Jochen; Kozlov, Serguei V.; Cinelli, Paolo; Rülicke, Thomas; Sonderegger, Peter

2007-01-01

Intracellular protein deposition due to aggregation caused by conformational alteration is the hallmark of a number of neurodegenerative disorders, including Parkinson’s disease, tauopathies, Huntington’s disease, and familial encephalopathy with neuroserpin inclusion bodies. The latter is an autosomal dominant disorder caused by point mutations in neuroserpin resulting in its destabilization. Mutant neuroserpin polymerizes and forms intracellular aggregates that eventually lead to neurodegeneration. We generated genetically modified mice expressing the late-onset S49P-Syracuse or the early-onset S52R-Portland mutation of neuroserpin in central nervous system neurons. Mice exhibited morphological, biochemical, and clinical features resembling those found in the human disease. Analysis of brains revealed large intraneuronal inclusions composed exclusively of mutant neuroserpin, accumulating long before the development of clinical symptoms in a time-dependent manner. Clinical symptoms and amount of neuroserpin inclusions correlated with the predicted instability of the protein. The presence of inclusion bodies in subclinical mice indicates that in humans the prevalence of the disease could be higher than anticipated. In addition to shedding light on the pathophysiology of the human disorder, these mice provide an excellent model to study mechanisms of neurodegeneration or establish novel therapies for familial encephalopathy with neuroserpin inclusion bodies and other neurodegenerative diseases with intracellular protein deposition. PMID:17392169

Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells.

PubMed

van Lidth de Jeude, J F; Meijer, B J; Wielenga, M C B; Spaan, C N; Baan, B; Rosekrans, S L; Meisner, S; Shen, Y H; Lee, A S; Paton, J C; Paton, A W; Muncan, V; van den Brink, G R; Heijmans, J

2017-06-15

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.

Immunogenicity and protection induced by a Mycobacterium tuberculosis sigE mutant in a BALB/c mouse model of progressive pulmonary tuberculosis.

PubMed

Hernandez Pando, Rogelio; Aguilar, Leon Diana; Smith, Issar; Manganelli, Riccardo

2010-07-01

Tuberculosis is still one of the main challenges to human global health, leading to about two million deaths every year. One of the reasons for its success is the lack of efficacy of the widely used vaccine Mycobacterium bovis BCG. In this article, we analyze the potential use of an attenuated mutant of Mycobacterium tuberculosis H37Rv lacking the sigma factor sigma(E) as a live vaccine. We have demonstrated that BALB/c mice infected by the intratracheal route with this mutant strain showed significantly higher survival rates and less tissue damage than animals infected with the parental or complemented mutant strain. Although animals infected with the sigE mutant had low bacillary loads, their lungs showed significantly higher production of the protective factors gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), inducible nitric oxide synthase (iNOS), and beta-defensins than those of animals infected with the parental or complemented mutant strain. Moreover, we demonstrate that the sigE mutant, when inoculated subcutaneously, was more attenuated than BCG in immunodeficient nude mice, thus representing a good candidate for a novel attenuated live vaccine strain. Finally, when we used the sigE mutant as a subcutaneous vaccine, it was able to induce a higher level of protection than did BCG with both H37Rv and a highly virulent strain of M. tuberculosis (Beijing code 9501000). Taken together, our findings suggest that the sigE mutant is a very promising strain for the development of a new vaccine against tuberculosis.

Attenuated Signature-Tagged Mutagenesis Mutants of Brucella melitensis Identified during the Acute Phase of Infection in Mice

PubMed Central

Lestrate, P.; Dricot, A.; Delrue, R.-M.; Lambert, C.; Martinelli, V.; De Bolle, X.; Letesson, J.-J.; Tibor, A.

2003-01-01

For this study, we screened 1,152 signature-tagged mutagenesis mutants of Brucella melitensis 16M in a mouse model of infection and found 36 of them to be attenuated in vivo. Molecular characterization of transposon insertion sites showed that for four mutants, the affected genes were only present in Rhizobiaceae. Another mutant contained a disruption in a gene homologous to mosA, which is involved in rhizopine biosynthesis in some strains of Rhizobium, suggesting that this sugar may be involved in Brucella pathogenicity. A mutant was disrupted in a gene homologous to fliF, a gene potentially coding for the MS ring, a basal component of the flagellar system. Surprisingly, a mutant was affected in the rpoA gene, coding for the essential α-subunit of the RNA polymerase. This disruption leaves a partially functional protein, impaired for the activation of virB transcription, as demonstrated by the absence of induction of the virB promoter in the Tn5::rpoA background. The results presented here highlight the fact that the ability of Brucella to induce pathogenesis shares similarities with the molecular mechanisms used by both Rhizobium and Agrobacterium to colonize their hosts. PMID:14638795

Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption.

PubMed

Canalis, Ernesto; Schilling, Lauren; Yee, Siu-Pok; Lee, Sun-Kyeong; Zanotti, Stefano

2016-01-22

Notch receptors are determinants of cell fate and function and play a central role in skeletal development and bone remodeling. Hajdu Cheney syndrome, a disease characterized by osteoporosis and fractures, is associated with NOTCH2 mutations resulting in a truncated stable protein and gain-of-function. We created a mouse model reproducing the Hajdu Cheney syndrome by introducing a 6955C→T mutation in the Notch2 locus leading to a Q2319X change at the amino acid level. Notch2(Q2319X) heterozygous mutants were smaller and had shorter femurs than controls; and at 1 month of age they exhibited cancellous and cortical bone osteopenia. As the mice matured, cancellous bone volume was restored partially in male but not female mice, whereas cortical osteopenia persisted in both sexes. Cancellous bone histomorphometry revealed an increased number of osteoclasts and bone resorption, without a decrease in osteoblast number or bone formation. Osteoblast differentiation and function were not affected in Notch2(Q2319X) cells. The pre-osteoclast cell pool, osteoclast differentiation, and bone resorption in response to receptor activator of nuclear factor κB ligand in vitro were increased in Notch2(Q2319X) mutants. These effects were suppressed by the γ-secretase inhibitor LY450139. In conclusion, Notch2(Q2319X) mice exhibit cancellous and cortical bone osteopenia, enhanced osteoclastogenesis, and increased bone resorption. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Endothelial cell leptin receptor mutant mice have hyperleptinemia and reduced tissue uptake

PubMed Central

Hsuchou, Hung; Jayaram, Bhavaani; Kastin, Abba J.; Wang, Yuping; Ouyang, Suidong; Pan, Weihong

2014-01-01

Hyperleptinemia is usually associated with obesity and leptin resistance. Endothelial cell leptin receptor knockout (ELKO) mice without a signaling membrane-bound leptin receptor in endothelia, however, have profound hyperleptinemia without signs of leptin resistance. Leptin mRNA in adipose tissue was unchanged. To test the hypothesis that the ELKO mutation results in delayed degradation and slowed excretion, we determined the kinetics of leptin transfer in groups of ELKO and wildtype mice after intravenous bolus injection of 125I-leptin and the reference substance 131I-albumin. The degradation pattern of 125I-leptin in serum and brain homogenates at different time points between 10-60 min was measured by HPLC and acid precipitation. Although ELKO mice had reduced uptake of 125I-leptin uptake by the brain and several peripheral organs, leptin was more stable in blood and tissue. There was no change in the rate of renal excretion. ELISA showed that serum soluble leptin receptor, known to antagonize leptin transport, had a 400-fold increase, probably contributing to the hyperleptinemia and reduced tissue uptake. Thus, the ELKO mutation unexpectedly increased the stability of leptin but suppressed its tissue uptake. These changes probably contribute to the known partial resistance of the ELKO mice to diet-induced obesity. PMID:23359322

Endothelial cell leptin receptor mutant mice have hyperleptinemia and reduced tissue uptake.

PubMed

Hsuchou, Hung; Jayaram, Bhavaani; Kastin, Abba J; Wang, Yuping; Ouyang, Suidong; Pan, Weihong

2013-07-01

Hyperleptinemia is usually associated with obesity and leptin resistance. Endothelial cell leptin receptor knockout (ELKO) mice without a signaling membrane-bound leptin receptor in endothelia, however, have profound hyperleptinemia without signs of leptin resistance. Leptin mRNA in adipose tissue was unchanged. To test the hypothesis that the ELKO mutation results in delayed degradation and slowed excretion, we determined the kinetics of leptin transfer in groups of ELKO and wildtype mice after intravenous bolus injection of (125) I-leptin and the reference substance (131) I-albumin. The degradation pattern of (125) I-leptin in serum and brain homogenates at different time points between 10 and 60 min was measured by HPLC and acid precipitation. Although ELKO mice had reduced uptake of (125) I-leptin uptake by the brain and several peripheral organs, leptin was more stable in blood and tissue. There was no change in the rate of renal excretion. ELISA showed that serum soluble leptin receptor, known to antagonize leptin transport, had a 400-fold increase, probably contributing to the hyperleptinemia and reduced tissue uptake. Thus, the ELKO mutation unexpectedly increased the stability of leptin but suppressed its tissue uptake. These changes probably contribute to the known partial resistance of the ELKO mice to diet-induced obesity. Copyright © 2013 Wiley Periodicals, Inc.

The orphan nuclear receptor small heterodimer partner is required for thiazolidinedione effects in leptin-deficient mice.

PubMed

Tseng, Hsiu-Ting; Park, Young Joo; Lee, Yoon Kwang; Moore, David D

2015-05-08

Small heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp(-/-)) double mutant mice. Both ob/ob and double mutant ob/ob;Shp(-/-) mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp(-/-) mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp(-/-) liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp(-/-) mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp(-/-) mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes. Our results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.

Virulence of Burkholderia mallei Quorum-Sensing Mutants

PubMed Central

Majerczyk, Charlotte; Kinman, Loren; Han, Tony; Bunt, Richard

2013-01-01

Many Proteobacteria use acyl-homoserine lactone-mediated quorum-sensing (QS) to activate specific sets of genes as a function of cell density. QS often controls the virulence of pathogenic species, and in fact a previous study indicated that QS was important for Burkholderia mallei mouse lung infections. To gain in-depth information on the role of QS in B. mallei virulence, we constructed and characterized a mutant of B. mallei strain GB8 that was unable to make acyl-homoserine lactones. The QS mutant showed virulence equal to that of its wild-type parent in an aerosol mouse infection model, and growth in macrophages was indistinguishable from that of the parent strain. Furthermore, we assessed the role of QS in B. mallei ATCC 23344 by constructing and characterizing a mutant strain producing AiiA, a lactonase enzyme that degrades acyl-homoserine lactones. Although acyl-homoserine lactone levels in cultures of this strain are very low, it showed full virulence. Contrary to the previous report, we conclude that QS is not required for acute B. mallei infections of mice. QS may be involved in some stage of chronic infections in the natural host of horses, or the QS genes may be remnants of the QS network in B. pseudomallei from which this host-adapted pathogen evolved. PMID:23429539

ketu mutant mice uncover an essential meiotic function for the ancient RNA helicase YTHDC2

PubMed Central

Jain, Devanshi; Puno, M Rhyan; Meydan, Cem; Lailler, Nathalie; Mason, Christopher E; Lima, Christopher D; Anderson, Kathryn V

2018-01-01

Mechanisms regulating mammalian meiotic progression are poorly understood. Here we identify mouse YTHDC2 as a critical component. A screen yielded a sterile mutant, ‘ketu’, caused by a Ythdc2 missense mutation. Mutant germ cells enter meiosis but proceed prematurely to aberrant metaphase and apoptosis, and display defects in transitioning from spermatogonial to meiotic gene expression programs. ketu phenocopies mutants lacking MEIOC, a YTHDC2 partner. Consistent with roles in post-transcriptional regulation, YTHDC2 is cytoplasmic, has 3′→5′ RNA helicase activity in vitro, and has similarity within its YTH domain to an N6-methyladenosine recognition pocket. Orthologs are present throughout metazoans, but are diverged in nematodes and, more dramatically, Drosophilidae, where Bgcn is descended from a Ythdc2 gene duplication. We also uncover similarity between MEIOC and Bam, a Bgcn partner unique to schizophoran flies. We propose that regulation of gene expression by YTHDC2-MEIOC is an evolutionarily ancient strategy for controlling the germline transition into meiosis. PMID:29360036

Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in mice.

PubMed

Monteiro, Gaby E R; Jansen van Vuren, Petrus; Wichgers Schreur, Paul J; Odendaal, Lieza; Clift, Sarah J; Kortekaas, Jeroen; Paweska, Janusz T

2018-04-02

The NSs protein encoded by the S segment of Rift Valley fever virus (RVFV) is the major virulence factor, counteracting the host innate antiviral defence. It contains five highly conserved cysteine residues at positions 39, 40, 149, 178 and 194, which are thought to stabilize the tertiary and quaternary structure of the protein. Here, we report significant differences between clinical, virological, histopathological and host gene responses in BALB/c mice infected with wild-type RVFV (wtRVFV) or a genetic mutant having a double cysteine-to-serine substitution at residues 39 and 40 of the NSs protein (RVFV-C39S/C40S). Mice infected with the wtRVFV developed a fatal acute disease; characterized by high levels of viral replication, severe hepatocellular necrosis, and massive up-regulation of transcription of genes encoding type I and -II interferons (IFN) as well as pro-apoptotic and pro-inflammatory cytokines. The RVFV-C39S/C40S mutant did not cause clinical disease and its attenuated virulence was consistent with virological, histopathological and host gene expression findings in BALB/c mice. Clinical signs in mice infected with viruses containing cysteine-to-serine substitutions at positions 178 or 194 were similar to those occurring in mice infected with the wtRVFV, while a mutant containing a substitution at position 149 caused mild, non-fatal disease in mice. As mutant RVFV-C39S/C40S showed an attenuated phenotype in mice, the molecular mechanisms behind this attenuation were further investigated. The results show that two mechanisms are responsible for the attenuation; (1) loss of the IFN antagonistic propriety characteristic of the wtRVFV NSs and (2) the inability of the attenuated mutant to degrade Proteine Kinase R (PKR). Copyright © 2018. Published by Elsevier B.V.

[Value of quantitative iodine-based material decomposition images with gemstone spectral CT imaging in the follow-up of patients with hepatocellular carcinoma after TACE treatment].

PubMed

Xing, Gusheng; Wang, Shuang; Li, Chenrui; Zhao, Xinming; Zhou, Chunwu

2015-03-01

To investigate the value of quantitative iodine-based material decomposition images with gemstone spectral CT imaging in the follow-up of patients with hepatocellular carcinoma (HCC) after transcatheter arterial chemoebolization (TACE). Consecutive 32 HCC patients with previous TACE treatment were included in this study. For the follow-up, arterial phase (AP) and venous phase (VP) dual-phase CT scans were performed with a single-source dual-energy CT scanner (Discovery CT 750HD, GE Healthcare). Iodine concentrations were derived from iodine-based material-decomposition images in the liver parenchyma, tumors and coagulation necrosis (CN) areas. The iodine concentration difference (ICD) between the arterial-phase (AP) and venal-phase (VP) were quantitatively evaluated in different tissues.The lesion-to-normal parenchyma iodine concentration ratio (LNR) was calculated. ROC analysis was performed for the qualitative evaluation, and the area under ROC (Az) was calculated to represent the diagnostic ability of ICD and LNR. In all the 32 HCC patients, the region of interesting (ROI) for iodine concentrations included liver parenchyma (n=42), tumors (n=28) and coagulation necrosis (n=24). During the AP the iodine concentration of CNs (median value 0.088 µg/mm(3)) appeared significantly higher than that of the tumors (0.064 µg/mm(3), P=0.022) and liver parenchyma (0.048 µg/mm(3), P=0.005). But it showed no significant difference between liver parenchyma and tumors (P=0.454). During the VP the iodine concentration in hepatic parenchyma (median value 0.181 µg/mm(3)) was significantly higher than that in CNs (0.140 µg/mm(3), P=0.042). There was no significant difference between liver parenchyma and tumors, CNs and tumors (both P>0.05). The median value of ICD in CNs was 0.006 µg/mm(3), significantly lower than that of the HCC (0.201 µg/mm(3), P<0.001) and hepatic parenchyma (0.117 µg/mm(3), P<0.001). The ICDs in tumors and hepatic parenchyma showed no significant

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

The effect of cerebellar transplantation and enforced physical activity on motor skills and spatial learning in adult Lurcher mutant mice.

PubMed

Cendelín, Jan; Korelusová, Ivana; Vozeh, Frantisek

2009-03-01

Lurcher mutant mice represent a model of olivocerebellar degeneration. They are used to investigate cerebellar functions, consequences of cerebellar degeneration and methods of therapy influencing them. The aim of the work was to assess the effect of foetal cerebellar graft transplantation, repeated enforced physical activity and the combination of both these types of treatment on motor skills, spontaneous motor activity and spatial learning ability in adult B6CBA Lurcher mice. Foetal cerebellar grafts were applied into the cerebellum of Lurchers in the form of solid tissue pieces. Enforced motor activity was realised through rotarod training. Motor functions were examined using bar, ladder and rotarod tests. Spatial learning was tested in the Morris water maze. Spontaneous motor activity in the open field was observed. The presence of the graft was examined histologically. Enforced physical activity led to moderate improvement of some motor skills and to a significant amelioration of spatial learning ability in Lurchers. The transplantation of cerebellar tissue did not influence motor functions significantly but led to an improvement of spatial learning ability. Mutual advancement of the effects of both types of treatment was not observed. Spontaneous motor activity was influenced neither by physical activity nor by the transplantation. Physical activity did not influence the graft survival and development. Because nerve sprouting and cell migration from the graft to the host cerebellum was poor, the functional effects of the graft should be explained with regard to its trophic influence rather than with any involvement of the grafted cells into neural circuitries.

Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice

PubMed Central

Kotajima-Murakami, Hiroko; Narumi, Sakae; Yuzaki, Michisuke; Yanagihara, Dai

2016-01-01

Lesions in the cerebellar vermis abolish acquisition of fear-conditioned bradycardia in animals and human patients. The δ2 glutamate receptor (GluD2) is predominantly expressed in cerebellar Purkinje cells. The mouse mutant ho15J carries a spontaneous mutation in GluD2 and these mice show a primary deficiency in parallel fiber-Purkinje cell synapses, multiple innervations of Purkinje cells by climbing fibers, and impairment of long-term depression. In the present study, we used ho15J mice to investigate the role of the cerebellum in fear-conditioned bradycardia. We recorded changes in heart rate of ho15J mice induced by repeated pairing of an acoustic (conditioned) stimulus (CS) with an aversive (unconditioned) stimulus (US). The mice acquired conditioned bradycardia on Day 1 of the CS-US phase, similarly to wild-type mice. However, the magnitude of the conditioned bradycardia was not stable in the mutant mice, but rather was exaggerated on Days 2–5 of the CS-US phase. We examined the effects of reversibly inactivating the cerebellum by injection of an antagonist against the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR). The antagonist abolished expression of conditioned responses in both wild-type and ho15J mice. We conclude that the GluD2 mutation in the ho15J mice affects stable retention of the acquired conditioned bradycardia. PMID:27820843

Prolonged longevity of hypopituitary dwarf mice.

PubMed

Bartke, A; Brown-Borg, H; Mattison, J; Kinney, B; Hauck, S; Wright, C

2001-01-01

In two types of mutant dwarf mice, congenital deficiencies in pituitary function are associated with remarkably increased life expectancy. In this review, we will describe the key phenotypic characteristics of these animals, the evidence that they exhibit delayed aging, and the mechanisms that are suspected to account for their prolonged longevity.

Selective internal radiotherapy (SIRT) versus transarterial chemoembolization (TACE) for the treatment of intrahepatic cholangiocellular carcinoma (CCC): study protocol for a randomized controlled trial.

PubMed

Kloeckner, Roman; Ruckes, Christian; Kronfeld, Kai; Wörns, Marcus Alexander; Weinmann, Arndt; Galle, Peter Robert; Lang, Hauke; Otto, Gerd; Eichhorn, Waltraud; Schreckenberger, Mathias; Dueber, Christoph; Pitton, Michael Bernhard

2014-08-06

Cholangiocellular carcinoma is the second most common primary liver cancer after hepatocellular carcinoma. Over the last 30 years, the incidence of intrahepatic cholangiocellular carcinoma has risen continuously worldwide. Meanwhile, the intrahepatic cholangiocellular carcinoma has become more common than the extrahepatic growth type and currently accounts for 10-15% of all primary hepatic malignancies. Intrahepatic cholangiocellular carcinoma is typically diagnosed in advanced stages due to late clinical symptoms and an absence of classic risk factors. A late diagnosis precludes curative surgical resection. There is evidence that transarterial chemoembolization leads to better local tumor control and prolongs survival compared to systemic chemotherapy. New data indicates that selective internal radiotherapy, also referred to as radioembolization, provides promising results for treating intrahepatic cholangiocellular carcinoma. This pilot study is a randomized, controlled, single center, phase II trial. Twenty-four patients with intrahepatic cholangiocellular carcinoma will be randomized in a 1:1 ratio to receive either chemoembolization or radioembolization. Randomization will be stratified according to tumor load. Progression-free survival is the primary endpoint; overall survival and time to progression are secondary endpoints. To evaluate treatment success, patients will receive contrast enhanced magnetic resonance imaging every 3 months. Currently, chemoembolization is routinely performed in many centers instead of systemic chemotherapy for treating intrahepatic cholangiocellular carcinoma confined to the liver. Recently, radioembolization has been increasingly applied to cholangiocellular carcinoma as second line therapy after TACE failure or even as an alternative first line therapy. Nonetheless, no randomized studies have compared radioembolization and chemoembolization. Considering all this background information, we recognized a strong need for a

Alanine–glyoxylate aminotransferase-deficient mice, a model for primary hyperoxaluria that responds to adenoviral gene transfer