Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers

PubMed Central

Fukada, So-ichiro; Yamaguchi, Masahiko; Kokubo, Hiroki; Ogawa, Ryo; Uezumi, Akiyoshi; Yoneda, Tomohiro; Matev, Miroslav M.; Motohashi, Norio; Ito, Takahito; Zolkiewska, Anna; Johnson, Randy L.; Saga, Yumiko; Miyagoe-Suzuki, Yuko; Tsujikawa, Kazutake; Takeda, Shin’ichi; Yamamoto, Hiroshi

2011-01-01

Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7+ MyoD– undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis. PMID:21989910

Absence of Tec Family Kinases Interleukin-2 Inducible T cell Kinase (Itk) and Bruton's Tyrosine Kinase (Btk) Severely Impairs FcϵRI-dependent Mast Cell Responses*

PubMed Central

Iyer, Archana S.; Morales, J. Luis; Huang, Weishan; Ojo, Folake; Ning, Gang; Wills, Elizabeth; Baines, Joel D.; August, Avery

2011-01-01

Mast cells are critical effector cells in the pathophysiology of allergic asthma and other IgE-mediated diseases. The Tec family of tyrosine kinases Itk and Btk serve as critical signal amplifiers downstream of antigen receptors. Although both kinases are expressed and activated in mast cells following FcϵRI stimulation, their individual contributions are not clear. To determine whether these kinases play unique and/or complementary roles in FcϵRI signaling and mast cell function, we generated Itk and Btk double knock-out mice. Analyses of these mice show decreased mast cell granularity and impaired passive systemic anaphylaxis responses. This impaired response is accompanied by a significant elevation in serum IgE in Itk/Btk double knock-out mice. In vitro analyses of bone marrow-derived mast cells (BMMCs) indicated that Itk/Btk double knock-out BMMCs are defective in degranulation and cytokine secretion responses downstream to FcϵRI activation. These responses were accompanied by a significant reduction in PLCγ2 phosphorylation and severely impaired calcium responses in these cells. This defect also results in altered NFAT1 nuclear localization in double knock-out BMMCs. Network analysis suggests that although they may share substrates, Itk plays both positive and negative roles, while Btk primarily plays a positive role in mast cell FcϵRI-induced cytokine secretion. PMID:21212279

Role of thin descending limb urea transport in renal urea handling and the urine concentrating mechanism

PubMed Central

Lei, Tianluo; Zhou, Lei; Layton, Anita T.; Zhou, Hong; Zhao, Xuejian; Bankir, Lise

2011-01-01

Urea transporters UT-A2 and UT-B are expressed in epithelia of thin descending limb of Henle's loop and in descending vasa recta, respectively. To study their role and possible interaction in the context of the urine concentration mechanism, a UT-A2 and UT-B double knockout (UT-A2/B knockout) mouse model was generated by targeted deletion of the UT-A2 promoter in embryonic stem cells with UT-B gene knockout. The UT-A2/B knockout mice lacked detectable UT-A2 and UT-B transcripts and proteins and showed normal survival and growth. Daily urine output was significantly higher in UT-A2/B knockout mice than that in wild-type mice and lower than that in UT-B knockout mice. Urine osmolality in UT-A2/B knockout mice was intermediate between that in UT-B knockout and wild-type mice. The changes in urine osmolality and flow rate, plasma and urine urea concentration, as well as non-urea solute concentration after an acute urea load or chronic changes in protein intake suggested that UT-A2 plays a role in the progressive accumulation of urea in the inner medulla. These results suggest that in wild-type mice UT-A2 facilitates urea absorption by urea efflux from the thin descending limb of short loops of Henle. Moreover, UT-A2 deletion in UT-B knockout mice partially remedies the urine concentrating defect caused by UT-B deletion, by reducing urea loss from the descending limbs to the peripheral circulation; instead, urea is returned to the inner medulla through the loops of Henle and the collecting ducts. PMID:21849488

Role of thin descending limb urea transport in renal urea handling and the urine concentrating mechanism.

PubMed

Lei, Tianluo; Zhou, Lei; Layton, Anita T; Zhou, Hong; Zhao, Xuejian; Bankir, Lise; Yang, Baoxue

2011-12-01

Urea transporters UT-A2 and UT-B are expressed in epithelia of thin descending limb of Henle's loop and in descending vasa recta, respectively. To study their role and possible interaction in the context of the urine concentration mechanism, a UT-A2 and UT-B double knockout (UT-A2/B knockout) mouse model was generated by targeted deletion of the UT-A2 promoter in embryonic stem cells with UT-B gene knockout. The UT-A2/B knockout mice lacked detectable UT-A2 and UT-B transcripts and proteins and showed normal survival and growth. Daily urine output was significantly higher in UT-A2/B knockout mice than that in wild-type mice and lower than that in UT-B knockout mice. Urine osmolality in UT-A2/B knockout mice was intermediate between that in UT-B knockout and wild-type mice. The changes in urine osmolality and flow rate, plasma and urine urea concentration, as well as non-urea solute concentration after an acute urea load or chronic changes in protein intake suggested that UT-A2 plays a role in the progressive accumulation of urea in the inner medulla. These results suggest that in wild-type mice UT-A2 facilitates urea absorption by urea efflux from the thin descending limb of short loops of Henle. Moreover, UT-A2 deletion in UT-B knockout mice partially remedies the urine concentrating defect caused by UT-B deletion, by reducing urea loss from the descending limbs to the peripheral circulation; instead, urea is returned to the inner medulla through the loops of Henle and the collecting ducts.

Double Knockout of the Na+-Driven Cl-/HCO3- Exchanger and Na+/Cl- Cotransporter Induces Hypokalemia and Volume Depletion.

PubMed

Sinning, Anne; Radionov, Nikita; Trepiccione, Francesco; López-Cayuqueo, Karen I; Jayat, Maximilien; Baron, Stéphanie; Cornière, Nicolas; Alexander, R Todd; Hadchouel, Juliette; Eladari, Dominique; Hübner, Christian A; Chambrey, Régine

2017-01-01

We recently described a novel thiazide-sensitive electroneutral NaCl transport mechanism resulting from the parallel operation of the Cl - /HCO 3 - exchanger pendrin and the Na + -driven Cl - /2HCO 3 - exchanger (NDCBE) in β-intercalated cells of the collecting duct. Although a role for pendrin in maintaining Na + balance, intravascular volume, and BP is well supported, there is no in vivo evidence for the role of NDCBE in maintaining Na + balance. Here, we show that deletion of NDCBE in mice caused only subtle perturbations of Na + homeostasis and provide evidence that the Na + /Cl - cotransporter (NCC) compensated for the inactivation of NDCBE. To unmask the role of NDCBE, we generated Ndcbe/Ncc double-knockout (dKO) mice. On a normal salt diet, dKO and single-knockout mice exhibited similar activation of the renin-angiotensin-aldosterone system, whereas only dKO mice displayed a lower blood K + concentration. Furthermore, dKO mice displayed upregulation of the epithelial sodium channel (ENaC) and the Ca 2+ -activated K + channel BKCa. During NaCl depletion, only dKO mice developed marked intravascular volume contraction, despite dramatically increased renin activity. Notably, the increase in aldosterone levels expected on NaCl depletion was attenuated in dKO mice, and single-knockout and dKO mice had similar blood K + concentrations under this condition. In conclusion, NDCBE is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation in mice. Furthermore, NDCBE has an important role in the prevention of hypokalemia. Because NCC and NDCBE are both thiazide targets, the combined inhibition of NCC and the NDCBE/pendrin system may explain thiazide-induced hypokalemia in some patients. Copyright © 2016 by the American Society of Nephrology.

Calorie restriction ameliorates neurodegenerative phenotypes in forebrain-specific presenilin-1 and presenilin-2 double knockout mice.

PubMed

Wu, Pu; Shen, Qian; Dong, Suzhen; Xu, Zhiliang; Tsien, Joe Z; Hu, Yinghe

2008-10-01

Conditional double knockout of presenilin-1 and presenilin-2 (cDKO) in forebrain of mice led to brain atrophy, tau hyperphosphorylation, synaptic dysfunction and cognitive deficit. These brain changes recapitulated most of the neurodegenerative phenotypes of Alzheimer's disease (AD). In this report, we have investigated the effects of 4-month calorie restriction (CR) regimen on different phenotypes in cDKO mice. We found that CR improved novel object recognition and contextual fear conditioning memory in the cDKO mice. Histological and biochemical analysis showed that CR attenuated ventricle enlargement, caspase-3 activation and astrogliosis. In addition, the induction of tau hyperphosphorylation in the cDKO mice was reduced by CR, possibly through reduction of p25 accumulation and aberrant CDK5 activation. Finally, DNA microarray analysis demonstrated that CR could increase the expression of neurogenesis related genes and decrease the expression of inflammation related genes in the hippocampus of cDKO mice. The possible molecular mechanisms of the CR effects on alleviating AD pathogenesis have been discussed.

Prostaglandin E2 Activates YAP and a Positive-Signaling Loop to Promote Colon Regeneration After Colitis but Also Carcinogenesis in Mice.

PubMed

Kim, Han-Byul; Kim, Minchul; Park, Young-Soo; Park, Intae; Kim, Tackhoon; Yang, Sung-Yeun; Cho, Charles J; Hwang, DaeHee; Jung, Jin-Hak; Markowitz, Sanford D; Hwang, Sung Wook; Yang, Suk-Kyun; Lim, Dae-Sik; Myung, Seung-Jae

2017-02-01

Prostaglandin E 2 (PGE 2 ) is mediator of inflammation that regulates tissue regeneration, but its continual activation has been associated with carcinogenesis. Little is known about factors in the PGE 2 signaling pathway that contribute to tumor formation. We investigated whether yes-associated protein 1 (YAP1), a transcriptional co-activator in the Hippo signaling pathway, mediates PGE 2 function. DLD-1 and SW480 colon cancer cell lines were transfected with vectors expressing transgenes or small hairpin RNAs and incubated with recombinant PGE 2 , with or without pharmacologic inhibitors of signaling proteins, and analyzed by immunoblot, immunofluorescence, quantitative reverse-transcription polymerase chain reaction, transcriptional reporter, and proliferation assays. Dextran sodium sulfate (DSS) was given to induce colitis in C57/BL6 (control) mice, as well as in mice with disruption of the hydroxyprostaglandin dehydrogenase 15 gene (15-PGDH-knockout mice), Yap1 gene (YAP-knockout mice), and double-knockout mice. Some mice also were given indomethacin to block PGE 2 synthesis. 15-PGDH knockout mice were crossed with mice with intestine-specific disruption of the salvador family WW domain containing 1 gene (Sav1), which encodes an activator of Hippo signaling. We performed immunohistochemical analyses of colon biopsy samples from 26 patients with colitis-associated cancer and 51 age-and sex-matched patients with colorectal cancer (without colitis). Incubation of colon cancer cell lines with PGE 2 led to phosphorylation of cyclic adenosine monophosphate-responsive element binding protein 1 and increased levels of YAP1 messenger RNA, protein, and YAP1 transcriptional activity. This led to increased transcription of the prostaglandin-endoperoxide synthase 2 gene (PTGS2 or cyclooxygenase 2) and prostaglandin E-receptor 4 gene (PTGER4 or EP4). Incubation with PGE 2 promoted proliferation of colon cancer cell lines, but not cells with knockdown of YAP1. Control mice developed colitis after administration of DSS, but injection of PGE 2 led to colon regeneration in these mice. However, YAP-knockout mice did not regenerate colon tissues and died soon after administration of DSS. 15-PGDH-knockout mice regenerated colon tissues more rapidly than control mice after withdrawal of DSS, and had faster recovery of body weight, colon length, and colitis histology scores. These effects were reversed by injection of indomethacin. SAV1-knockout or 15-PGDH-knockout mice did not develop spontaneous tumors after colitis induction, but SAV1/15-PGDH double-knockout mice developed polyps that eventually progressed to carcinoma in situ. Administration of indomethacin to these mice prevented spontaneous tumor formation. Levels of PGE 2 correlated with those of YAP levels in human sporadic colorectal tumors and colitis-associated tumors. PGE 2 signaling increases the expression and transcriptional activities of YAP1, leading to increased expression of cyclooxygenase 2 and EP4 to activate a positive signaling loop. This pathway promotes proliferation of colon cancer cell lines and colon tissue regeneration in mice with colitis. Constitutive activation of this pathway led to formation of polyps and colon tumors in mice. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

The Role of mDia1 in the Aberrant Innate Immune Signaling in del(5q) Myelodysplastic Syndromes

DTIC Science & Technology

2016-10-01

Myeloproliferative Neoplasms The goal of this project will be to identify transcriptional pathways that are dysregulated in PMF megakaryocytes and... myeloproliferative phenotype, as previously reported in miR-146 knockout mice. Here we propose that the mDia1/miR-146a double knockout mice phenocopy...ineffective erythropoiesis and represent a model of anemia that is commonly seen MDS. However, the possibility of a myeloproliferative phenotype cannot be

TRPV2 KNOCKOUT MICE ARE SUSCEPTIBLE TO PERINATAL LETHALITY BUT DISPLAY NORMAL THERMAL AND MECHANICAL NOCICEPTION

PubMed Central

Park, Una; Vastani, Nisha; Guan, Yun; Raja, Srinivasa N.; Koltzenburg, Martin; Caterina, Michael J.

2011-01-01

TRPV2 is a nonselective cation channel expressed prominently in medium- to large-diameter sensory neurons that can be activated by extreme heat (>52°C). These features suggest that TRPV2 might be a transducer of noxious heat in vivo. TRPV2 can also be activated by hypoosmolarity or cell stretch, suggesting potential roles in mechanotransduction. To address the physiological functions of TRPV2 in somatosensation, we generated TRPV2 knockout mice and examined their behavioral and electrophysiological responses to heat and mechanical stimuli. TRPV2 knockout mice showed reduced embryonic weight and perinatal viability. As adults, surviving knockout mice also exhibited a slightly reduced body weight. TRPV2 knockout mice showed normal behavioral responses to noxious heat over a broad range of temperatures and normal responses to punctate mechanical stimuli, both in the basal state and under hyperalgesic conditions such as peripheral inflammation and L5 spinal nerve ligation. Moreover, behavioral assays of TRPV1/TRPV2 double knockout mice or of TRPV2 knockout mice treated with resiniferatoxin to desensitize TRPV1-expressing afferents revealed no thermosensory consequences of TRPV2 absence. In line with behavioral findings, electrophysiological recordings from skin afferents showed that C-fiber responses to heat and C- and Aδ-fiber responses to noxious mechanical stimuli were unimpaired in the absence of TRPV2. The prevalence of thermosensitive Aδ-fibers was too low to permit comparison between genotypes. Thus, TRPV2 is important for perinatal viability but is not essential for heat or mechanical nociception or hypersensitivity in the adult mouse. PMID:21832173

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study.

PubMed

Chen, Yanyan; Xu, Yuanyuan; Zheng, Hongzhi; Fu, Jingqi; Hou, Yongyong; Wang, Huihui; Zhang, Qiang; Yamamoto, Masayuki; Pi, Jingbo

2016-09-09

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. Copyright © 2016 Elsevier Inc. All rights reserved.

Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice.

PubMed

Vandenbeuch, Aurelie; Larson, Eric D; Anderson, Catherine B; Smith, Steven A; Ford, Anthony P; Finger, Thomas E; Kinnamon, Sue C

2015-03-01

Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca(2+) transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 μm or 100 μm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish

PubMed Central

Ishikawa, Tokiro; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Kamei, Yasuhiro; Shigenobu, Shuji; Tanaka, Minoru; Saito, Taro L.; Yoshimura, Jun; Morishita, Shinichi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Taniguchi, Yoshihito; Takeda, Shunichi; Mori, Kazutoshi

2013-01-01

ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra. PMID:23447699

ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish.

PubMed

Ishikawa, Tokiro; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Kamei, Yasuhiro; Shigenobu, Shuji; Tanaka, Minoru; Saito, Taro L; Yoshimura, Jun; Morishita, Shinichi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Taniguchi, Yoshihito; Takeda, Shunichi; Mori, Kazutoshi

2013-05-01

ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.

Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure.

PubMed

Soleimani, Manoocher; Barone, Sharon; Xu, Jie; Shull, Gary E; Siddiqui, Faraz; Zahedi, Kamyar; Amlal, Hassane

2012-08-14

The Na-Cl cotransporter (NCC), which is the target of inhibition by thiazides, is located in close proximity to the chloride-absorbing transporter pendrin in the kidney distal nephron. Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these transporters are predominantly active during salt depletion or in response to excess aldosterone. We hypothesized that pendrin and NCC compensate for loss of function of the other under basal conditions, thereby masking the role that each plays in salt absorption. To test our hypothesis, we generated pendrin/NCC double knockout (KO) mice by crossing pendrin KO mice with NCC KO mice. Pendrin/NCC double KO mice displayed severe salt wasting and sharp increase in urine output under basal conditions. As a result, animals developed profound volume depletion, renal failure, and metabolic alkalosis without hypokalemia, which were all corrected with salt replacement. We propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without causing hypokalemia for patients with fluid overload, including patients with congestive heart failure, nephrotic syndrome, diuretic resistance, or generalized edema.

Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure

PubMed Central

Soleimani, Manoocher; Barone, Sharon; Xu, Jie; Shull, Gary E.; Siddiqui, Faraz; Zahedi, Kamyar; Amlal, Hassane

2012-01-01

The Na-Cl cotransporter (NCC), which is the target of inhibition by thiazides, is located in close proximity to the chloride-absorbing transporter pendrin in the kidney distal nephron. Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these transporters are predominantly active during salt depletion or in response to excess aldosterone. We hypothesized that pendrin and NCC compensate for loss of function of the other under basal conditions, thereby masking the role that each plays in salt absorption. To test our hypothesis, we generated pendrin/NCC double knockout (KO) mice by crossing pendrin KO mice with NCC KO mice. Pendrin/NCC double KO mice displayed severe salt wasting and sharp increase in urine output under basal conditions. As a result, animals developed profound volume depletion, renal failure, and metabolic alkalosis without hypokalemia, which were all corrected with salt replacement. We propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without causing hypokalemia for patients with fluid overload, including patients with congestive heart failure, nephrotic syndrome, diuretic resistance, or generalized edema. PMID:22847418

Toll-Like Receptor 3 Is Critical for Coxsackievirus B4-Induced Type 1 Diabetes in Female NOD Mice

PubMed Central

Thuma, Jean R.; Courreges, Maria C.; Benencia, Fabian; James, Calvin B.L.; Malgor, Ramiro; Kantake, Noriko; Mudd, William; Denlinger, Nathan; Nolan, Bret; Wen, Li; Schwartz, Frank L.

2015-01-01

Group B coxsackieviruses (CVBs) are involved in triggering some cases of type 1 diabetes mellitus (T1DM). However, the molecular mechanism(s) responsible for this remain elusive. Toll-like receptor 3 (TLR3), a receptor that recognizes viral double-stranded RNA, is hypothesized to play a role in virus-induced T1DM, although this hypothesis is yet to be substantiated. The objective of this study was to directly investigate the role of TLR3 in CVB-triggered T1DM in nonobese diabetic (NOD) mice, a mouse model of human T1DM that is widely used to study both spontaneous autoimmune and viral-induced T1DM. As such, we infected female wild-type (TLR3+/+) and TLR3 knockout (TLR3−/−) NOD mice with CVB4 and compared the incidence of diabetes in CVB4-infected mice with that of uninfected counterparts. We also evaluated the islets of uninfected and CVB4-infected wild-type and TLR3 knockout NOD mice by immunohistochemistry and insulitis scoring. TLR3 knockout mice were markedly protected from CVB4-induced diabetes compared with CVB4-infected wild-type mice. CVB4-induced T-lymphocyte-mediated insulitis was also significantly less severe in TLR3 knockout mice compared with wild-type mice. No differences in insulitis were observed between uninfected animals, either wild-type or TLR3 knockout mice. These data demonstrate for the first time that TLR3 is 1) critical for CVB4-induced T1DM, and 2) modulates CVB4-induced insulitis in genetically prone NOD mice. PMID:25422874

Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment

PubMed Central

Robinson, Stephen D.; Frenette, Paul S.; Rayburn, Helen; Cummiskey, Marge; Ullman-Culleré, Mollie; Wagner, Denisa D.; Hynes, Richard O.

1999-01-01

We extend our previous analyses of mice deficient in selectins by describing the generation and comparative phenotype of mice lacking one, two, or three selectins after sequential ablation of the murine genes encoding P-, E-, and L-selectins. All mice deficient in selectins are viable and fertile as homozygotes. However, mice missing both P- and E-selectins (PE−/−), and mice missing all three selectins (ELP−/−) develop mucocutaneous infections that eventually lead to death. Mice deficient in multiple selectins display varying degrees of leukocytosis, resulting in part from alterations in leukocyte rolling and recruitment. PE−/− mice, ELP−/− mice, and mice missing both P- and L-selectins (PL−/−) show drastic reductions in leukocyte rolling and in extravasation of neutrophils in thioglycollate-induced peritonitis. In a separate inflammatory model (ragweed-induced peritoneal eosinophilia), we demonstrate P-selectin to be both necessary and sufficient for the recruitment of eosinophils. The phenotype of mice missing both E- and L-selectins (EL−/−) is less severe than those seen in the other double knockouts. Comparisons among the double knockouts suggest that P-selectin normally cooperates with both E- and L-selectins. Our results indicate a preeminent role for P-selectin in regulating leukocyte behavior in mice. Data from the ELP−/− mice indicate, however, that all three selectins are important to leukocyte homeostasis and efficient neutrophil recruitment. PMID:10500197

Generating double knockout mice to model genetic intervention for diabetic cardiomyopathy in humans.

PubMed

Chavali, Vishalakshi; Nandi, Shyam Sundar; Singh, Shree Ram; Mishra, Paras Kumar

2014-01-01

Diabetes is a rapidly increasing disease that enhances the chances of heart failure twofold to fourfold (as compared to age and sex matched nondiabetics) and becomes a leading cause of morbidity and mortality. There are two broad classifications of diabetes: type1 diabetes (T1D) and type2 diabetes (T2D). Several mice models mimic both T1D and T2D in humans. However, the genetic intervention to ameliorate diabetic cardiomyopathy in these mice often requires creating double knockout (DKO). In order to assess the therapeutic potential of a gene, that specific gene is either overexpressed (transgenic expression) or abrogated (knockout) in the diabetic mice. If the genetic mice model for diabetes is used, it is necessary to create DKO with transgenic/knockout of the target gene to investigate the specific role of that gene in pathological cardiac remodeling in diabetics. One of the important genes involved in extracellular matrix (ECM) remodeling in diabetes is matrix metalloproteinase-9 (Mmp9). Mmp9 is a collagenase that remains latent in healthy hearts but induced in diabetic hearts. Activated Mmp9 degrades extracellular matrix (ECM) and increases matrix turnover causing cardiac fibrosis that leads to heart failure. Insulin2 mutant (Ins2+/-) Akita is a genetic model for T1D that becomes diabetic spontaneously at the age of 3-4 weeks and show robust hyperglycemia at the age of 10-12 weeks. It is a chronic model of T1D. In Ins2+/- Akita, Mmp9 is induced. To investigate the specific role of Mmp9 in diabetic hearts, it is necessary to create diabetic mice where Mmp9 gene is deleted. Here, we describe the method to generate Ins2+/-/Mmp9-/- (DKO) mice to determine whether the abrogation of Mmp9 ameliorates diabetic cardiomyopathy.

Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.

PubMed

Yan, C; Wang, P; DeMayo, J; DeMayo, F J; Elvin, J A; Carino, C; Prasad, S V; Skinner, S S; Dunbar, B S; Dube, J L; Celeste, A J; Matzuk, M M

2001-06-01

Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-beta superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15(-/-)) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15(-/-)Gdf9(-/-)) display oocyte loss and cysts and resemble Gdf9(-/-) mutants. In contrast, Bmp15(-/-)Gdf9(+/-) female mice have more severe fertility defects than Bmp15(-/-) females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and normal ovarian physiology.

Uterine Dysfunction in Biglycan and Decorin Deficient Mice Leads to Dystocia during Parturition

PubMed Central

Wu, Zhiping; Aron, Abraham W.; Macksoud, Elyse E.; Iozzo, Renato V.; Hai, Chi-Ming; Lechner, Beatrice E.

2012-01-01

Cesarean birth rates are rising. Uterine dysfunction, the exact mechanism of which is unknown, is a common indication for Cesarean delivery. Biglycan and decorin are two small leucine-rich proteoglycans expressed in the extracellular matrix of reproductive tissues and muscle. Mice deficient in biglycan display a mild muscular dystrophy, and, along with mice deficient in decorin, are models of Ehlers-Danlos Syndrome, a connective tissue anomaly associated with uterine rupture. As a variant of Ehlers-Danlos Syndrome is caused by a genetic mutation resulting in abnormal biglycan and decorin secretion, we hypothesized that biglycan and decorin play a role in uterine function. Thus, we assessed wild-type, biglycan, decorin and double knockout pregnancies for timing of birth and uterine function. Uteri were harvested at embryonic days 12, 15 and 18. Nonpregnant uterine samples of the same genotypes were assessed for tissue failure rate and spontaneous and oxytocin-induced contractility. We discovered that biglycan/decorin mixed double-knockout dams displayed dystocia, were at increased risk of delayed labor onset, and showed increased tissue failure in a predominantly decorin-dependent manner. In vitro spontaneous uterine contractile amplitude and oxytocin-induced contractile force were decreased in all biglycan and decorin knockout genotypes compared to wild-type. Notably, we found no significant compensation between biglycan and decorin using quantitative real time PCR or immunohistochemistry. We conclude that the biglycan/decorin mixed double knockout mouse is a model of dystocia and delayed labor onset. Moreover, decorin is necessary for uterine function in a dose-dependent manner, while biglycan exhibits partial compensatory mechanisms in vivo. Thus, this model is poised for use as a model for testing novel targets for preventive or therapeutic manipulation of uterine dysfunction. PMID:22253749

Increased 4-hydroxynonenal protein adducts in male GSTA4–4/PPAR-alpha double knockout mice enhance injury during early stages of alcoholic liver disease

USDA-ARS?s Scientific Manuscript database

To test the significance of lipid peroxidation in the development of alcoholic liver injury, an ethanol (EtOH) liquid diet was fed to male wild type 129/SvJ mice, and glutathione S-transferase A4-4 null (GSTA4-/-) mice for 40 d. GSTA4-/- mice were also crossed with peroxisome proliferator-activated ...

Hypothalamic-pituitary cytokine network.

PubMed

Kariagina, Anastasia; Romanenko, Dmitry; Ren, Song-Guang; Chesnokova, Vera

2004-01-01

Cytokines expressed in the brain and involved in regulating the hypothalamus-pituitary-adrenal (HPA) axis contribute to the neuroendocrine interface. Leukemia inhibitory factor (LIF) and LIF receptors are expressed in human pituitary cells and murine hypothalamus and pituitary. LIF potently induces pituitary proopiomelanocortin (POMC) gene transcription and ACTH secretion and potentiates CRH induction of POMC. In vivo, LIF, along with CRH, enhances POMC expression and ACTH secretion in response to emotional and inflammatory stress. To further elucidate specific roles for both CRH and LIF in activating the inflammatory HPA response, double-knockout mice (CRH/LIFKO) were generated by breeding the null mutants for each respective single gene. Inflammation produced by ip injection of lipopolysaccharide (1 microg/mouse) to double CRH and LIF-deficient mice elicited pituitary POMC induction similar to wild type and markedly higher than in single null animals (P<0.0.01). Double-knockout mice also demonstrated robust corticosterone response to inflammation. High pituitary POMC mRNA levels may reflect abundant TNFalpha, IL-1beta, and IL-6 activation observed in the hypothalamus and pituitary of these animals. Our results suggest that increased central proinflammatory cytokine expression can compensate for the impaired HPA axis function and activates inflammatory ACTH and corticosterone responses in mice-deficient in both CRH and LIF.

Alcoholic fatty liver is enhanced in CYP2A5 knockout mice: The role of the PPARα-FGF21 axis.

PubMed

Chen, Xue; Ward, Stephen C; Cederbaum, Arthur I; Xiong, Huabao; Lu, Yongke

2017-03-15

Cytochrome P450 2A5 (CYP2A5) is induced by ethanol, and the ethanol induction of CYP2A5 is regulated by nuclear factor-erythroid 2-related factor 2 (NRF2). Cyp2a5 knockout (Cyp2a5 -/- ) mice develop more severe alcoholic fatty liver than Cyp2a5 +/+ mice. Fibroblast growth factor 21 (FGF21), a PPARα-regulated liver hormone, is involved in hepatic lipid metabolism. Alcoholic and non-alcoholic fatty liver are enhanced in Pparα knockout (Pparα -/- ) mice. This study investigates the relationship between the PPARα-FGF21 axis and the enhanced alcoholic fatty liver in Cyp2a5 -/- mice. Mice were fed the Lieber-Decarli ethanol diet to induce alcoholic fatty liver. More severe alcoholic fatty liver disease was developed in Cyp2a5 -/- mice than in Cyp2a5 +/+ mice. Basal FGF21 levels were higher in Cyp2a5 -/- mice than in Cyp2a5 +/+ mice, but ethanol did not further increase the elevated FGF21 levels in Cyp2a5 -/- mice while FGF21 was induced by ethanol in Cyp2a5 +/+ mice. Basal levels of serum FGF21 were lower in Pparα -/- mice than in Pparα +/+ mice; ethanol induced FGF21 in Pparα +/+ mice but not in Pparα -/- mice, whereas ethanol induced hypertriglyceridemia in Pparα -/- mice but not in Pparα +/+ mice. Administration of recombinant FGF21 normalized serum FGF21 and triglyceride in Pparα -/- mice. Alcoholic fatty liver was enhanced in liver-specific Fgf21 knockout mice. Pparα and Cyp2a5 double knockout (Pparα -/- /Cyp2a5 -/- ) mice developed more severe alcoholic fatty liver than Pparα +/+ /Cyp2a5 -/- mice. These results suggest that CYP2A5 protects against the development of alcoholic fatty liver disease, and the PPARα-FGF21 axis contributes to the protective effects of CYP2A5 on alcoholic fatty liver disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel In Vivo Model for Combinatorial Fluorescence Labeling in Mouse Prostate

PubMed Central

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

2015-01-01

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

Novel In Vivo model for combinatorial fluorescence labeling in mouse prostate.

PubMed

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

2015-06-15

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

Iron misregulation and neurodegenerative disease in mouse models that lack iron regulatory proteins

PubMed Central

Ghosh, Manik C.; Zhang, De-Liang; Rouault, Tracey A.

2015-01-01

Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are two cytosolic proteins that maintain cellular iron homeostasis by binding to RNA stem loops known as iron responsive elements (IREs) that are found in the untranslated regions of target mRNAs that encode proteins involved in iron metabolism. IRPs modify expression of iron metabolism genes, and global and tissue-specific knockout mice have been made to evaluate the physiological significance of these iron regulatory proteins (Irps). Here, we will discuss the results of the studies that have been performed with mice engineered to lack expression of one or both Irps, and made in different strains using different methodologies. Both Irp1 and Irp2 knockout mice are viable, but the double knockout (Irp1−/−Irp2−/−) mice die before birth, indicating that these Irps play a crucial role in maintaining iron homeostasis. Irp1−/− mice develop polycythemia and pulmonary hypertension, and when these mice are challenged with a low iron diet, they die early of abdominal hemorrhages, suggesting that Irp1 plays an essential role in erythropoiesis and in the pulmonary and cardiovascular systems. Irp2−/− mice develop microcytic anemia, erythropoietic protoporphyria and a progressive neurological disorder, indicating that Irp2 has important functions in the nervous system and erythropoietic homeostasis. Several excellent review articles have recently been published on Irp knockout mice that mainly focus on Irp1−/− mice (referenced in the introduction). In this review, we will briefly describe the phenotypes and physiological implications of Irp1−/− mice, and will discuss the phenotypes observed for Irp2−/− mice in detail with a particular emphasis on the neurological problems of these mice. PMID:25771171

Attenuation of neurodegenerative phenotypes in Alzheimer-like presenilin 1/presenilin 2 conditional double knockout mice by EUK1001, a promising derivative of xanomeline.

PubMed

Wang, Dong; Yang, Liguo; Su, Jingjing; Niu, Yan; Lei, Xiaoping; Xiong, Juan; Cao, Xiaohua; Hu, Yinghe; Mei, Bing; Hu, Jin-Feng

2011-07-01

The M1/M4-preferring muscarinic agonist xanomeline was found to have some benefit in the treatment of the memory impairment of Alzheimer's disease (AD), but side effects precluded further development. EUK1001, a fluorinated derivative of xanomeline, because of greater affinity for M1 muscarinic receptors, is likely to have a significantly better side effect profile than xanomeline. We have now studied the effects of 3-month chronic administration of EUK1001 and xanomeline (0.5mg/kg/day) in AD-like presenilin 1/presenilin 2 conditional double knockout (PS cDKO) mice. Only EUK1001 was found to significantly ameliorate the deficit in recognition memory. Histological analysis demonstrated partial attenuation of the brain atrophy in EUK1001-treated PS cDKO mice and minimal effect in the xanomeline-treated mice. Both compounds effectively suppressed the elevation of brain tau phosphorylation in the PS cDKO mice, but neither inhibited the increased inflammatory responses. These results indicate that EUK1001 showed superiority to xanomeline with regard to attenuation of several AD-like neurodegenerative phenotypes in PS cDKO mice. These results suggest further investigation of the development of EUK1001 for the treatment of AD is indicated. Copyright © 2011 Elsevier Inc. All rights reserved.

A miniature mechanical ventilator for newborn mice.

PubMed

Kolandaivelu, K; Poon, C S

1998-02-01

Transgenic/knockout mice with pre-defined mutations have become increasingly popular in biomedical research as models of human diseases. In some instances, the resulting mutation may cause cardiorespiratory distress in the neonatal or adult animals and may necessitate resuscitation. Here we describe the design and testing of a miniature and versatile ventilator that can deliver varying ventilatory support modes, including conventional mechanical ventilation and high-frequency ventilation, to animals as small as the newborn mouse. With a double-piston body chamber design, the device circumvents the problem of air leakage and obviates the need for invasive procedures such as endotracheal intubation, which are particularly important in ventilating small animals. Preliminary tests on newborn mice as early as postnatal day O demonstrated satisfactory restoration of pulmonary ventilation and the prevention of respiratory failure in mutant mice that are prone to respiratory depression. This device may prove useful in the postnatal management of transgenic/knockout mice with genetically inflicted respiratory disorders.

Epistatic interaction between the lipase-encoding genes Pnpla2 and Lipe causes liposarcoma in mice

PubMed Central

Wang, Shu Pei; Yang, Hao; Ji, Bo; Gladdy, Rebecca; Andelfinger, Gregor; Mitchell, Grant A.

2017-01-01

Liposarcoma is an often fatal cancer of fat cells. Mechanisms of liposarcoma development are incompletely understood. The cleavage of fatty acids from acylglycerols (lipolysis) has been implicated in cancer. We generated mice with adipose tissue deficiency of two major enzymes of lipolysis, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), encoded respectively by Pnpla2 and Lipe. Adipocytes from double adipose knockout (DAKO) mice, deficient in both ATGL and HSL, showed near-complete deficiency of lipolysis. All DAKO mice developed liposarcoma between 11 and 14 months of age. No tumors occurred in single knockout or control mice. The transcriptome of DAKO adipose tissue showed marked differences from single knockout and normal controls as early as 3 months. Gpnmb and G0s2 were among the most highly dysregulated genes in premalignant and malignant DAKO adipose tissue, suggesting a potential utility as early markers of the disease. Similar changes of GPNMB and G0S2 expression were present in a human liposarcoma database. These results show that a previously-unknown, fully penetrant epistatic interaction between Pnpla2 and Lipe can cause liposarcoma in mice. DAKO mice provide a promising model for studying early premalignant changes that lead to late-onset malignant disease. PMID:28459858

Oxidant stress in mitochondrial DNA damage, autophagy and inflammation in atherosclerosis

PubMed Central

Ding, Zufeng; Liu, Shijie; Wang, Xianwei; Khaidakov, Magomed; Dai, Yao; Mehta, Jawahar L.

2013-01-01

Our studies in HUVECs show that ox-LDL induced autophagy and damaged mtDNA leading to TLR9 expression. LOX-1 antibody or the ROS inhibitor apocynin attenuated ox-LDL-mediated autophagy, mtDNA damage and TLR9 expression, suggesting that these events are LOX-1 and ROS-dependent phenomena. Experiments using siRNA to DNase II indicated that DNase II digests mtDNA to protect the tissue from inflammation. Next, we studied and found intense autophagy, TLR9 expression and inflammatory signals (CD45 and CD68) in the aortas of LDLR knockout mice fed high cholesterol diet. Deletion of LOX-1 (LDLR/LOX-1 double knockout mice) attenuated autophagy, TLR9 expression as well as CD45 and CD68. Damaged mtDNA signal was also very high in LDLR knockout mice aortas, and this signal was attenuated by LOX-1 deletion. Thus, it appears that oxidative stress-mediated damaged mtDNA that escapes autophagy induces a potent inflammatory response in atherosclerosis. PMID:23326634

Probucol prevents early coronary heart disease and death in the high-density lipoprotein receptor SR-BI/apolipoprotein E double knockout mouse

PubMed Central

Braun, Anne; Zhang, Songwen; Miettinen, Helena E.; Ebrahim, Shamsah; Holm, Teresa M.; Vasile, Eliza; Post, Mark J.; Yoerger, Danita M.; Picard, Michael H.; Krieger, Joshua L.; Andrews, Nancy C.; Simons, Michael; Krieger, Monty

2003-01-01

Mice with homozygous null mutations in the high-density lipoprotein receptor SR-BI (scavenger receptor class B, type I) and apolipoprotein E genes fed a low-fat diet exhibit a constellation of pathologies shared with human atherosclerotic coronary heart disease (CHD): hypercholesterolemia, occlusive coronary atherosclerosis, myocardial infarctions, cardiac dysfunction (heart enlargement, reduced systolic function and ejection fraction, and ECG abnormalities), and premature death (mean age 6 weeks). They also exhibit a block in RBC maturation and abnormally high plasma unesterified-to-total cholesterol ratio (0.8) with associated abnormal lipoprotein morphology (lamellar/vesicular and stacked discoidal particles reminiscent of those in lecithin/cholesterol acyltransferase deficiency and cholestasis). Treatment with the lipid-lowering, antiatherosclerosis, and antioxidation drug probucol extended life to as long as 60 weeks (mean 36 weeks), and at 5–6 weeks of age, virtually completely reversed the cardiac and most RBC pathologies and corrected the unesterified to total cholesterol ratio (0.3) and associated distinctive abnormal lipoprotein morphologies. Manipulation of the timing of administration and withdrawal of probucol could control the onset of death and suggested that critical pathological changes usually occurred in untreated double knockout mice between ≈3 (weaning) and 5 weeks of age and that probucol delayed heart failure even after development of substantial CHD. The ability of probucol treatment to modulate pathophysiology in the double knockout mice enhances the potential of this murine system for analysis of the pathophysiology of CHD and preclinical testing of new approaches for the prevention and treatment of cardiovascular disease. PMID:12771386

Ibuprofen partially attenuates neurodegenerative symptoms in presenilin conditional double-knockout mice.

PubMed

Dong, Z; Yan, L; Huang, G; Zhang, L; Mei, B; Meng, B

2014-06-13

Ibuprofen is a widely used nonsteroidal anti-inflammatory drug that reportedly reduces the risk of Alzheimer's disease (AD) development. The anti-inflammatory effect of ibuprofen occurred via inhibition of cyclooxygenases and anti-amyloidogenesis through modulation of γ-secretase. Presenilin 1 and 2 conditional double-knockout (cDKO) mice exhibited age-dependent memory impairment and forebrain degeneration without elevation of amyloid β deposition. Therefore, cDKO mice can be an ideal animal model on which to independently test the effects of ibuprofen anti-inflammatory properties on the prevention of AD. Three- and six-month-old cDKO mice were fed diet containing 375 ppm ibuprofen for six months. After multiple, well-validated behavioral tests, treatment with ibuprofen improved cognition-related behavioral performance, and drug efficacy was correlated with the timing of administration. Ibuprofen was more effective on six-month-old than on three-month-old cDKO mice. Biochemical analysis demonstrated that the effects of ibuprofen on glial fibrillary acidic protein and CD68 expression levels were uneven in different brain regions of cDKO mice and that age also influenced such effects. Tau hyperphosphorylation and the cleavage of caspase-3 decreased after ibuprofen treatment, and this effect was more significant in the older than the younger group of mice, which was consistent with the results of behavioral tests. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Alcoholic fatty liver is enhanced in CYP2A5 knockout mice: the role of the PPARα-FGF21 axis

PubMed Central

Chen, Xue; Ward, Stephen C.; Cederbaum, Arthur I.; Xiong, Huabao; Lu, Yongke

2017-01-01

Background & Aims Cytochrome P450 2A5 (CYP2A5) is induced by ethanol, and the ethanol induction of CYP2A5 is regulated by nuclear factor-erythroid 2-related factor 2 (NRF2). Cyp2a5 knockout (Cyp2a5−/−) mice develop more severe alcoholic fatty liver than Cyp2a5+/+ mice. Fibroblast growth factor 21 (FGF21), a PPARα-regulated liver hormone, is involved in hepatic lipid metabolism. Alcoholic and non-alcoholic fatty liver are enhanced in Pparα knockout (Pparα−/−) mice. This study investigates the relationship between the PPARα-FGF21 axis and the enhanced alcoholic fatty liver in Cyp2a5−/− mice. Methods Mice were fed the Lieber-Decarli ethanol diet to induce alcoholic fatty liver. Results More severe alcoholic fatty liver disease was developed in Cyp2a5−/− mice than in Cyp2a5+/+ mice. Basal FGF21 levels were higher in Cyp2a5−/− mice than in Cyp2a5+/+ mice, but ethanol did not further increase the elevated FGF21 levels in Cyp2a5−/− mice while FGF21 was induced by ethanol in Cyp2a5+/+ mice. Basal levels of serum FGF21 were lower in Pparα−/− mice than in Pparα+/+ mice; ethanol induced FGF21 in Pparα+/+ mice but not in Pparα−/− mice, whereas ethanol induced hypertriglyceridemia in Pparα−/− mice but not in Pparα+/+ mice. Administration of recombinant FGF21 normalized serum FGF21 and triglyceride in Pparα−/− mice. Alcoholic fatty liver was enhanced in liver-specific Fgf21 knockout mice. Pparα and Cyp2a5 double knockout (Pparα−/−/Cyp2a5−/−) mice developed more severe alcoholic fatty liver than Pparα+/+/Cyp2a5−/− mice. Conclusions These results suggest that CYP2A5 protects against the development of alcoholic fatty liver disease, and the PPARα-FGF21 axis contributes to the protective effects of CYP2A5 on alcoholic fatty liver disease. PMID:28131861

Double Knockout of the Na+-Driven Cl−/HCO3− Exchanger and Na+/Cl− Cotransporter Induces Hypokalemia and Volume Depletion

PubMed Central

Sinning, Anne; Radionov, Nikita; Trepiccione, Francesco; López-Cayuqueo, Karen I.; Jayat, Maximilien; Baron, Stéphanie; Cornière, Nicolas; Alexander, R. Todd; Hadchouel, Juliette; Eladari, Dominique; Hübner, Christian A.

2017-01-01

We recently described a novel thiazide–sensitive electroneutral NaCl transport mechanism resulting from the parallel operation of the Cl−/HCO3− exchanger pendrin and the Na+–driven Cl−/2HCO3− exchanger (NDCBE) in β-intercalated cells of the collecting duct. Although a role for pendrin in maintaining Na+ balance, intravascular volume, and BP is well supported, there is no in vivo evidence for the role of NDCBE in maintaining Na+ balance. Here, we show that deletion of NDCBE in mice caused only subtle perturbations of Na+ homeostasis and provide evidence that the Na+/Cl− cotransporter (NCC) compensated for the inactivation of NDCBE. To unmask the role of NDCBE, we generated Ndcbe/Ncc double–knockout (dKO) mice. On a normal salt diet, dKO and single-knockout mice exhibited similar activation of the renin-angiotensin-aldosterone system, whereas only dKO mice displayed a lower blood K+ concentration. Furthermore, dKO mice displayed upregulation of the epithelial sodium channel (ENaC) and the Ca2+–activated K+ channel BKCa. During NaCl depletion, only dKO mice developed marked intravascular volume contraction, despite dramatically increased renin activity. Notably, the increase in aldosterone levels expected on NaCl depletion was attenuated in dKO mice, and single-knockout and dKO mice had similar blood K+ concentrations under this condition. In conclusion, NDCBE is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation in mice. Furthermore, NDCBE has an important role in the prevention of hypokalemia. Because NCC and NDCBE are both thiazide targets, the combined inhibition of NCC and the NDCBE/pendrin system may explain thiazide-induced hypokalemia in some patients. PMID:27151921

Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

PubMed

Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie

2014-10-01

Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure. © 2014 American Heart Association, Inc.

Genetic induction of phosphate toxicity significantly reduces the survival of hypercholesterolemic obese mice

PubMed Central

Ohnishi, Mutsuko; Kato, Shigeko; Razzaque, M. Shawkat

2013-01-01

Objective The adverse effects of metabolic disorders in obesity have been extensively studied; however, the pathologic effects of hyperphosphatemia or phosphate toxicity in obesity have not been studied in similar depth and detail, chiefly because such an association is thought to be uncommon. Studies have established that the incidence of obesity-associated nephropathy is increasing. Because hyperphosphatemia is a major consequence of renal impairment, this study determines the in vivo effects of hyperphosphatemia in obesity. Methods and results We genetically induced hyperphosphatemia in leptin-deficient obese (ob/ob) mice by generating ob/ob and klotho double knockout [ob/ob-klotho−/−] mice. As a control, we made ob/ob mice with hypophosphatemia by generating ob/ob and 1-alpha hydroxylase double knockout [ob/ob-1α(OH)ase−/−] mice. Compared to the wild-type mice, all three obese background mice, namely ob/ob, ob/ob-klotho−/−, and ob/ob-1α(OH)ase−/− mice developed hypercholesterolemia. In addition, the hyperphosphatemic, ob/ob-klotho−/− genetic background induced generalized tissue atrophy and widespread soft-tissue and vascular calcifications, which led to a shorter lifespan; no such changes were observed in the hypophosphatemic, ob/ob-1α(OH)ase−/− mice. Significantly, in contrast to the reduced survival of the ob/ob-klotho−/− mice, lowering serum phosphate levels in ob/ob-1α(OH)ase−/− mice showed no such compromised survival, despite both mice being hypercholesterolemic. Conclusion These genetic manipulation studies suggest phosphate toxicity is an important risk factor in obesity that can adversely affect survival. PMID:22037453

Genetic induction of phosphate toxicity significantly reduces the survival of hypercholesterolemic obese mice.

PubMed

Ohnishi, Mutsuko; Kato, Shigeko; Razzaque, M Shawkat

2011-11-25

The adverse effects of metabolic disorders in obesity have been extensively studied; however, the pathologic effects of hyperphosphatemia or phosphate toxicity in obesity have not been studied in similar depth and detail, chiefly because such an association is thought to be uncommon. Studies have established that the incidence of obesity-associated nephropathy is increasing. Because hyperphosphatemia is a major consequence of renal impairment, this study determines the in vivo effects of hyperphosphatemia in obesity. We genetically induced hyperphosphatemia in leptin-deficient obese (ob/ob) mice by generating ob/ob and klotho double knockout [ob/ob-klotho(-/-)] mice. As a control, we made ob/ob mice with hypophosphatemia by generating ob/ob and 1-alpha hydroxylase double knockout [ob/ob-1α(OH)ase(-/-)] mice. Compared to the wild-type mice, all three obese background mice, namely ob/ob, ob/ob-klotho(-/-), and ob/ob-1α(OH)ase(-/-) mice developed hypercholesterolemia. In addition, the hyperphosphatemic, ob/ob-klotho(-/-) genetic background induced generalized tissue atrophy and widespread soft-tissue and vascular calcifications, which led to a shorter lifespan; no such changes were observed in the hypophosphatemic, ob/ob-1α(OH)ase(-/-) mice. Significantly, in contrast to the reduced survival of the ob/ob-klotho(-/-) mice, lowering serum phosphate levels in ob/ob-1α(OH)ase(-/-) mice showed no such compromised survival, despite both mice being hypercholesterolemic. These genetic manipulation studies suggest phosphate toxicity is an important risk factor in obesity that can adversely affect survival. Copyright © 2011 Elsevier Inc. All rights reserved.

Heteromeric Canonical Transient Receptor Potential 1 and 4 Channels Play a Critical Role in Epileptiform Burst Firing and Seizure-Induced Neurodegeneration

PubMed Central

Phelan, Kevin D.; Mock, Matthew M.; Kretz, Oliver; Shwe, U. Thaung; Kozhemyakin, Maxim; Greenfield, L. John; Dietrich, Alexander; Birnbaumer, Lutz; Freichel, Marc; Flockerzi, Veit

2012-01-01

Canonical transient receptor potential channels (TRPCs) are receptor-operated cation channels that are activated in response to phospholipase C signaling. Although TRPC1 is ubiquitously expressed in the brain, TRPC4 expression is the most restrictive, with the highest expression level limited to the lateral septum. The subunit composition of neuronal TRPC channels remains uncertain because of conflicting data from recombinant expression systems. Here we report that the large depolarizing plateau potential that underlies the epileptiform burst firing induced by metabotropic glutamate receptor agonists in lateral septal neurons was completely abolished in TRPC1/4 double-knockout mice, and was abolished in 74% of lateral septal neurons in TRPC1 knockout mice. Furthermore, neuronal cell death in the lateral septum and the cornu ammonis 1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in TRPC1/4 double-knockout mice. Our data suggest that both TRPC1 and TRPC4 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons, possibly as heteromeric channels. Moreover, excitotoxic neuronal cell death, an underlying process for many neurological diseases, is not mediated merely by ionotropic glutamate receptors but also by heteromeric TRPC channels activated by metabotropic glutamate receptors. TRPC channels could be an unsuspected but critical molecular target for clinical intervention for excitotoxicity. PMID:22144671

Heteromeric canonical transient receptor potential 1 and 4 channels play a critical role in epileptiform burst firing and seizure-induced neurodegeneration.

PubMed

Phelan, Kevin D; Mock, Matthew M; Kretz, Oliver; Shwe, U Thaung; Kozhemyakin, Maxim; Greenfield, L John; Dietrich, Alexander; Birnbaumer, Lutz; Freichel, Marc; Flockerzi, Veit; Zheng, Fang

2012-03-01

Canonical transient receptor potential channels (TRPCs) are receptor-operated cation channels that are activated in response to phospholipase C signaling. Although TRPC1 is ubiquitously expressed in the brain, TRPC4 expression is the most restrictive, with the highest expression level limited to the lateral septum. The subunit composition of neuronal TRPC channels remains uncertain because of conflicting data from recombinant expression systems. Here we report that the large depolarizing plateau potential that underlies the epileptiform burst firing induced by metabotropic glutamate receptor agonists in lateral septal neurons was completely abolished in TRPC1/4 double-knockout mice, and was abolished in 74% of lateral septal neurons in TRPC1 knockout mice. Furthermore, neuronal cell death in the lateral septum and the cornu ammonis 1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in TRPC1/4 double-knockout mice. Our data suggest that both TRPC1 and TRPC4 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons, possibly as heteromeric channels. Moreover, excitotoxic neuronal cell death, an underlying process for many neurological diseases, is not mediated merely by ionotropic glutamate receptors but also by heteromeric TRPC channels activated by metabotropic glutamate receptors. TRPC channels could be an unsuspected but critical molecular target for clinical intervention for excitotoxicity.

Hypervitaminosis D mediates compensatory Ca2+ hyperabsorption in TRPV5 knockout mice.

PubMed

Renkema, Kirsten Y; Nijenhuis, Tom; van der Eerden, Bram C J; van der Kemp, Annemiete W C M; Weinans, Harrie; van Leeuwen, Johannes P T M; Bindels, René J M; Hoenderop, Joost G J

2005-11-01

Vitamin D plays an important role in Ca(2+) homeostasis by controlling Ca(2+) (re)absorption in intestine, kidney, and bone. The epithelial Ca(2+) channel TRPV5 mediates the Ca(2+) entry step in active Ca(2+) reabsorption. TRPV5 knockout (TRPV5(-/-)) mice show impaired Ca(2+) reabsorption, hypercalciuria, hypervitaminosis D, and intestinal hyperabsorption of Ca(2+). Moreover, these mice demonstrate upregulation of intestinal TRPV6 and calbindin-D(9K) expression compared with wild-type mice. For addressing the role of the observed hypervitaminosis D in the maintenance of Ca(2+) homeostasis and the regulation of expression levels of the Ca(2+) transport proteins in kidney and intestine, TRPV5/25-hydroxyvitamin-D(3)-1alpha-hydroxylase double knockout (TRPV5(-/-)/1alpha-OHase(-/-)) mice, which show undetectable serum 1,25(OH)(2)D(3) levels, were generated. TRPV5(-/-)/1alpha-OHase(-/-) mice displayed a significant hypocalcemia compared with wild-type mice (1.10 +/- 0.02 and 2.54 +/- 0.01 mM, respectively; P < 0.05). mRNA levels of renal calbindin-D(28K) (7 +/- 2%), calbindin-D(9K) (32 +/- 4%), Na(+)/Ca(2+) exchanger (12 +/- 2%), and intestinal TRPV6 (40 +/- 8%) and calbindin-D(9K) (26 +/- 4%) expression levels were decreased compared with wild-type mice. Hyperparathyroidism and rickets were present in TRPV5(-/-)/1alpha-OHase(-/-) mice, more pronounced than observed in single TRPV5 or 1alpha-OHase knockout mice. It is interesting that a renal Ca(2+) leak, as demonstrated in TRPV5(-/-) mice, persisted in TRPV5(-/-)/1alpha-OHase(-/-) mice, but a compensatory upregulation of intestinal Ca(2+) transporters was abolished. In conclusion, the elevation of serum 1,25(OH)(2)D(3) levels in TRPV5(-/-) mice is responsible for the upregulation of intestinal Ca(2+) transporters and Ca(2+) hyperabsorption. Hypervitaminosis D, therefore, is of crucial importance to maintain normocalcemia in impaired Ca(2+) reabsorption in TRPV5(-/-) mice.

Differential regulation of primary afferent input to spinal cord by muscarinic receptor subtypes delineated using knockout mice.

PubMed

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2014-05-16

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential Regulation of Primary Afferent Input to Spinal Cord by Muscarinic Receptor Subtypes Delineated Using Knockout Mice*

PubMed Central

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2014-01-01

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs. PMID:24695732

Roles of HAUSP-mediated p53 regulation in central nervous system development.

PubMed

Kon, N; Zhong, J; Kobayashi, Y; Li, M; Szabolcs, M; Ludwig, T; Canoll, P D; Gu, W

2011-08-01

The deubiquitinase HAUSP (herpesvirus-associated ubiquitin-specific protease; also called USP7) has a critical role in regulating the p53-Mdm2 (murine double minute 2) pathway. By using the conventional knockout approach, we previously showed that hausp inactivation leads to early embryonic lethality. To fully understand the physiological functions of hausp, we have generated mice lacking hausp specifically in the brain and examined the impacts of this manipulation on brain development. We found that deletion of hausp in neural cells resulted in neonatal lethality. The brains from these mice displayed hypoplasia and deficiencies in development, which were mainly caused by p53-mediated apoptosis. Detailed analysis also showed an increase of both p53 levels and p53-dependent transcriptional activation in hausp knockout brains. Notably, neural cell survival and brain development of hausp-mutant mice can largely be restored in the p53-null background. Nevertheless, in contrast to the case of mdm2- and mdm4 (murine double minute 4)-mutant mice, inactivation of p53 failed to completely rescue the neonatal lethality of these hausp-mutant mice. These results indicate that HAUSP-mediated p53 regulation is crucial for brain development, and also suggest that both the p53-dependent and the p53-independent functions of HAUSP contribute to the neonatal lethality of hausp-mutant mice.

Deoxynucleoside stress exacerbates the phenotype of a mouse model of mitochondrial neurogastrointestinal encephalopathy

PubMed Central

Garcia-Diaz, Beatriz; Garone, Caterina; Barca, Emanuele; Mojahed, Hamed; Gutierrez, Purification; Pizzorno, Giuseppe; Tanji, Kurenai; Arias-Mendoza, Fernando; Quinzii, Caterina M.

2014-01-01

Balanced pools of deoxyribonucleoside triphosphate precursors are required for DNA replication, and alterations of this balance are relevant to human mitochondrial diseases including mitochondrial neurogastrointestinal encephalopathy. In this disease, autosomal recessive TYMP mutations cause severe reductions of thymidine phosphorylase activity; marked elevations of the pyrimidine nucleosides thymidine and deoxyuridine in plasma and tissues, and somatic multiple deletions, depletion and site-specific point mutations of mitochondrial DNA. Thymidine phosphorylase and uridine phosphorylase double knockout mice recapitulated several features of these patients including thymidine phosphorylase activity deficiency, elevated thymidine and deoxyuridine in tissues, mitochondrial DNA depletion, respiratory chain defects and white matter changes. However, in contrast to patients with this disease, mutant mice showed mitochondrial alterations only in the brain. To test the hypothesis that elevated levels of nucleotides cause unbalanced deoxyribonucleoside triphosphate pools and, in turn, pathogenic mitochondrial DNA instability, we have stressed double knockout mice with exogenous thymidine and deoxyuridine, and assessed clinical, neuroradiological, histological, molecular, and biochemical consequences. Mutant mice treated with exogenous thymidine and deoxyuridine showed reduced survival, body weight, and muscle strength, relative to untreated animals. Moreover, in treated mutants, leukoencephalopathy, a hallmark of the disease, was enhanced and the small intestine showed a reduction of smooth muscle cells and increased fibrosis. Levels of mitochondrial DNA were depleted not only in the brain but also in the small intestine, and deoxyribonucleoside triphosphate imbalance was observed in the brain. The relative proportion, rather than the absolute amount of deoxyribonucleoside triphosphate, was critical for mitochondrial DNA maintenance. Thus, our results demonstrate that stress of exogenous pyrimidine nucleosides enhances the mitochondrial phenotype of our knockout mice. Our mouse studies provide insights into the pathogenic role of thymidine and deoxyuridine imbalance in mitochondrial neurogastrointestinal encephalopathy and an excellent model to study new therapeutic approaches. PMID:24727567

Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.

PubMed

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

2016-08-15

Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. © 2016. Published by The Company of Biologists Ltd.

Txnip ablation reduces vascular smooth muscle cell inflammation and ameliorates atherosclerosis in apolipoprotein E knockout mice.

PubMed

Byon, Chang Hyun; Han, Tieyan; Wu, Judy; Hui, Simon T

2015-08-01

Inflammation of vascular smooth muscle cells (VSMC) is intimately linked to atherosclerosis and other vascular inflammatory disease. Thioredoxin interacting protein (Txnip) is a key regulator of cellular sulfhydryl redox and a mediator of inflammasome activation. The goals of the present study were to examine the impact of Txnip ablation on inflammatory response to oxidative stress in VSMC and to determine the effect of Txnip ablation on atherosclerosis in vivo. Using cultured VSMC, we showed that ablation of Txnip reduced cellular oxidative stress and increased protection from oxidative stress when challenged with oxidized phospholipids and hydrogen peroxide. Correspondingly, expression of inflammatory markers and adhesion molecules were diminished in both VSMC and macrophages from Txnip knockout mice. The blunted inflammatory response was associated with a decrease in NF-ĸB nuclear translocation. Loss of Txnip in VSMC also led to a dramatic reduction in macrophage adhesion to VSMC. In vivo data from Txnip-ApoE double knockout mice showed that Txnip ablation led to 49% reduction in atherosclerotic lesion in the aortic root and 71% reduction in the abdominal aorta, compared to control ApoE knockout mice. Our data show that Txnip plays an important role in oxidative inflammatory response and atherosclerotic lesion development in mice. The atheroprotective effect of Txnip ablation implicates that modulation of Txnip expression may serve as a potential target for intervention of atherosclerosis and inflammatory vascular disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Impaired TFEB-mediated Lysosome Biogenesis and Autophagy Promote Chronic Ethanol-induced Liver Injury and Steatosis in Mice.

PubMed

Chao, Xiaojuan; Wang, Shaogui; Zhao, Katrina; Li, Yuan; Williams, Jessica A; Li, Tiangang; Chavan, Hemantkumar; Krishnamurthy, Partha; He, Xi C; Li, Linheng; Ballabio, Andrea; Ni, Hong-Min; Ding, Wen-Xing

2018-05-18

Defects in lysosome function and autophagy contribute to pathogenesis of alcoholic liver disease. We investigated the mechanisms by which alcohol consumption affects these processes, evaluating the functions transcription factor EB (TFEB), which regulates lysosomal biogenesis. We performed studies with GFP-LC3 mice, mice with liver-specific deletion of transcription factor EB (TFEB), mice with disruption of the transcription factor E3 gene (TFE3-knockout mice), mice with disruption of the Tefb and Tfe3 genes (TFEB, TFE3 double-knockout mice), and Tfeb flox/flox albumin cre-negative mice (controls). TFEB was overexpressed from adenoviral vectors or knocked down with small interfering RNAs in mouse livers. Mice were placed on diets of chronic ethanol feeding plus an acute binge to induce liver damage (ethanol diet); some mice were also given injections of torin1, an inhibitor of the kinase activity of the mechanistic target of rapamycin (mTOR). Liver tissues were collected and analyzed by immunohistochemistry, immunoblots, and quantitative real-time PCR to monitor lysosome biogenesis. We analyzed levels of TFEB in liver tissues from patients with alcoholic hepatitis and from healthy donors (controls) by immunohistochemistry. Liver tissues from mice on the ethanol diet had lower levels of total and nuclear TFEB, compared with control mice, and hepatocytes had reduced lysosome biogenesis and autophagy. Hepatocytes from mice on the ethanol diet had increased translocation of mTOR into lysosomes, resulting increased mTOR activation. Administration of torin1 increased liver levels of TFEB and reduced steatosis and liver injury induced by ethanol. Mice that overexpressed TFEB in liver developed less-severe ethanol-induced liver injury and had increased lysosomal biogenesis and mitochondrial bioenergetics compared to mice carrying a control vector. Mice with knockdown of TFEB, as well as TFEB, TFE3 double-knockout mice, developed more severe liver injury in response to the ethanol diet than control mice. Liver tissues from patients with alcohol-induced hepatitis had lower nuclear levels of TFEB than control tissues CONCLUSIONS: We found chronic ethanol feeding plus an acute binge to reduce hepatic expression of the transcription factor TFEB, which is required for lysosomal biogenesis and autophagy. Strategies to block mTOR activity or increase levels of TFEB might be developed to protect liver from ethanol-induced damage. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

BAD knockout provides metabolic seizure resistance in a genetic model of epilepsy with sudden unexplained death in epilepsy.

PubMed

Foley, Jeannine; Burnham, Veronica; Tedoldi, Meghan; Danial, Nika N; Yellen, Gary

2018-01-01

Metabolic alteration, either through the ketogenic diet (KD) or by genetic alteration of the BAD protein, can produce seizure protection in acute chemoconvulsant models of epilepsy. To assess the seizure-protective role of knocking out (KO) the Bad gene in a chronic epilepsy model, we used the Kcna1 -/- model of epilepsy, which displays progressively increased seizure severity and recapitulates the early death seen in sudden unexplained death in epilepsy (SUDEP). Beginning on postnatal day 24 (P24), we continuously video monitored Kcna1 -/- and Kcna1 -/- Bad -/- double knockout mice to assess survival and seizure severity. We found that Kcna1 -/- Bad -/- mice outlived Kcna1 -/- mice by approximately 2 weeks. Kcna1 -/- Bad -/- mice also spent significantly less time in seizure than Kcna1 -/- mice on P24 and the day of death, showing that BadKO provides seizure resistance in a genetic model of chronic epilepsy. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

WWOX and p53 Dysregulation Synergize to Drive the Development of Osteosarcoma.

PubMed

Del Mare, Sara; Husanie, Hussam; Iancu, Ortal; Abu-Odeh, Mohammad; Evangelou, Konstantinos; Lovat, Francesca; Volinia, Stefano; Gordon, Jonathan; Amir, Gail; Stein, Janet; Stein, Gary S; Croce, Carlo M; Gorgoulis, Vassilis; Lian, Jane B; Aqeilan, Rami I

2016-10-15

Osteosarcoma is a highly metastatic form of bone cancer in adolescents and young adults that is resistant to existing treatments. Development of an effective therapy has been hindered by very limited understanding of the mechanisms of osteosarcomagenesis. Here, we used genetically engineered mice to investigate the effects of deleting the tumor suppressor Wwox selectively in either osteoblast progenitors or mature osteoblasts. Mice with conditional deletion of Wwox in preosteoblasts (Wwox Δosx1 ) displayed a severe inhibition of osteogenesis accompanied by p53 upregulation, effects that were not observed in mice lacking Wwox in mature osteoblasts. Deletion of p53 in Wwox Δosx1 mice rescued the osteogenic defect. In addition, the Wwox;p53 Δosx1 double knockout mice developed poorly differentiated osteosarcomas that resemble human osteosarcoma in histology, location, metastatic behavior, and gene expression. Strikingly, the development of osteosarcomas in these mice was greatly accelerated compared with mice lacking p53 only. In contrast, combined WWOX and p53 inactivation in mature osteoblasts did not accelerate osteosarcomagenesis compared with p53 inactivation alone. These findings provide evidence that a WWOX-p53 network regulates normal bone formation and that disruption of this network in osteoprogenitors results in accelerated osteosarcoma. The Wwox;p53 Δosx1 double knockout establishes a new osteosarcoma model with significant advancement over existing models. Cancer Res; 76(20); 6107-17. ©2016 AACR. ©2016 American Association for Cancer Research.

SPAK and OSR1 play essential roles in potassium homeostasis through actions on the distal convoluted tubule

PubMed Central

Ferdaus, Mohammed Z.; Barber, Karl W.; López‐Cayuqueo, Karen I.; Terker, Andrew S.; Argaiz, Eduardo R.; Gassaway, Brandon M.; Chambrey, Régine; Gamba, Gerardo; Rinehart, Jesse

2016-01-01

Key points STE20 (Sterile 20)/SPS‐1 related proline/alanine‐rich kinase (SPAK) and oxidative stress‐response kinase‐1 (OSR1) phosphorylate and activate the renal Na+–K+–2Cl− cotransporter 2 (NKCC2) and Na+Cl− cotransporter (NCC).Mouse models suggest that OSR1 mainly activates NKCC2‐mediated sodium transport along the thick ascending limb, while SPAK mainly activates NCC along the distal convoluted tubule, but the kinases may compensate for each other. We hypothesized that disruption of both kinases would lead to polyuria and severe salt‐wasting, and generated SPAK/OSR1 double knockout mice to test this.Despite a lack of SPAK and OSR1, phosphorylated NKCC2 abundance was still high, suggesting the existence of an alternative activating kinase.Compensatory changes in SPAK/OSR1‐independent phosphorylation sites on both NKCC2 and NCC and changes in sodium transport along the collecting duct were also observed.Potassium restriction revealed that SPAK and OSR1 play essential roles in the emerging model that NCC activation is central to sensing changes in plasma [K+]. Abstract STE20 (Sterile 20)/SPS‐1 related proline/alanine‐rich kinase (SPAK) and oxidative stress‐response kinase‐1 (OSR1) activate the renal cation cotransporters Na+–K+–2Cl− cotransporter (NKCC2) and Na+–Cl− cotransporter (NCC) via phosphorylation. Knockout mouse models suggest that OSR1 mainly activates NKCC2, while SPAK mainly activates NCC, with possible cross‐compensation. We tested the hypothesis that disrupting both kinases causes severe polyuria and salt‐wasting by generating SPAK/OSR1 double knockout (DKO) mice. DKO mice displayed lower systolic blood pressure compared with SPAK knockout (SPAK‐KO) mice, but displayed no severe phenotype even after dietary salt restriction. Phosphorylation of NKCC2 at SPAK/OSR1‐dependent sites was lower than in SPAK‐KO mice, but still significantly greater than in wild type mice. In the renal medulla, there was significant phosphorylation of NKCC2 at SPAK/OSR1‐dependent sites despite a complete absence of SPAK and OSR1, suggesting the existence of an alternative activating kinase. The distal convoluted tubule has been proposed to sense plasma [K+], with NCC activation serving as the primary effector pathway that modulates K+ secretion, by metering sodium delivery to the collecting duct. Abundance of phosphorylated NCC (pNCC) is dramatically lower in SPAK‐KO mice than in wild type mice, and the additional disruption of OSR1 further reduced pNCC. SPAK‐KO and kidney‐specific OSR1 single knockout mice maintained plasma [K+] following dietary potassium restriction, but DKO mice developed severe hypokalaemia. Unlike mice lacking SPAK or OSR1 alone, DKO mice displayed an inability to phosphorylate NCC under these conditions. These data suggest that SPAK and OSR1 are essential components of the effector pathway that maintains plasma [K+]. PMID:27068441

Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice

PubMed Central

Niksch, Paul D.; Webber, Roxanna M.; Garcia-Gonzalez, Miguel; Watnick, Terry; Zhou, Jing; Vollrath, Melissa A.; Corey, David P.

2016-01-01

Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction. PMID:27196058

Shadoo/PrP (Sprn0/0/Prnp0/0) double knockout mice

PubMed Central

Daude, Nathalie; Westaway, David

2012-01-01

Shadoo (Sho) is a brain glycoprotein with similarities to the unstructured region of PrPC. Frameshift alleles of the Sho gene, Sprn, are reported in variant Creutzfeldt-Jakob disease (vCJD) patients while Sprn mRNA knockdown in PrP-null (Prnp0/0) embryos produces lethality, advancing Sho as the hypothetical PrP-like “pi” protein. Also, Sho levels are reduced as misfolded PrP accumulates during prion infections. To penetrate these issues we created Sprn null alleles (Daude et al., Proc. Natl. Acad. Sci USA 2012; 109(23): 9035–40). Results from the challenge of Sprn null and TgSprn transgenic mice with rodent-adapted prions coalesce to define downregulation of Sho as a “tracer” for the formation of misfolded PrP. However, classical BSE and rodent-adapted BSE isolates may behave differently, as they do for other facets of the pathogenic process, and this intriguing variation warrants closer scrutiny. With regards to physiological function, double knockout mice (Sprn0/0/Prnp0/0) mice survived to over 600 d of age. This suggests that Sho is not pi, or, given the accumulating data for many activities for PrPC, that the pi hypothesis invoking a discrete signaling pathway to maintain neuronal viability is no longer tenable. PMID:22929230

FGF signaling in the osteoprogenitor lineage non-autonomously regulates postnatal chondrocyte proliferation and skeletal growth

PubMed Central

Karuppaiah, Kannan; Yu, Kai; Lim, Joohyun; Chen, Jianquan; Smith, Craig; Long, Fanxin

2016-01-01

ABSTRACT Fibroblast growth factor (FGF) signaling is important for skeletal development; however, cell-specific functions, redundancy and feedback mechanisms regulating bone growth are poorly understood. FGF receptors 1 and 2 (Fgfr1 and Fgfr2) are both expressed in the osteoprogenitor lineage. Double conditional knockout mice, in which both receptors were inactivated using an osteoprogenitor-specific Cre driver, appeared normal at birth; however, these mice showed severe postnatal growth defects that include an ∼50% reduction in body weight and bone mass, and impaired longitudinal bone growth. Histological analysis showed reduced cortical and trabecular bone, suggesting cell-autonomous functions of FGF signaling during postnatal bone formation. Surprisingly, the double conditional knockout mice also showed growth plate defects and an arrest in chondrocyte proliferation. We provide genetic evidence of a non-cell-autonomous feedback pathway regulating Fgf9, Fgf18 and Pthlh expression, which led to increased expression and signaling of Fgfr3 in growth plate chondrocytes and suppression of chondrocyte proliferation. These observations show that FGF signaling in the osteoprogenitor lineage is obligately coupled to chondrocyte proliferation and the regulation of longitudinal bone growth. PMID:27052727

Increase in SGLT1-mediated transport explains renal glucose reabsorption during genetic and pharmacological SGLT2 inhibition in euglycemia

PubMed Central

Rieg, Timo; Masuda, Takahiro; Gerasimova, Maria; Mayoux, Eric; Platt, Kenneth; Powell, David R.; Thomson, Scott C.; Koepsell, Hermann

2013-01-01

In the kidney, the sodium-glucose cotransporters SGLT2 and SGLT1 are thought to account for >90 and ∼3% of fractional glucose reabsorption (FGR), respectively. However, euglycemic humans treated with an SGLT2 inhibitor maintain an FGR of 40–50%, mimicking values in Sglt2 knockout mice. Here, we show that oral gavage with a selective SGLT2 inhibitor (SGLT2-I) dose dependently increased urinary glucose excretion (UGE) in wild-type (WT) mice. The dose-response curve was shifted leftward and the maximum response doubled in Sglt1 knockout (Sglt1−/−) mice. Treatment in diet with the SGLT2-I for 3 wk maintained 1.5- to 2-fold higher urine glucose/creatinine ratios in Sglt1−/− vs. WT mice, associated with a temporarily greater reduction in blood glucose in Sglt1−/− vs. WT after 24 h (−33 vs. −11%). Subsequent inulin clearance studies under anesthesia revealed free plasma concentrations of the SGLT2-I (corresponding to early proximal concentration) close to the reported IC50 for SGLT2 in mice, which were associated with FGR of 64 ± 2% in WT and 17 ± 2% in Sglt1−/−. Additional intraperitoneal application of the SGLT2-I (maximum effective dose in metabolic cages) increased free plasma concentrations ∼10-fold and reduced FGR to 44 ± 3% in WT and to −1 ± 3% in Sglt1−/−. The absence of renal glucose reabsorption was confirmed in male and female Sglt1/Sglt2 double knockout mice. In conclusion, SGLT2 and SGLT1 account for renal glucose reabsorption in euglycemia, with 97 and 3% being reabsorbed by SGLT2 and SGLT1, respectively. When SGLT2 is fully inhibited by SGLT2-I, the increase in SGLT1-mediated glucose reabsorption explains why only 50–60% of filtered glucose is excreted. PMID:24226519

Dietary and genetic evidence for phosphate toxicity accelerating mammalian aging

PubMed Central

Ohnishi, Mutsuko; Razzaque, M. Shawkat

2010-01-01

Identifying factors that accelerate the aging process can provide important therapeutic targets for slowing down this process. Misregulation of phosphate homeostasis has been noted in various skeletal, cardiac, and renal diseases, but the exact role of phosphate toxicity in mammalian aging is not clearly defined. Phosphate is widely distributed in the body and is involved in cell signaling, energy metabolism, nucleic acid synthesis, and the maintenance of acid-base balance by urinary buffering. In this study, we used an in vivo genetic approach to determine the role of phosphate toxicity in mammalian aging. Klotho-knockout mice (klotho−/−) have a short life span and show numerous physical, biochemical, and morphological features consistent with premature aging, including kyphosis, uncoordinated movement, hypogonadism, infertility, severe skeletal muscle wasting, emphysema, and osteopenia, as well as generalized atrophy of the skin, intestine, thymus, and spleen. Molecular and biochemical analyses suggest that increased renal activity of sodium-phosphate cotransporters (NaPi2a) leads to severe hyperphosphatemia in klotho−/− mice. Genetically reducing serum phosphate levels in klotho−/− mice by generating a NaPi2a and klotho double-knockout (NaPi2a−/−/klotho−/−) strain resulted in amelioration of premature aging-like features. The NaPi2a−/−/klotho−/− double-knockout mice regained reproductive ability, recovered their body weight, reduced their organ atrophy, and suppressed ectopic calcifications, with the resulting effect being prolonged survival. More important, when hyperphosphatemia was induced in NaPi2a−/−/klotho−/− mice by feeding with a high-phosphate diet, premature aging-like features reappeared, clearly suggesting that phosphate toxicity is the main cause of premature aging in klotho−/− mice. The results of our dietary and genetic manipulation studies provide in vivo evidence for phosphate toxicity accelerating the aging process and suggest a novel role for phosphate in mammalian aging.—Ohnishi, M., Razzaque, M. S. Dietary and genetic evidence for phosphate toxicity accelerating mammalian aging. PMID:20418498

HJV and HFE Play Distinct Roles in Regulating Hepcidin.

PubMed

Wu, Qian; Wang, Hao; An, Peng; Tao, Yunlong; Deng, Jiali; Zhang, Zhuzhen; Shen, Yuanyuan; Chen, Caiyong; Min, Junxia; Wang, Fudi

2015-05-20

Hereditary hemochromatosis (HH) is an iron overload disease that is caused by mutations in HFE, HJV, and several other genes. However, whether HFE-HH and HJV-HH share a common pathway via hepcidin regulation is currently unclear. Recently, some HH patients have been reported to carry concurrent mutations in both the HFE and HJV genes. To dissect the roles and molecular mechanisms of HFE and/or HJV in the pathogenesis of HH, we studied Hfe(-/-), Hjv(-/-), and Hfe(-/-)Hjv(-/-) double-knockout mouse models. Hfe(-/-)Hjv(-/-) mice developed iron overload in multiple organs at levels comparable to Hjv(-/-) mice. After an acute delivery of iron, the expression of hepcidin (i.e., Hamp1 mRNA) was increased in the livers of wild-type and Hfe(-/-) mice, but not in either Hjv(-/-) or Hfe(-/-)Hjv(-/-) mice. Furthermore, iron-induced phosphorylation of Smad1/5/8 was not detected in the livers of Hjv(-/-) or Hfe(-/-)Hjv(-/-) mice. We generated and phenotypically characterized Hfe(-/-)Hjv(-/-) double-knockout mice. In addition, because they faithfully phenocopy clinical HH patients, these mouse models are an invaluable tool for mechanistically dissecting how HFE and HJV regulate hepcidin expression. Based on our results, we conclude that HFE may depend on HJV for transferrin-dependent hepcidin regulation. The presence of residual hepcidin in the absence of HFE suggests either the presence of an unknown regulator (e.g., TFR2) that is synergistic with HJV or that HJV is sufficient to maintain basal levels of hepcidin.

Canonical Transient Receptor Channel 5 (TRPC5) and TRPC1/4 Contribute to Seizure and Excitotoxicity by Distinct Cellular Mechanisms

PubMed Central

Phelan, Kevin D.; Shwe, U Thaung; Abramowitz, Joel; Wu, Hong; Rhee, Sung W.; Howell, Matthew D.; Gottschall, Paul E.; Freichel, Marc; Flockerzi, Veit; Birnbaumer, Lutz

2013-01-01

Seizures are the manifestation of highly synchronized burst firing of a large population of cortical neurons. Epileptiform bursts with an underlying plateau potential in neurons are a cellular correlate of seizures. Emerging evidence suggests that the plateau potential is mediated by neuronal canonical transient receptor potential (TRPC) channels composed of members of the TRPC1/4/5 subgroup. We previously showed that TRPC1/4 double-knockout (DKO) mice lack epileptiform bursting in lateral septal neurons and exhibit reduced seizure-induced neuronal cell death, but surprisingly have unaltered pilocarpine-induced seizures. Here, we report that TRPC5 knockout (KO) mice exhibit both significantly reduced seizures and minimal seizure-induced neuronal cell death in the hippocampus. Interestingly, epileptiform bursting induced by agonists for metabotropic glutamate receptors in the hippocampal CA1 area is unaltered in TRPC5 KO mice, but is abolished in TRPC1 KO and TRPC1/4 DKO mice. In contrast, long-term potentiation is greatly reduced in TRPC5 KO mice, but is normal in TRPC1 KO and TRPC1/4 DKO mice. The distinct changes from these knockouts suggest that TRPC5 and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms. Furthermore, the reduced seizure and excitotoxicity and normal spatial learning exhibited in TRPC5 KO mice suggest that TRPC5 is a promising novel molecular target for new therapy. PMID:23188715

Canonical transient receptor channel 5 (TRPC5) and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms.

PubMed

Phelan, Kevin D; Shwe, U Thaung; Abramowitz, Joel; Wu, Hong; Rhee, Sung W; Howell, Matthew D; Gottschall, Paul E; Freichel, Marc; Flockerzi, Veit; Birnbaumer, Lutz; Zheng, Fang

2013-02-01

Seizures are the manifestation of highly synchronized burst firing of a large population of cortical neurons. Epileptiform bursts with an underlying plateau potential in neurons are a cellular correlate of seizures. Emerging evidence suggests that the plateau potential is mediated by neuronal canonical transient receptor potential (TRPC) channels composed of members of the TRPC1/4/5 subgroup. We previously showed that TRPC1/4 double-knockout (DKO) mice lack epileptiform bursting in lateral septal neurons and exhibit reduced seizure-induced neuronal cell death, but surprisingly have unaltered pilocarpine-induced seizures. Here, we report that TRPC5 knockout (KO) mice exhibit both significantly reduced seizures and minimal seizure-induced neuronal cell death in the hippocampus. Interestingly, epileptiform bursting induced by agonists for metabotropic glutamate receptors in the hippocampal CA1 area is unaltered in TRPC5 KO mice, but is abolished in TRPC1 KO and TRPC1/4 DKO mice. In contrast, long-term potentiation is greatly reduced in TRPC5 KO mice, but is normal in TRPC1 KO and TRPC1/4 DKO mice. The distinct changes from these knockouts suggest that TRPC5 and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms. Furthermore, the reduced seizure and excitotoxicity and normal spatial learning exhibited in TRPC5 KO mice suggest that TRPC5 is a promising novel molecular target for new therapy.

Not So Giants: Mice Lacking Both Somatostatin and Cortistatin Have High GH Levels but Show No Changes in Growth Rate or IGF-1 Levels.

PubMed

Pedraza-Arévalo, S; Córdoba-Chacón, J; Pozo-Salas, A I; L-López, F; de Lecea, L; Gahete, M D; Castaño, J P; Luque, R M

2015-06-01

Somatostatin (SST) and cortistatin (CORT) are two highly related neuropeptides involved in the regulation of various endocrine secretions. In particular, SST and CORT are two primary negative regulators of GH secretion. Consequently, single SST or CORT knockout mice exhibit elevated GH levels; however, this does not lead to increased IGF-1 levels or somatic growth. This apparent lack of correspondence has been suggested to result from compensatory mechanisms between both peptides. To test this hypothesis, in this study we explored, for the first time, the consequences of simultaneously deleting endogenous SST and CORT by generating a double SST/CORT knockout mouse model and exploring its endocrine and metabolic phenotype. Our results demonstrate that simultaneous deletion of SST and CORT induced a drastic elevation of endogenous GH levels, which, surprisingly, did not lead to changes in growth rate or IGF-1 levels, suggesting the existence of additional factors/systems that, in the absence of endogenous SST and CORT, could counteract GH actions. Notably, elevation in circulating GH levels were not accompanied by changes in pituitary GH expression or by alterations in the expression of its main regulators (GHRH and ghrelin) or their receptors (GHRH receptor, GHS receptor, or SST/CORT receptors) at the hypothalamic or pituitary level. However, although double-SST/CORT knockout male mice exhibited normal glucose and insulin levels, they had improved insulin sensitivity compared with the control mice. Therefore, these results suggest the existence of an intricate interplay among the known (SST/CORT), and likely unknown, inhibitory components of the GH/IGF-1 axis to regulate somatic growth and glucose/insulin homeostasis.

Neuronal Nitric Oxide Synthase and NADPH Oxidase Interact to Affect Cognitive, Affective, and Social Behaviors in Mice

PubMed Central

Walton, James C.; Selvakumar, Balakrishnan; Weil, Zachary M.; Snyder, Solomon H.; Nelson, Randy J.

2013-01-01

Both nitric oxide (NO) and reactive oxygen species (ROS) generated by nNOS and NADPH oxidase (NOX), respectively, in the brain have been implicated in an array of behaviors ranging from learning and memory to social interactions. Although recent work has elucidated how these separate redox pathways regulate neural function and behavior, the interaction of these two pathways in the regulation of neural function and behavior remains unspecified. Toward this end, the p47phox subunit of NOX, and nNOS were deleted to generate double knockout mice that were used to characterize the behavioral outcomes of concurrent impairment of the NO and ROS pathways in the brain. Mice were tested in a battery of behavioral tasks to evaluate learning and memory, as well as social, affective, and cognitive behaviors. p47phox deletion did not affect depressive-like behavior, whereas nNOS deletion abolished it. Both p47phox and nNOS deletion singly reduced anxiety-like behavior, increased general locomotor activity, impaired spatial learning and memory, and impaired preference for social novelty. Deletion of both genes concurrently had synergistic effects to elevate locomotor activity, impair spatial learning and memory, and disrupt prepulse inhibition of acoustic startle. Although preference for social novelty was impaired in single knockouts, double knockout mice displayed elevated levels of preference for social novelty above that of wild type littermates. These data demonstrate that, depending upon modality, deletion of p47phox and nNOS genes have dissimilar, similar, or additive effects. The current findings provide evidence that the NOX and nNOS redox signaling cascades interact in the brain to affect both cognitive function and social behavior. PMID:23948215

Global deletion of glutathione S-Transferase A4 exacerbates developmental nonalcoholic steatohepatitis

USDA-ARS?s Scientific Manuscript database

We established a mouse model of developmental nonalcoholic steatohepatitis (NASH) by feeding a high polyunsaturated fat liquid diet to female glutathione-S-transferase 4-4 (Gsta4-/-)/peroxisome proliferator activated receptor a (Ppara-/-) double knockout 129/SvJ mice for 12 weeks from weaning. We us...

Evidence for complete epistasis of null mutations in murine Fanconi anemia genes Fanca and Fancg.

PubMed

van de Vrugt, Henri J; Koomen, Mireille; Bakker, Sietske; Berns, Mariska A D; Cheng, Ngan Ching; van der Valk, Martin A; de Vries, Yne; Rooimans, Martin A; Oostra, Anneke B; Hoatlin, Maureen E; Te Riele, Hein; Joenje, Hans; Arwert, Fré

2011-12-10

Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a(-/-)/g(-/-) mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a(-/-)/g(-/-) mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a(-/-)/g(-/-) males and females. During the first year of life a(-/-)/g(-/-) did not develop malignancies or bone marrow failure. At the cellular level a(-/-)/g(-/-), Fanca(-/-), and Fancg(-/-) cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a(-/-)/g(-/-) double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely. 2011 Elsevier B.V. All rights reserved.

Control of glycinergic input to spinal dorsal horn neurons by distinct muscarinic receptor subtypes revealed using knockout mice.

PubMed

Zhang, Hong-Mei; Zhou, Hong-Yi; Chen, Shao-Rui; Gautam, Dinesh; Wess, Jürgen; Pan, Hui-Lin

2007-12-01

Muscarinic acetylcholine receptors (mAChRs) play an important role in the tonic regulation of nociceptive transmission in the spinal cord. However, how mAChR subtypes contribute to the regulation of synaptic glycine release is unknown. To determine their role, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons by using whole-cell recordings in spinal cord slices of wild-type (WT) and mAChR subtype knockout (KO) mice. In WT mice, the mAChR agonist oxotremorine-M dose-dependently decreased the frequency of sIPSCs in most neurons, but it had variable effects in other neurons. In contrast, in M3-KO mice, oxotremorine-M consistently decreased the glycinergic sIPSC frequency in all neurons tested, and in M2/M4 double-KO mice, it always increased the sIPSC frequency. In M2/M4 double-KO mice, the potentiating effect of oxotremorine-M was attenuated by higher concentrations in some neurons through activation of GABA(B) receptors. In pertussis toxin-treated WT mice, oxotremorine-M also consistently increased the sIPSC frequency. In M2-KO and M4-KO mice, the effect of oxotremorine-M on sIPSCs was divergent because of the opposing functions of the M3 subtype and the M2 and M4 subtypes. This study demonstrates that stimulation of the M2 and M4 subtypes inhibits glycinergic inputs to spinal dorsal horn neurons of mice, whereas stimulation of the M3 subtype potentiates synaptic glycine release. Furthermore, GABA(B) receptors are involved in the feedback regulation of glycinergic synaptic transmission in the spinal cord. This study revealed distinct functions of mAChR subtypes in controlling glycinergic input to spinal dorsal horn neurons.

Running Promotes Wakefulness and Increases Cataplexy in Orexin Knockout Mice

PubMed Central

España, Rodrigo A.; McCormack, Sarah L.; Mochizuki, Takatoshi; Scammell, Thomas E.

2007-01-01

Study Objective: People with narcolepsy and mice lacking orexin/hypocretin have disrupted sleep/wake behavior and reduced physical activity. Our objective was to identify physiologic mechanisms through which orexin deficiency reduces locomotor activity. Design: We examined spontaneous wheel running activity and its relationship to sleep/wake behavior in wild type (WT) and orexin knockout (KO) mice. Additionally, given that physical activity promotes alertness, we also studied whether orexin deficiency reduces the wake-promoting effects of exercise. Measurements and Results: Orexin KO mice ran 42% less than WT mice. Their ability to run appeared normal as they initiated running as often as WT mice and ran at normal speeds. However, their running bouts were considerably shorter, and they often had cataplexy or quick transitions into sleep after running. Wheel running increased the total amount of wakefulness in WT and orexin KO mice similarly, however, KO mice continued to have moderately fragmented sleep/wake behavior. Wheel running also doubled the amount of cataplexy by increasing the probability of transitioning into cataplexy. Conclusions: Orexin KO mice run significantly less than normal, likely due to sleepiness, imminent cataplexy, or a reduced motivation to run. Orexin is not required for the wake-promoting effects of wheel running given that both WT and KO mice had similar increases in wakefulness with running wheels. In addition, the clear increase in cataplexy with wheel running suggests the possibility that positive emotions or reward can trigger murine cataplexy, similar to that seen in people and dogs with narcolepsy. Citation: España RA; McCormack SL; Mochizuki T; Scammell TE. Running promotes wakefulness and increases cataplexy in orexin knockout mice. SLEEP 2007;30(11):1417-1425. PMID:18041476

Role of Aquaporin Water Channels in Airway Fluid Transport, Humidification, and Surface Liquid Hydration

PubMed Central

Song, Yuanlin; Jayaraman, Sujatha; Yang, Baoxue; Matthay, Michael A.; Verkman, A.S.

2001-01-01

Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in microvascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in alveolar epithelia. Novel quantitative methods were developed to compare airway fluid transport–related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from the moisture content of expired air during mechanical ventilation with dry air through a tracheotomy, was 54–56% efficient in wild-type mice, and reduced by only 3–4% in AQP1/AQP5 or AQP3/AQP4 double knockout mice. Upper airway humidification, measured from the moisture gained by dry air passed through the upper airways in mice breathing through a tracheotomy, decreased from 91 to 50% with increasing ventilation from 20 to 220 ml/min, and reduced by 3–5% in AQP3/AQP4 knockout mice. The depth and salt concentration of the airway surface liquid in trachea was measured in vivo using fluorescent probes and confocal and ratio imaging microscopy. Airway surface liquid depth was 45 ± 5 μm and [Na+] was 115 ± 4 mM in wild-type mice, and not significantly different in AQP3/AQP4 knockout mice. Osmotic water permeability in upper airways, measured by an in vivo instillation/sample method, was reduced by ∼40% by AQP3/AQP4 deletion. In doing these measurements, we discovered a novel amiloride-sensitive isosmolar fluid absorption process in upper airways (13% in 5 min) that was not affected by aquaporin deletion. These results establish the fluid transporting properties of mouse airways, and indicate that aquaporins play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption. PMID:11382807

Thyroid Hormone Transporters MCT8 and OATP1C1 Control Skeletal Muscle Regeneration.

PubMed

Mayerl, Steffen; Schmidt, Manuel; Doycheva, Denica; Darras, Veerle M; Hüttner, Sören S; Boelen, Anita; Visser, Theo J; Kaether, Christoph; Heuer, Heike; von Maltzahn, Julia

2018-06-05

Thyroid hormone (TH) transporters are required for the transmembrane passage of TH in target cells. In humans, inactivating mutations in the TH transporter MCT8 cause the Allan-Herndon-Dudley syndrome, characterized by severe neuromuscular symptoms and an abnormal TH serum profile, which is fully replicated in Mct8 knockout mice and Mct8/Oatp1c1 double-knockout (M/O DKO) mice. Analysis of tissue TH content and expression of TH-regulated genes indicate a thyrotoxic state in Mct8-deficient skeletal muscles. Both TH transporters are upregulated in activated satellite cells (SCs). In M/O DKO mice, we observed a strongly reduced number of differentiated SCs, suggesting an impaired stem cell function. Moreover, M/O DKO mice and mice lacking both transporters exclusively in SCs showed impaired skeletal muscle regeneration. Our data provide solid evidence for a unique gate-keeper function of MCT8 and OATP1C1 in SC activation, underscoring the importance of a finely tuned TH signaling during myogenesis. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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.

Genetic deletion of regulator of G-protein signaling 4 (RGS4) rescues a subset of fragile X related phenotypes in the FMR1 knockout mouse.

PubMed

Pacey, Laura K K; Doss, Lilian; Cifelli, Carlo; van der Kooy, Derek; Heximer, Scott P; Hampson, David R

2011-03-01

Fragile X syndrome (FXS), the most common cause of inherited mental retardation, is caused by the loss of the mRNA binding protein, FMRP. Persons with FXS also display epileptic seizures, social anxiety, hyperactivity, and autistic behaviors. The metabotropic glutamate receptor theory of FXS postulates that in the absence of FMRP, enhanced signaling though G-protein coupled group I metabotropic glutamate receptors in the brain contributes to many of the abnormalities observed in the disorder. However, recent evidence suggests that alterations in cellular signaling through additional G-protein coupled receptors may also be involved in the pathogenesis of FXS, thus providing impetus for examining downstream molecules. One group of signaling molecules situated downstream of the receptors is the regulator of G-protein signaling (RGS) proteins. Notably, RGS4 is highly expressed in brain and has been shown to negatively regulate signaling through Group I mGluRs and GABA(B) receptors. To examine the potential role for RGS4 in the pathogenesis of FXS, we generated FXS/RGS4 double knockout mice. Characterization of these mice revealed that a subset of FXS related phenotypes, including increased body weight, altered synaptic protein expression, and abnormal social behaviors, were rescued in the double knockout mice. Other phenotypes, such as hyperactivity and macroorchidism, were not affected by the loss of RGS4. These findings suggest that tissue and cell-type specific differences in GPCR signaling and RGS function may contribute to the spectrum of phenotypic differences observed in FXS. Copyright © 2010 Elsevier Inc. All rights reserved.

Ceruloplasmin and hephaestin jointly protect the exocrine pancreas against oxidative damage by facilitating iron efflux.

PubMed

Chen, Min; Zheng, Jiashuo; Liu, Guohao; Xu, En; Wang, Junzhuo; Fuqua, Brie K; Vulpe, Chris D; Anderson, Gregory J; Chen, Huijun

2018-05-31

Little is known about the iron efflux from the pancreas, but it is likely that multicopper ferroxidases (MCFs) are involved in this process. We thus used hephaestin (Heph) and ceruloplasmin (Cp) single-knockout mice and Heph/Cp double-knockout mice to investigate the roles of MCFs in pancreatic iron homeostasis. We found that both HEPH and CP were expressed in the mouse pancreas, and that ablation of either MCF had limited effect on the pancreatic iron levels. However, ablation of both MCFs together led to extensive pancreatic iron deposition and severe oxidative damage. Perls' Prussian blue staining revealed that this iron deposition was predominantly in the exocrine pancreas, while the islets were spared. Consistent with these results, plasma lipase and trypsin were elevated in Heph/Cp knockout mice, indicating damage to the exocrine pancreas, while insulin secretion was not affected. These data indicate that HEPH and CP play mutually compensatory roles in facilitating iron efflux from the exocrine pancreas, and show that MCFs are able to protect the pancreas against iron-induced oxidative damage. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

PubMed

Shi, Wei; Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K; Straube, Jasmin; Boyle, Glen M; Al-Ejeh, Fares; Nag, Purba; Jeffery, Jessie; Harris, Janelle L; Bain, Amanda L; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J; Finnie, John; Skaar, Jeffrey R; Walkley, Carl R; Pandita, Tej K; Rowicka, Maga; Ginalski, Krzysztof; Lane, Steven W; Khanna, Kum Kum

2017-05-04

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. © 2017 by The American Society of Hematology.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress

PubMed Central

Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K.; Straube, Jasmin; Boyle, Glen M.; Al-Ejeh, Fares; Jeffery, Jessie; Harris, Janelle L.; Bain, Amanda L.; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J.; Finnie, John; Skaar, Jeffrey R.; Walkley, Carl R.; Pandita, Tej K.; Rowicka, Maga; Ginalski, Krzysztof

2017-01-01

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. PMID:28270450

An abnormal bone marrow microenvironment contributes to hematopoietic dysfunction in Fanconi anemia.

PubMed

Zhou, Yuan; He, Yongzheng; Xing, Wen; Zhang, Peng; Shi, Hui; Chen, Shi; Shi, Jun; Bai, Jie; Rhodes, Steven D; Zhang, Fengqui; Yuan, Jin; Yang, Xianlin; Zhu, Xiaofan; Li, Yan; Hanenberg, Helmut; Xu, Mingjiang; Robertson, Kent A; Yuan, Weiping; Nalepa, Grzegorz; Cheng, Tao; Clapp, D Wade; Yang, Feng-Chun

2017-06-01

Fanconi anemia is a complex heterogeneous genetic disorder with a high incidence of bone marrow failure, clonal evolution to acute myeloid leukemia and mesenchymal-derived congenital anomalies. Increasing evidence in Fanconi anemia and other genetic disorders points towards an interdependence of skeletal and hematopoietic development, yet the impact of the marrow microenvironment in the pathogenesis of the bone marrow failure in Fanconi anemia remains unclear. Here we demonstrated that mice with double knockout of both Fancc and Fancg genes had decreased bone formation at least partially due to impaired osteoblast differentiation from mesenchymal stem/progenitor cells. Mesenchymal stem/progenitor cells from the double knockout mice showed impaired hematopoietic supportive activity. Mesenchymal stem/progenitor cells of patients with Fanconi anemia exhibited similar cellular deficits, including increased senescence, reduced proliferation, impaired osteoblast differentiation and defective hematopoietic stem/progenitor cell supportive activity. Collectively, these studies provide unique insights into the physiological significance of mesenchymal stem/progenitor cells in supporting the marrow microenvironment, which is potentially of broad relevance in hematopoietic stem cell transplantation. Copyright© Ferrata Storti Foundation.

An abnormal bone marrow microenvironment contributes to hematopoietic dysfunction in Fanconi anemia

PubMed Central

Zhou, Yuan; He, Yongzheng; Xing, Wen; Zhang, Peng; Shi, Hui; Chen, Shi; Shi, Jun; Bai, Jie; Rhodes, Steven D.; Zhang, Fengqui; Yuan, Jin; Yang, Xianlin; Zhu, Xiaofan; Li, Yan; Hanenberg, Helmut; Xu, Mingjiang; Robertson, Kent A.; Yuan, Weiping; Nalepa, Grzegorz; Cheng, Tao; Clapp, D. Wade; Yang, Feng-Chun

2017-01-01

Fanconi anemia is a complex heterogeneous genetic disorder with a high incidence of bone marrow failure, clonal evolution to acute myeloid leukemia and mesenchymal-derived congenital anomalies. Increasing evidence in Fanconi anemia and other genetic disorders points towards an interdependence of skeletal and hematopoietic development, yet the impact of the marrow microenvironment in the pathogenesis of the bone marrow failure in Fanconi anemia remains unclear. Here we demonstrated that mice with double knockout of both Fancc and Fancg genes had decreased bone formation at least partially due to impaired osteoblast differentiation from mesenchymal stem/progenitor cells. Mesenchymal stem/progenitor cells from the double knockout mice showed impaired hematopoietic supportive activity. Mesenchymal stem/progenitor cells of patients with Fanconi anemia exhibited similar cellular deficits, including increased senescence, reduced proliferation, impaired osteoblast differentiation and defective hematopoietic stem/progenitor cell supportive activity. Collectively, these studies provide unique insights into the physiological significance of mesenchymal stem/progenitor cells in supporting the marrow microenvironment, which is potentially of broad relevance in hematopoietic stem cell transplantation. PMID:28341737

Pituitary-adrenal responses to oxotremorine and acute stress in male and female M1 muscarinic receptor knockout mice: comparisons to M2 muscarinic receptor knockout mice.

PubMed

Rhodes, M E; Rubin, R T; McKlveen, J M; Karwoski, T E; Fulton, B A; Czambel, R K

2008-05-01

Both within the brain and in the periphery, M(1) muscarinic receptors function primarily as postsynaptic receptors and M(2) muscarinic receptors function primarily as presynaptic autoreceptors. In addition to classical parasympathetic effectors, cholinergic stimulation of central muscarinic receptors influences the release of adrenocorticotrophic hormone (ACTH) and corticosterone. We previously reported that oxotremorine administration to male and female M(2) receptor knockout and wild-type mice increased ACTH to a significantly greater degree in knockout males compared to all other groups, and that M(2) knockout mice of both sexes were significantly more responsive to the mild stress of saline injection than were wild-type mice. These results accord with the primary function of M(2) receptors as presynaptic autoreceptors. In the present study, we explored the role of the M(1) receptor in pituitary-adrenal responses to oxotremorine and saline in male and female M(1) knockout and wild-type mice. Because these mice responded differently to the mild stress of saline injection than did the M(2) knockout and wild-type mice, we also determined hormone responses to restraint stress in both M(1) and M(2) knockout and wild-type mice. Male and female M(1) knockout and wild-type mice were equally unresponsive to the stress of saline injection. Oxotremorine increased both ACTH and corticosterone in M(1) wild-type mice to a significantly greater degree than in knockout mice. In both M(1) knockout and wild-type animals, ACTH responses were greater in males compared to females, and corticosterone responses were greater in females compared to males. Hormone responses to restraint stress were increased in M(2) knockout mice and decreased in M(1) knockout mice compared to their wild-type counterparts. These findings suggest that M(1) and M(2) muscarinic receptor subtypes differentially influence male and female pituitary-adrenal responses to cholinergic stimulation and stress. The decreased pituitary-adrenal sensitivity to oxotremorine and restraint stress noted in M(1) knockout mice is consistent with M(1) being primarily a postsynaptic receptor. Conversely, the increased pituitary-adrenal sensitivity to these challenges noted in M(2) knockout mice is consistent with M(2) being primarily a presynaptic autoreceptor.

An ATF4-ATG5 signaling in hypothalamic POMC neurons regulates obesity.

PubMed

Xiao, Yuzhong; Deng, Yalan; Yuan, Feixiang; Xia, Tingting; Liu, Hao; Li, Zhigang; Chen, Shanghai; Liu, Zhixue; Ying, Hao; Liu, Yi; Zhai, Qiwei; Guo, Feifan

2017-06-03

ATF4 (activating transcription factor 4) is an important transcription factor that has many biological functions, while its role in hypothalamic POMC (pro-opiomelanocortin-α) neurons in the regulation of energy homeostasis has not been explored. We recently discovered that mice with an Atf4 deletion specific to POMC neurons (PAKO mice) are lean and have higher energy expenditure. Furthermore, these mice are resistant to high-fat diet (HFD)-induced obesity and obesity-related metabolic disorders. Mechanistically, we found the expression of ATG5 (autophagy-related 5) is upregulated in POMC neurons of PAKO mice, and ATF4 regulates ATG5 expression by binding directly to its promoter. Mice with Atf4 and Atg5 double knockout in POMC neurons have reduced energy expenditure and gain more fat mass compared with PAKO mice under a HFD. Finally, the effect of Atf4 knockout in POMC neurons is possibly mediated by enhanced ATG5-dependent macroautophagy/autophagy and α-melanocyte-stimulating hormone (α-MSH) production in the hypothalamus. Together, this work not only identifies a beneficial role for ATF4 in hypothalamic POMC neurons in the regulation of obesity, but also provides a new potential therapeutic target for obesity and obesity-related metabolic diseases.

Monoglyceride lipase deficiency affects hepatic cholesterol metabolism and lipid-dependent gut transit in ApoE−/− mice

PubMed Central

Vujic, Nemanja; Korbelius, Melanie; Leopold, Christina; Duta-Mare, Madalina; Rainer, Silvia; Schlager, Stefanie; Goeritzer, Madeleine; Kolb, Dagmar; Eichmann, Thomas O.; Diwoky, Clemens; Zimmer, Andreas; Zimmermann, Robert; Lass, Achim; Radovic, Branislav; Kratky, Dagmar

2017-01-01

Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among various MG species MGL also degrades 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid and potent activator of cannabinoid receptors (CBR) 1 and 2. MGL-knockout (−/−) mice exhibit pronounced 2-AG accumulation, but lack central cannabimimetic effects due to CB1R desensitization. We have previously shown that MGL affects plaque stability in apolipoprotein E (ApoE)−/− mice, an established animal model for dyslipidemia and atherosclerosis. In the current study, we investigated functional consequences of MGL deficiency on lipid and energy metabolism in ApoE/MGL double knockout (DKO) mice. MGL deficiency affected hepatic cholesterol metabolism by causing increased cholesterol elimination via the biliary pathway. Moreover, DKO mice exhibit lipid-triggered delay in gastric emptying without major effects on overall triglyceride and cholesterol absorption. The observed phenotype of DKO mice is likely not a consequence of potentiated CB1R signaling but rather dependent on the activation of alternative signaling pathways. We conclude that MGL deficiency causes complex metabolic changes including cholesterol metabolism and regulation of gut transit independent of the endocannabinoid system. PMID:28380440

Monoglyceride lipase deficiency affects hepatic cholesterol metabolism and lipid-dependent gut transit in ApoE-/- mice.

PubMed

Vujic, Nemanja; Korbelius, Melanie; Leopold, Christina; Duta-Mare, Madalina; Rainer, Silvia; Schlager, Stefanie; Goeritzer, Madeleine; Kolb, Dagmar; Eichmann, Thomas O; Diwoky, Clemens; Zimmer, Andreas; Zimmermann, Robert; Lass, Achim; Radovic, Branislav; Kratky, Dagmar

2017-05-16

Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among various MG species MGL also degrades 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid and potent activator of cannabinoid receptors (CBR) 1 and 2. MGL-knockout (-/-) mice exhibit pronounced 2-AG accumulation, but lack central cannabimimetic effects due to CB1R desensitization. We have previously shown that MGL affects plaque stability in apolipoprotein E (ApoE)-/- mice, an established animal model for dyslipidemia and atherosclerosis. In the current study, we investigated functional consequences of MGL deficiency on lipid and energy metabolism in ApoE/MGL double knockout (DKO) mice. MGL deficiency affected hepatic cholesterol metabolism by causing increased cholesterol elimination via the biliary pathway. Moreover, DKO mice exhibit lipid-triggered delay in gastric emptying without major effects on overall triglyceride and cholesterol absorption. The observed phenotype of DKO mice is likely not a consequence of potentiated CB1R signaling but rather dependent on the activation of alternative signaling pathways. We conclude that MGL deficiency causes complex metabolic changes including cholesterol metabolism and regulation of gut transit independent of the endocannabinoid system.

Bax and Bak are required for apoptosis induction by sulforaphane, a cruciferous vegetable-derived cancer chemopreventive agent.

PubMed

Choi, Sunga; Singh, Shivendra V

2005-03-01

Sulforaphane, a constituent of many edible cruciferous vegetables, including broccoli, effectively suppresses proliferation of cancer cells in culture and in vivo by causing apoptosis induction, but the sequence of events leading to cell death is poorly defined. Here, we show that multidomain proapoptotic Bcl-2 family members Bax and Bak play a critical role in apoptosis induction by sulforaphane. This conclusion is based on the following observations: (a) sulforaphane treatment caused a dose- and time-dependent increase in the protein levels of both Bax and Bak and conformational change and mitochondrial translocation of Bax in SV40-transformed mouse embryonic fibroblasts (MEF) derived from wild-type mice to trigger cytosolic release of apoptogenic molecules (cytochrome c and Smac/DIABLO), activation of caspase-9 and caspase-3, and ultimately cell death; (b) MEFs derived from Bax or Bak knockout mice resisted cell death by sulforaphane, and (c) MEFs derived from Bax and Bak double knockout mice exhibited even greater protection against sulforaphane-induced cytochrome c release, caspase activation, and apoptosis compared with wild-type or single knockout cells. Interestingly, sulforaphane treatment also caused a dose- and time-dependent increase in the protein level of Apaf-1 in wild-type, Bax-/-, and Bak-/- MEFs but not in double knockout, suggesting that Bax and Bak might regulate sulforaphane-mediated induction of Apaf-1 protein. A marked decline in the protein level of X-linked inhibitor of apoptosis on treatment with sulforaphane was also observed. Thus, it is reasonable to postulate that sulforaphane-induced apoptosis is amplified by a decrease in X-linked inhibitor of apoptosis level, which functions to block cell death by inhibiting activities of caspases. In conclusion, the results of the present study indicate that Bax and Bak proteins play a critical role in initiation of cell death by sulforaphane.

Comprehensive phenotypic analysis of knockout mice deficient in cyclin G1 and cyclin G2

PubMed Central

Ohno, Shouichi; Ikeda, Jun-ichiro; Naito, Yoko; Okuzaki, Daisuke; Sasakura, Towa; Fukushima, Kohshiro; Nishikawa, Yukihiro; Ota, Kaori; Kato, Yorika; Wang, Mian; Torigata, Kosuke; Kasama, Takashi; Uchihashi, Toshihiro; Miura, Daisaku; Yabuta, Norikazu; Morii, Eiichi; Nojima, Hiroshi

2016-01-01

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B’γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan–Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer. PMID:27982046

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.

Effect of the bradykinin 1 receptor antagonist SSR240612 after oral administration in Mycobacterium tuberculosis-infected mice.

PubMed

Rodrigues-Junior, Valnês S; Pail, Priscilla B; Villela, Anne D; Falcão, Virgínia C A; Dadda, Adílio S; Abbadi, Bruno L; Pesquero, João B; Santos, Diógenes S; Basso, Luiz A; Campos, Maria M

2018-03-01

The role, if any, played by the kinin system in tuberculosis infection models, either in vivo or in vitro, was investigated. The effects of Mycobacterium tuberculosis infection on C57BL/6 wild type, B 1 R-/-, B 2 R-/- and double B 1 R/B 2 R knockout mice were evaluated. Immunohistochemistry analysis was carried out to assess B 1 R and B 2 R expression in spleens and lungs of M. tuberculosis-infected mice. In addition, in vitro experiments with M. tuberculosis-infected macrophages were performed. The in vivo effects of HOE-140 and SSR240612 on the mice model of infection were also evaluated. Infected B 2 R-/- mice exhibited increased splenomegaly, whereas decreased spleen weight in infected double B 1 R/B 2 R knockout mice was observed. The bacterial load, determined as colony-forming units, did not differ in the spleens and lungs of the studied mouse strains. Importantly, immunohistochemical analysis revealed that B 1 R was upregulated in both spleens and lungs of infected mice. M. tuberculosis-infected macrophages incubated with SSR240612, alone or in combination with des-Arg 9 -BK, for four days, displayed a marked inhibitory effect on CFU counts. However, the pre-incubation of the selective B 1 R (des-Arg 9 -BK and SSR240612) and B 2 R (BK and HOE-140) agonists and antagonists, respectively, did not significantly affect the bacterial loads. A statistically significant reduction in the CFU of M. tuberculosis in lungs and spleens of animals treated with SSR240612, but not with HOE-140, was observed. Further efforts should be pursued to clarify whether or not SSR240612 might be considered an option for the treatment of tuberculosis. Copyright © 2018. Published by Elsevier Ltd.

Differential Modulation of Retinal Degeneration by Ccl2 and Cx3cr1 Chemokine Signalling

PubMed Central

Luhmann, Ulrich F. O.; Lange, Clemens A.; Robbie, Scott; Munro, Peter M. G.; Cowing, Jill A.; Armer, Hannah E. J.; Luong, Vy; Carvalho, Livia S.; MacLaren, Robert E.; Fitzke, Frederick W.; Bainbridge, James W. B.; Ali, Robin R.

2012-01-01

Microglia and macrophages are recruited to sites of retinal degeneration where local cytokines and chemokines determine protective or neurotoxic microglia responses. Defining the role of Ccl2-Ccr2 and Cx3cl1-Cx3cr1 signalling for retinal pathology is of particular interest because of its potential role in age-related macular degeneration (AMD). Ccl2, Ccr2, and Cx3cr1 signalling defects impair macrophage trafficking, but have, in several conflicting studies, been reported to show different degrees of age-related retinal degeneration. Ccl2/Cx3cr1 double knockout (CCDKO) mice show an early onset retinal degeneration and have been suggested as a model for AMD. In order to understand phenotypic discrepancies in different chemokine knockout lines and to study how defects in Ccl2 and/or Cx3cr1 signalling contribute to the described early onset retinal degeneration, we defined primary and secondary pathological events in CCDKO mice. To control for genetic background variability, we compared the original phenotype with that of single Ccl2, Cx3cr1 and Ccl2/Cx3cr1 double knockout mice obtained from backcrosses of CCDKO with C57Bl/6 mice. We found that the primary pathological event in CCDKO mice develops in the inferior outer nuclear layer independently of light around postnatal day P14. RPE and vascular lesions develop secondarily with increasing penetrance with age and are clinically similar to retinal telangiectasia not to choroidal neovascularisation. Furthermore, we provide evidence that a third autosomal recessive gene causes the degeneration in CCDKO mice and in all affected re-derived lines and subsequently demonstrated co-segregation of the naturally occurring RD8 mutation in the Crb1 gene. By comparing CCDKO mice with re-derived CCl2−/−/Crb1Rd8/RD8, Cx3cr1−/−/Crb1Rd8/RD8 and CCl2−/−/Cx3cr1−/−/Crb1Rd8/RD8 mice, we observed a differential modulation of the retinal phenotype by genetic background and both chemokine signalling pathways. These findings indicate that CCDKO mice are not a model of AMD, but a model for an inherited retinal degeneration that is differentially modulated by Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling. PMID:22545116

The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice

PubMed Central

Atochina-Vasserman, Elena N.; Massa, Christopher B.; Birkelbach, Bastian; Guo, Chang-Jiang; Scott, Pamela; Haenni, Beat; Beers, Michael F.; Ochs, Matthias; Gow, Andrew J.

2015-01-01

Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd−/−) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd−/− mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd−/− mice. These changes were reduced in DiNOS, and compared with Sftpd−/− mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd−/−. Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces. PMID:26320150

Retinal accumulation of zeaxanthin, lutein, and β-carotene in mice deficient in carotenoid cleavage enzymes.

PubMed

Li, Binxing; Vachali, Preejith P; Shen, Zhengqing; Gorusupudi, Aruna; Nelson, Kelly; Besch, Brian M; Bartschi, Alexis; Longo, Simone; Mattinson, Ty; Shihab, Saeed; Polyakov, Nikolay E; Suntsova, Lyubov P; Dushkin, Alexander V; Bernstein, Paul S

2017-06-01

Carotenoid supplementation can prevent and reduce the risk of age-related macular degeneration (AMD) and other ocular disease, but until now, there has been no validated and well-characterized mouse model which can be employed to investigate the protective mechanism and relevant metabolism of retinal carotenoids. β-Carotene oxygenases 1 and 2 (BCO1 and BCO2) are the only two carotenoid cleavage enzymes found in animals. Mutations of the bco2 gene may cause accumulation of xanthophyll carotenoids in animal tissues, and BCO1 is involved in regulation of the intestinal absorption of carotenoids. To determine whether or not mice deficient in BCO1 and/or BCO2 can serve as a macular pigment mouse model, we investigated the retinal accumulation of carotenoids in these mice when fed with zeaxanthin, lutein, or β-carotene using an optimized carotenoid feeding method. HPLC analysis revealed that all three carotenoids were detected in sera, livers, retinal pigment epithelium (RPE)/choroids, and retinas of all of the mice, except that no carotenoid was detectable in the retinas of wild type (WT) mice. Significantly higher amounts of zeaxanthin and lutein accumulated in the retinas of BCO2 knockout (bco2 -/- ) mice and BCO1/BCO2 double knockout (bco1 -/- /bco2 -/- ) mice relative to BCO1 knockout (bco1 -/- ) mice, while bco1 -/- mice preferred to take up β-carotene. The levels of zeaxanthin and lutein were higher than β-carotene levels in the bco1 -/- /bco2 -/- retina, consistent with preferential uptake of xanthophyll carotenoids by retina. Oxidative metabolites were detected in mice fed with lutein or zeaxanthin but not in mice fed with β-carotene. These results indicate that bco2 -/- and bco1 -/- /bco2 -/- mice could serve as reasonable non-primate models for macular pigment function in the vertebrate eye, while bco1 -/- mice may be more useful for studies related to β-carotene. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of supplementation on food intake, body weight and hepatic metabolites in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mouse model of human citrin deficiency.

PubMed

Saheki, Takeyori; Inoue, Kanako; Ono, Hiromi; Katsura, Natsumi; Yokogawa, Mana; Yoshidumi, Yukari; Furuie, Sumie; Kuroda, Eishi; Ushikai, Miharu; Asakawa, Akihiro; Inui, Akio; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Yamamura, Ken-Ichi; Kobayashi, Keiko

2012-11-01

The C57BL/6:Slc23a13(-/-);Gpd2(-/-) double-knockout (a.k.a., citrin/mitochondrial glycerol 3-phosphate dehydrogenase double knockout or Ctrn/mGPD-KO) mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2), making it a suitable model of human citrin deficiency. In the present study, we show that when mature Ctrn/mGPD-KO mice are switched from a standard chow diet (CE-2) to a purified maintenance diet (AIN-93M), this resulted in a significant loss of body weight as a result of reduced food intake compared to littermate mGPD-KO mice. However, supplementation of the purified maintenance diet with additional protein (from 14% to 22%; and concomitant reduction or corn starch), or with specific supplementation with alanine, sodium glutamate, sodium pyruvate or medium-chain triglycerides (MCT), led to increased food intake and body weight gain near or back to that on chow diet. No such effect was observed when supplementing the diet with other sources of fat that contain long-chain fatty acids. Furthermore, when these supplements were added to a sucrose solution administered enterally to the mice, which has been shown previously to lead to elevated blood ammonia as well as altered hepatic metabolite levels in Ctrn/mGPP-KO mice, this led to metabolic correction. The elevated hepatic glycerol 3-phosphate and citrulline levels after sucrose administration were suppressed by the administration of sodium pyruvate, alanine, sodium glutamate and MCT, although the effect of MCT was relatively small. Low hepatic citrate and increased lysine levels were only found to be corrected by sodium pyruvate, while alanine and sodium glutamate both corrected hepatic glutamate and aspartate levels. Overall, these results suggest that dietary factors including increased protein content, supplementation of specific amino acids like alanine and sodium glutamate, as well as sodium pyruvate and MCT all show beneficial effects on citrin deficiency by increasing the carbohydrate tolerance of Ctrn/mGPD-KO mice, as observed through increased food intake and maintenance of body weight. Copyright © 2012 Elsevier Inc. All rights reserved.

Rescue of a recombinant Machupo virus from cloned cDNAs and in vivo characterization in interferon (αβ/γ) receptor double knockout mice.

PubMed

Patterson, Michael; Seregin, Alexey; Huang, Cheng; Kolokoltsova, Olga; Smith, Jennifer; Miller, Milagros; Smith, Jeanon; Yun, Nadezhda; Poussard, Allison; Grant, Ashley; Tigabu, Bersabeh; Walker, Aida; Paessler, Slobodan

2014-02-01

Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates.

Increased accumulation of 4-hydroxynonenal adducts in female GSTA4/PPAR alpha double knockout mice enhance steatosis and inflammation in a model of pediatric nonalcoholic fatty liver disease

USDA-ARS?s Scientific Manuscript database

Hepatocellular injury resulting from increased lipid peroxidation products and oxidative stress is considered a potential mechanism driving the progression of nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitsis (NASH). To test the significance of lipid peroxidation and protein...

FoxO1 integrates direct and indirect effects of insulin on hepatic glucose production and glucose utilization

PubMed Central

O-Sullivan, InSug; Zhang, Wenwei; Wasserman, David H.; Liew, Chong Wee; Liu, Jonathan; Paik, Jihye; DePinho, Ronald A.; Stolz, Donna Beer; Kahn, C. Ronald; Schwartz, Michael W.; Unterman, Terry G.

2016-01-01

FoxO proteins are major targets of insulin action. To better define the role of FoxO1 in mediating insulin effects in the liver, we generated liver-specific insulin receptor knockout (LIRKO) and IR/FoxO1 double knockout (LIRFKO) mice. Here we show that LIRKO mice are severely insulin resistant based on glucose, insulin and C-peptide levels, and glucose and insulin tolerance tests, and genetic deletion of hepatic FoxO1 reverses these effects. 13C-glucose and insulin clamp studies indicate that regulation of both hepatic glucose production (HGP) and glucose utilization is impaired in LIRKO mice, and these defects are also restored in LIRFKO mice corresponding to changes in gene expression. We conclude that (1) inhibition of FoxO1 is critical for both direct (hepatic) and indirect effects of insulin on HGP and utilization, and (2) extrahepatic effects of insulin are sufficient to maintain normal whole-body and hepatic glucose metabolism when liver FoxO1 activity is disrupted. PMID:25963540

Dynamic control of glutamatergic synaptic input in the spinal cord by muscarinic receptor subtypes defined using knockout mice.

PubMed

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2010-12-24

Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M(2), M(3), and M(4)) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M(3)-single KO and M(1)/M(3) double-KO mice. In addition, the M(3) antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M(5)-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M(2)/M(4) double-KO mice, but not M(2)- or M(4)-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M(2)/M(4) antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M(2) and M(4) receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M(5) is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord.

Dynamic Control of Glutamatergic Synaptic Input in the Spinal Cord by Muscarinic Receptor Subtypes Defined Using Knockout Mice*

PubMed Central

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2010-01-01

Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M2, M3, and M4) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M3-single KO and M1/M3 double-KO mice. In addition, the M3 antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M5-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M2/M4 double-KO mice, but not M2- or M4-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M2/M4 antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M2 and M4 receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M5 is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord. PMID:20940295

Evidence of reactive astrocytes but not peripheral immune system activation in a mouse model of Fragile X Syndrome

PubMed Central

Yuskaitis, Christopher J.; Beurel, Eleonore; Jope, Richard S.

2010-01-01

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is one of the few known genetic causes of autism. FXS results from the loss of Fmr1 gene function, thus Fmr1 knockout mice provide a model to study impairments associated with FXS and autism and to test potential therapeutic interventions. The inhibitory serine-phosphorylation of glycogen synthase kinase-3 (GSK3) is lower in brain regions of Fmr1 knockout mice than wild-type mice and the GSK3 inhibitor lithium rescues several behavioral impairments in Fmr1 knockout mice. Therefore, we examined if the serine-phosphorylation of GSK3 in Fmr1 knockout mice also was altered outside the brain and if administration of lithium ameliorated the macroorchidism phenotype. Additionally, since GSK3 regulates numerous functions of the immune system and immune alterations have been associated with autism, we tested if immune function is altered in Fmr1 knockout mice. The inhibitory serine-phosphorylation of GSK3 was significantly lower in the testis and liver of Fmr1 knockout mice than wild-type mice, and chronic lithium treatment reduced macroorchidism in Fmr1 knockout mice. No alterations in peripheral immune function were identified in Fmr1 knockout mice. However, examination of glia, the immune cells of the brain, revealed reactive astrocytes in several brain regions of Fmr1 knockout mice and treatment with lithium reduced this in the striatum and cerebellum. These results provide further evidence of the involvement of dysregulated GSK3 in FXS, and demonstrate that lithium administration reduces macroorchidism and reactive astrocytes in Fmr1 knockout mice. PMID:20600866

Deletion of Numb/Numblike in glutamatergic neurons leads to anxiety-like behavior in mice.

PubMed

Qian, Wenyu; Hong, Yang; Zhu, Minyan; Zhou, Liang; Li, Hongchang; Li, Huashun

2017-06-15

Endocytic adaptor protein Numb is the first identified cell fate determinant in Drosophila melanogaster. It has been implicated in Notch signaling pathway and regulation of neural stem cells proliferation in the central nervous system. Numb is also expressed in postmitotic neurons, in vitro studies showed that Numb is involved in neuronal morphologic development, such as neurite growth, axonal growth and spine development. However, in vivo functions of Numb in the postmitotic neurons are largely unknown. Here we show that deletion of Numb/Numblike in glutamatergic neurons causes anxiety-like behavior in mouse. In this study, we conditionally deleted Numb and its homologous gene Numblike in the glutamatergic neurons in dorsal forebrain, and thoroughly characterized the behavioral phenotypes of mutant mice. On a battery of tests for anxiety-like behavior, the conditional double knockout mice showed increased anxiety-like behavior on light/dark exploration and novel open field tests, but not on elevated zero maze tests. The conditional double knockout mice also displayed novelty induced hyperactivity in novel open field test. Control measures of general health, motor functions, startle response, sensorimotor gating, depression-related behaviors did not show differences between genotypes. Our present findings provide new insight into the indispensable functions of Numb/Numblike in the brain and behavior, and suggest that Numb/Numblike may play a role in mediating neuronal functions that underlie behaviors related to anxiety. Copyright © 2017. Published by Elsevier B.V.

R/L, a double reporter mouse line that expresses luciferase gene upon Cre-mediated excision, followed by inactivation of mRFP expression.

PubMed

Jia, Junshuang; Lin, Xiaolin; Lin, Xia; Lin, Taoyan; Chen, Bangzhu; Hao, Weichao; Cheng, Yushuang; Liu, Yu; Dian, Meijuan; Yao, Kaitai; Xiao, Dong; Gu, Weiwang

2016-10-01

The Cre/loxP system has become an important tool for the conditional gene knockout and conditional gene expression in genetically engineered mice. The applications of this system depend on transgenic reporter mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. To develop a sensitive assay for monitoring Cre-mediated DNA excisions in mice, we generated Cre-mediated excision reporter mice, designated R/L mice (R/L: mRFP(monomeric red fluorescent protein)/luciferase), express mRFP throughout embryonic development and adult stages, while Cre-mediated excision deletes a loxP-flanked mRFP reporter gene and STOP sequence, thereby activating the expression of the second reporter gene luciferase, as assayed by in vivo and ex vivo bioluminescence imaging. After germ line deletion of the floxed mRFP and STOP sequence in R/L mice by EIIa-Cre mice, the resulting luciferase transgenic mice in which the loxP-mRFP-STOP-loxP cassette is excised from all cells express luciferase in all tissues and organs examined. The expression of luciferase transgene was activated in liver of RL/Alb-Cre double transgenic mice and in brain of RL/Nestin-Cre double transgenic mice when R/L reporter mice were mated with Alb-Cre mice and Nestin-Cre mice, respectively. Our findings reveal that the double reporter R/L mouse line is able to indicate the occurrence of Cre-mediated excision from early embryonic to adult lineages. Taken together, these findings demonstrate that the R/L mice serve as a sensitive reporter for Cre-mediated DNA excision both in living animals and in organs, tissues, and cells following necropsy.

HJV and HFE Play Distinct Roles in Regulating Hepcidin

PubMed Central

Wu, Qian; Wang, Hao; An, Peng; Tao, Yunlong; Deng, Jiali; Zhang, Zhuzhen; Shen, Yuanyuan; Chen, Caiyong

2015-01-01

Abstract Aims: Hereditary hemochromatosis (HH) is an iron overload disease that is caused by mutations in HFE, HJV, and several other genes. However, whether HFE-HH and HJV-HH share a common pathway via hepcidin regulation is currently unclear. Recently, some HH patients have been reported to carry concurrent mutations in both the HFE and HJV genes. To dissect the roles and molecular mechanisms of HFE and/or HJV in the pathogenesis of HH, we studied Hfe−/−, Hjv−/−, and Hfe−/−Hjv−/− double-knockout mouse models. Results: Hfe−/−Hjv−/− mice developed iron overload in multiple organs at levels comparable to Hjv−/− mice. After an acute delivery of iron, the expression of hepcidin (i.e., Hamp1 mRNA) was increased in the livers of wild-type and Hfe−/− mice, but not in either Hjv−/− or Hfe−/−Hjv−/− mice. Furthermore, iron-induced phosphorylation of Smad1/5/8 was not detected in the livers of Hjv−/− or Hfe−/−Hjv−/− mice. Innovation: We generated and phenotypically characterized Hfe−/−Hjv−/− double-knockout mice. In addition, because they faithfully phenocopy clinical HH patients, these mouse models are an invaluable tool for mechanistically dissecting how HFE and HJV regulate hepcidin expression. Conclusions: Based on our results, we conclude that HFE may depend on HJV for transferrin-dependent hepcidin regulation. The presence of residual hepcidin in the absence of HFE suggests either the presence of an unknown regulator (e.g., TFR2) that is synergistic with HJV or that HJV is sufficient to maintain basal levels of hepcidin. Antioxid. Redox Signal. 22, 1325–1336. PMID:25608116

Elevated glutaric acid levels in Dhtkd1-/Gcdh- double knockout mice challenge our current understanding of lysine metabolism.

PubMed

Biagosch, Caroline; Ediga, Raga Deepthi; Hensler, Svenja-Viola; Faerberboeck, Michael; Kuehn, Ralf; Wurst, Wolfgang; Meitinger, Thomas; Kölker, Stefan; Sauer, Sven; Prokisch, Holger

2017-09-01

Glutaric aciduria type I (GA-I) is a rare organic aciduria caused by the autosomal recessive inherited deficiency of glutaryl-CoA dehydrogenase (GCDH). GCDH deficiency leads to disruption of l-lysine degradation with characteristic accumulation of glutarylcarnitine and neurotoxic glutaric acid (GA), glutaryl-CoA, 3-hydroxyglutaric acid (3-OHGA). DHTKD1 acts upstream of GCDH, and its deficiency leads to none or often mild clinical phenotype in humans, 2-aminoadipic 2-oxoadipic aciduria. We hypothesized that inhibition of DHTKD1 may prevent the accumulation of neurotoxic dicarboxylic metabolites suggesting DHTKD1 inhibition as a possible treatment strategy for GA-I. In order to validate this hypothesis we took advantage of an existing GA-I (Gcdh -/- ) mouse model and established a Dhtkd1 deficient mouse model. Both models reproduced the biochemical and clinical phenotype observed in patients. Under challenging conditions of a high lysine diet, only Gcdh -/- mice but not Dhtkd1 -/- mice developed clinical symptoms such as lethargic behaviour and weight loss. However, the genetic Dhtkd1 inhibition in Dhtkd1 -/- /Gcdh -/- mice could not rescue the GA-I phenotype. Biochemical results confirm this finding with double knockout mice showing similar metabolite accumulations as Gcdh -/- mice with high GA in brain and liver. This suggests that DHTKD1 inhibition alone is not sufficient to treat GA-I, but instead a more complex strategy is needed. Our data highlights the many unresolved questions within the l-lysine degradation pathway and provides evidence for a so far unknown mechanism leading to glutaryl-CoA. Copyright © 2017 Elsevier B.V. All rights reserved.

Rosuvastatin reduces atherosclerotic lesions and promotes progenitor cell mobilisation and recruitment in apolipoprotein E knockout mice.

PubMed

Schroeter, Marco R; Humboldt, Tim; Schäfer, Katrin; Konstantinides, Stavros

2009-07-01

Statins enhance incorporation of bone marrow-derived cells into experimental neointimal lesions. However, the contribution of progenitor cells to progression of spontaneous atherosclerotic plaques, and the possible modulatory role of statins in this process, remain poorly understood. We compared the effects of rosuvastatin (1 and 10mg/kg BW) and pravastatin (10mg/kg) on progenitor cell mobilisation, recruitment into atherosclerotic plaques, and lesion growth. Statins were administered over 8 weeks to apolipoprotein E knockout mice on atherogenic diet. In addition, mice were lethally irradiated, followed by transplantation of bone marrow from LacZ transgenic mice. Rosuvastatin reduced lesion area and intima-to-media ratio at the brachiocephalic artery compared to vehicle, while both parameters were not significantly altered by pravastatin. Rosuvastatin also augmented endothelialisation (P<0.05) and reduced the smooth muscle cells (SMC) content (P=0.042) of lesions. Numbers of c-kit, sca-1 and flk-1, sca-1 double-positive progenitor cells were significantly increased in rosuvastatin compared to control-treated mice, both in the bone marrow and the peripheral blood. Similarly, the number of spleen-derived acLDL, lectin double-positive progenitor cells (P=0.001) and colony-forming units (P=0.0104) was significantly increased in mice treated with rosuvastatin compared to vehicle alone. In the bone marrow, increased Akt and p42/44 MAP kinase phosphorylation and upregulated SDF1alpha mRNA expression were observed. Importantly, rosuvastatin treatment also increased the plasma levels of c-kit ligand (P=0.003), and the number of c-kit-positive cells within atherosclerotic lesions (P=0.041). Our findings suggest that rosuvastatin reduces the size of atherosclerotic plaques, and this effect appears to involve progenitor cell mobilisation and recruitment into vascular lesions.

Structural and Functional Analysis of HIV-1 Coreceptors: Roles of Charged Residues and Posttranslational Modifications on Coreceptor Activity

DTIC Science & Technology

2000-01-01

various organs and to sites of inflammation. They may have additional functions. For example analysis of CXCR4 knockout mice show that CXCR4, which...SDF-1 knockout mice had similar phenotypes (195). Homozygous knockout of CXCR4 or SDF-1 results in embyonic lethality. Though CCR5 appears to be...dispensable, other chemokine receptors have vital functions. CXCR5 knockout mice have B-cell homing defects (118), and CXCR2 knockout mice

Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum.

PubMed

Unger, Tina; Bette, Stefanie; Zhang, Jiong; Theis, Martin; Engele, Jürgen

2012-01-06

The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy.

PubMed

Zhou, Jibin; Ahmad, Firdos; Parikh, Shan; Hoffman, Nichole E; Rajan, Sudarsan; Verma, Vipin K; Song, Jianliang; Yuan, Ancai; Shanmughapriya, Santhanam; Guo, Yuanjun; Gao, Erhe; Koch, Walter; Woodgett, James R; Madesh, Muniswamy; Kishore, Raj; Lal, Hind; Force, Thomas

2016-04-15

Cardiac myocyte-specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3β leads to cardiac protection after myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration because of the fact that all GSK-3-targeted drugs, including the drugs already in clinical trial target both isoforms of GSK-3, and none are isoform specific. To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3β in heart function. We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, double-knockout hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from double-knockout implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. Double-knockout cardiac myocytes showed cell cycle progression resulting in increased DNA content and multinucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe-induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis, and its loss is incompatible with life because of cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy. © 2016 American Heart Association, Inc.

Functional characterization of double-knockout mouse sperm lacking SPAM1 and ACR or SPAM1 and PRSS21 in fertilization.

PubMed

Zhou, Chong; Kang, Woojin; Baba, Tadashi

2012-01-01

Mammalian fertilization requires sperm to penetrate the cumulus to reach the oocyte. Although sperm hyaluronidase has long been believed to participate in the penetration process, our previous works revealed that neither of two sperm hyaluronidases, SPAM1 and HYAL5, are essential for fertilization. In this study, we have produced double-knockout mice lacking SPAM1 and either one of two sperm serine proteases, ACR and PRSS21, and characterized the mutant sperm. The SPAM1/ACR- and SPAM1/PRSS21-deficient males were fertile, whereas epididymal sperm of the mutant mice exhibited a reduced capacity to fertilize the oocytes in vitro. Despite normal motility, the ability of sperm to traverse the cumulus matrix was more severely impaired by the loss of SPAM1 and ACR or SPAM1 and PRSS21 than by the loss of only SPAM1. Moreover, SPAM1/ACR- and SPAM1/PRSS21-deficient sperm accumulated on the surface (outer edge) of the cumulus more abundantly than SPAM1-deficient sperm. These results suggest that ACR or PRSS21 or both may function cooperatively with SPAM1 in sperm/cumulus penetration.

Behavioral and neurochemical characterization of mice deficient in the phosphodiesterase-1B (PDE1B) enzyme.

PubMed

Siuciak, J A; McCarthy, S A; Chapin, D S; Reed, T M; Vorhees, C V; Repaske, D R

2007-07-01

PDE1B is a calcium-dependent cyclic nucleotide phosphodiesterase that is highly expressed in the striatum. In order to investigate the physiological role of PDE1B in the central nervous system, PDE1B knockout mice (C57BL/6N background) were assessed in behavioral tests and their brains were assayed for monoamine content. In a variety of well-characterized behavioral tasks, including the elevated plus maze (anxiety-like behavior), forced swim test (depression-like behavior), hot plate (nociception) and two cognition models (passive avoidance and acquisition of conditioned avoidance responding), PDE1B knockout mice performed similarly to wild-type mice. PDE1B knockout mice showed increased baseline exploratory activity when compared to wild-type mice. When challenged with amphetamine (AMPH) and methamphetamine (METH), male and female PDE1B knockout mice showed an exaggerated locomotor response. Male PDE1B knockout mice also showed increased locomotor responses to higher doses of phencyclidine (PCP) and MK-801; however, this effect was not consistently observed in female knockout mice. In the striatum, increased dopamine turnover (DOPAC/DA and HVA/DA ratios) was found in both male and female PDE1B knockout mice. Striatal serotonin (5-HT) levels were also decreased in PDE1B knockout mice, although levels of the metabolite, 5HIAA, were unchanged. The present studies demonstrate increased striatal dopamine turnover in PDE1B knockout mice associated with increased baseline motor activity and an exaggerated locomotor response to dopaminergic stimulants such as methamphetamine and amphetamine. These data further support a role for PDE1B in striatal function.

High susceptibility to liver injury in IL-27 p28 conditional knockout mice involves intrinsic interferon-γ dysregulation of CD4+ T cells.

PubMed

Zhang, Song; Liang, Ruifang; Luo, Wei; Liu, Chang; Wu, Xiaoli; Gao, Yanan; Hao, Jianlei; Cao, Guangchao; Chen, Xi; Wei, Jun; Xia, Siyuan; Li, Zheng; Wen, Ti; Wu, Yunyun; Zhou, Xinglong; Wang, Puyue; Zhao, Liqing; Wu, Zhengzhou; Xiong, Sidong; Gao, Xiaoming; Gao, Xiang; Chen, Yongyan; Ge, Qing; Tian, Zhigang; Yin, Zhinan

2013-04-01

Interleukin (IL)-27, a newly discovered IL-12 family cytokine, is composed of p28 and EBI3. In this study, CD11c-p28(f/f) conditional knockout mice were generated to delete p28 specifically in dendritic cells (DCs). We demonstrated that in the absence of DC-derived p28, these mice were highly susceptible to both low and higher concentrations of concanavalin A (ConA) (5 mg/kg or 10 mg/kg), with extremely early and steady high levels of interferon-γ (IFN-γ) in sera. Neutralizing IFN-γ prevented ConA-induced liver damage in these mice, indicating a critical role of IFN-γ in this pathological process. Interestingly, the main source of the increased IFN-γ in CD11c-p28(f/f) mice was CD4+ T cells, but not natural killer T (NKT) cells. Depletion of CD4+ , but not NK1.1+ , cells completely abolished liver damage, whereas transferring CD4+ T cells from CD11c-p28(f/f) mice, but not from wild-type mice or CD11c-p28(f/f) -IFN-γ(-/-) double knockout mice to CD4(-/-) mice, restored the increased liver damage. Further studies defined higher levels of IFN-γ and T-bet messenger RNA in naïve CD4+ T cells from CD11c-p28(f/f) mice, and these CD4+ T cells were highly responsive to both low and higher concentrations of anti-CD3, indicating a programmed functional alternation of CD4+ T cells. We provide a unique model for studying the pathology of CD4+ T cell-mediated liver injury and reveal a novel function of DC-derived p28 on ConA-induced fulminant hepatitis through regulation of the intrinsic ability for IFN-γ production by CD4+ T cells. Copyright © 2012 American Association for the Study of Liver Diseases.

Estrogen receptors in skeletal metabolism: lessons from genetically modified models of receptor function.

PubMed

McCauley, Laurie K; Tözüm, Tolga F; Rosol, Thomas J

2002-01-01

Estrogens have long been known to be important for skeletal homeostasis, but their precise mechanisms of action in bone are still unclear. Mice with targeted deletions of the estrogen receptors alpha (ERalpha) and beta (ERbeta) have been generated by two research groups and several studies performed characterizing the phenotype of ERalpha knockout (ERKOalpha), ERbeta knockout (ERKObeta), or double deletion of ERalpha and ERbeta (DERKO) mice. Initial studies reported a reduction in bone mineral density in male ERKOalpha mice. More extensive analyses have been puzzling, likely because of compensatory mechanisms in ERKO mice. Furthermore, the existence of a third ER continues to be a potential explanation for some actions of estrogen in bone. Other rodent models, including the testicular feminized mouse and rat, the aromatase knockout mouse, and a rat with a dominant negative ER mutation, have added information regarding estrogen's actions in bone. This review summarizes many reports characterizing available rodent models with genetic alterations relevant to estrogen action. The sum of these reports suggests that the ERbeta is not highly protective in bone because loss of its function results in minimal alterations in the skeleton. Furthermore, loss of both the ERalpha and the ERbeta does not account for loss of estrogen action in bone, because the impact of DERKO is seemingly not as great as the impact of gonadectomy on the skeleton. Finally, through studies of ERKO mice and other rodent models of altered sex steroid action, it appears that estrogen may be more protective in the skeleton than androgens.

Mouse model of fragile X syndrome: behavioral and hormonal response to stressors.

PubMed

Nielsen, Darci M; Evans, Jeffrey J; Derber, William J; Johnston, Kenzie A; Laudenslager, Mark L; Crnic, Linda S; Maclean, Kenneth N

2009-06-01

Fragile X syndrome, a form of mental retardation caused by inadequate levels of fragile X mental retardation protein (FMRP), is characterized by extreme sensitivity to sensory stimuli and increased behavioral and hormonal reactivity to stressors. Fmr1 knockout mice lack FMRP and exhibit abnormal responses to auditory stimuli. This study sought to determine whether Fmr1 knockout mice on an F1 hybrid background are normal in their response to footshock. Knockout mice were also examined for signs of hyperexcitation across an extended trial range, and serum corticosterone levels were evaluated in response to various stressors. The ability to acquire conditioned taste aversion was also assessed. Knockout mice exhibited no impairment in associative aversive learning or memory, since they successfully expressed conditioned taste aversion. Footshock-sensitivity, freezing behavior, and corticosterone response to various stressors did not differ between knockout and wild-type mice. However, knockout mice exhibited significantly increased responses during the extended test. The knockout mice's increased responsiveness to footshock in the extended test may be an indication of increased vulnerability to stress or enhanced emotional reactivity. Copyright (c) 2009 APA, all rights reserved.

High Levels of S100A8/A9 Proteins Aggravate Ventilator-Induced Lung Injury via TLR4 Signaling

PubMed Central

Aslami, Hamid; Jongsma, Geartsje; van den Berg, Elske; Vlaar, Alexander P. J.; Roelofs, Joris J. T. H.; Juffermans, Nicole P.; Schultz, Marcus J.; van der Poll, Tom; Roth, Johannes; Wieland, Catharina W.

2013-01-01

Background Bacterial products add to mechanical ventilation in enhancing lung injury. The role of endogenous triggers of innate immunity herein is less well understood. S100A8/A9 proteins are released by phagocytes during inflammation. The present study investigates the role of S100A8/A9 proteins in ventilator-induced lung injury. Methods Pulmonary S100A8/A9 levels were measured in samples obtained from patients with and without lung injury. Furthermore, wild-type and S100A9 knock-out mice, naive and with lipopolysaccharide-induced injured lungs, were randomized to 5 hours of spontaneously breathing or mechanical ventilation with low or high tidal volume (VT). In addition, healthy spontaneously breathing and high VT ventilated mice received S100A8/A9, S100A8 or vehicle intratracheal. Furthermore, the role of Toll-like receptor 4 herein was investigated. Results S100A8/A9 protein levels were elevated in patients and mice with lung injury. S100A8/A9 levels synergistically increased upon the lipopolysaccharide/high VT MV double hit. Markers of alveolar barrier dysfunction, cytokine and chemokine levels, and histology scores were attenuated in S100A9 knockout mice undergoing the double-hit. Exogenous S100A8/A9 and S100A8 induced neutrophil influx in spontaneously breathing mice. In ventilated mice, these proteins clearly amplified inflammation: neutrophil influx, cytokine, and chemokine levels were increased compared to ventilated vehicle-treated mice. In contrast, administration of S100A8/A9 to ventilated Toll-like receptor 4 mutant mice did not augment inflammation. Conclusion S100A8/A9 proteins increase during lung injury and contribute to inflammation induced by HVT MV combined with lipopolysaccharide. In the absence of lipopolysaccharide, high levels of extracellular S100A8/A9 still amplify ventilator-induced lung injury via Toll-like receptor 4. PMID:23874727

Opposing functions of spinal M2, M3, and M4 receptor subtypes in regulation of GABAergic inputs to dorsal horn neurons revealed by muscarinic receptor knockout mice.

PubMed

Zhang, Hong-Mei; Chen, Shao-Rui; Matsui, Minoru; Gautam, Dinesh; Wess, Jürgen; Pan, Hui-Lin

2006-03-01

Spinal muscarinic acetylcholine receptors (mAChRs) play an important role in the regulation of nociception. To determine the role of individual mAChR subtypes in control of synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in lamina II neurons using whole-cell recordings in spinal cord slices of wild-type and mAChR subtype knockout (KO) mice. The mAChR agonist oxotremorine-M (3-10 microM) dose-dependently decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. However, in the presence of the M2 and M4 subtype-preferring antagonist himbacine, oxotremorine-M caused a large increase in the sIPSC frequency. In M3 KO and M1/M3 double-KO mice, oxotremorine-M produced a consistent decrease in the frequency of sIPSCs, and this effect was abolished by himbacine. We were surprised to find that in M2/M4 double-KO mice, oxotremorine-M consistently increased the frequency of sIPSCs and mIPSCs in all neurons tested, and this effect was completely abolished by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 subtype-preferring antagonist. In M2 or M4 single-KO mice, oxotremorine-M produced a variable effect on sIPSCs; it increased the frequency of sIPSCs in some cells but decreased the sIPSC frequency in other neurons. Taken together, these data strongly suggest that activation of the M3 subtype increases synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M2 and M4 subtypes predominantly attenuates GABAergic inputs to dorsal horn neurons in mice, an action that is opposite to the role of M2 and M4 subtypes in the spinal cord of rats.

Excess 25-hydroxyvitamin D3 exacerbates tubulointerstitial injury in mice by modulating macrophage phenotype.

PubMed

Kusunoki, Yasuo; Matsui, Isao; Hamano, Takayuki; Shimomura, Akihiro; Mori, Daisuke; Yonemoto, Sayoko; Takabatake, Yoshitsugu; Tsubakihara, Yoshiharu; St-Arnaud, René; Isaka, Yoshitaka; Rakugi, Hiromi

2015-11-01

Vitamin D hydroxylated at carbon 25 (25(OH)D) is generally recognized as a precursor of active vitamin D. Despite its low affinity for the vitamin D receptor (VDR), both deficient and excessive 25(OH)D levels are associated with poor clinical outcomes. Here we studied direct effects of 25(OH)D3 on the kidney using 25(OH)D-1α-hydroxylase (CYP27B1) knockout mice. The effects of 25(OH)D3 on unilateral ureteral obstruction were analyzed as proximal tubular cells and macrophages are two major cell types that take up 25(OH)D and contribute to the pathogenesis of kidney injury. Excess 25(OH)D3 in obstructed mice worsened oxidative stress and tubulointerstitial fibrosis, whereas moderate levels of 25(OH)D3 had no effects. The exacerbating effects of excess 25(OH)D3 were abolished in CYP27B1/VDR double-knockout mice and in macrophage-depleted CYP27B1 knockout mice. Excess 25(OH)D3 upregulated both M1 marker (TNF-α) and M2 marker (TGF-β1) levels of kidney-infiltrating macrophages. In vitro analyses verified that excess 25(OH)D3 directly upregulated TNF-α and TGF-β1 in cultured macrophages but not in tubular cells. TNF-α and 25(OH)D3 cooperatively induced oxidative stress by upregulating iNOS in tubular cells. Aggravated tubulointerstitial fibrosis in mice with excess 25(OH)D3 indicated that macrophage-derived TGF-β1 also had a key role in the pathogenesis of surplus 25(OH)D3. Thus, excess 25(OH)D3 worsens tubulointerstitial injury by modulating macrophage phenotype.

Running promotes wakefulness and increases cataplexy in orexin knockout mice.

PubMed

España, Rodrigo A; McCormack, Sarah L; Mochizuki, Takatoshi; Scammell, Thomas E

2007-11-01

People with narcolepsy and mice lacking orexin/hypocretin have disrupted sleep/wake behavior and reduced physical activity. Our objective was to identify physiologic mechanisms through which orexin deficiency reduces locomotor activity. We examined spontaneous wheel running activity and its relationship to sleep/wake behavior in wild type (WT) and orexin knockout (KO) mice. Additionally, given that physical activity promotes alertness, we also studied whether orexin deficiency reduces the wake-promoting effects of exercise. Orexin KO mice ran 42% less than WT mice. Their ability to run appeared normal as they initiated running as often as WT mice and ran at normal speeds. However, their running bouts were considerably shorter, and they often had cataplexy or quick transitions into sleep after running. Wheel running increased the total amount of wakefulness in WT and orexin KO mice similarly, however, KO mice continued to have moderately fragmented sleep/wake behavior. Wheel running also doubled the amount of cataplexy by increasing the probability of transitioning into cataplexy. Orexin KO mice run significantly less than normal, likely due to sleepiness, imminent cataplexy, or a reduced motivation to run. Orexin is not required for the wake-promoting effects of wheel running given that both WT and KO mice had similar increases in wakefulness with running wheels. In addition, the clear increase in cataplexy with wheel running suggests the possibility that positive emotions or reward can trigger murine cataplexy, similar to that seen in people and dogs with narcolepsy.

Synergistic function of Smad4 and PTEN in suppressing forestomach squamous cell carcinoma in the mouse.

PubMed

Teng, Yan; Sun, An-Na; Pan, Xiao-Chen; Yang, Guan; Yang, Lei-Lei; Wang, Ming-Rong; Yang, Xiao

2006-07-15

The genetic bases underlying esophageal tumorigenesis are poorly understood. Our previous studies have shown that coordinated deletion of the Smad4 and PTEN genes results in accelerated hair loss and skin tumor formation in mice. Herein, we exemplify that the concomitant inactivation of Smad4 and PTEN accelerates spontaneous forestomach carcinogenesis at complete penetrance during the first 2 months of age. All of the forestomach tumors were invasive squamous cell carcinomas (SCCs), which recapitulated the natural history and pathologic features of human esophageal SCCs. A small population of the SCC lesions was accompanied by adenocarcinomas at the adjacent submucosa region in the double mutant mice. The rapid progression of forestomach tumor formation in the Smad4 and PTEN double knockout mice corresponded to a dramatic increase in esophageal and forestomach epithelial proliferation. The decreased expression of p27, p21, and p16 together with the overexpression of cyclin D1 contributed cooperatively to the accelerated forestomach tumorigenesis in the double mutant mice. Our results point strongly to the crucial relevance of synergy between Smad4 and PTEN to suppress forestomach tumorigenesis through the cooperative induction of cell cycle inhibitors.

COL4A6 is dispensable for autosomal recessive Alport syndrome.

PubMed

Murata, Tomohiro; Katayama, Kan; Oohashi, Toshitaka; Jahnukainen, Timo; Yonezawa, Tomoko; Sado, Yoshikazu; Ishikawa, Eiji; Nomura, Shinsuke; Tryggvason, Karl; Ito, Masaaki

2016-07-05

Alport syndrome is caused by mutations in the genes encoding α3, α4, or α5 (IV) chains. Unlike X-linked Alport mice, α5 and α6 (IV) chains are detected in the glomerular basement membrane of autosomal recessive Alport mice, however, the significance of this finding remains to be investigated. We therefore generated mice lacking both α3 and α6 (IV) chains and compared their renal function and survival with Col4a3 knockout mice of 129 × 1/Sv background. No significant difference was observed in the renal function or survival of the two groups, or when the mice were backcrossed once to C57BL/6 background. However, the survival of backcrossed double knockout mice was significantly longer than that of the mice of 129 × 1/Sv background, which suggests that other modifier genes were involved in this phenomenon. In further studies we identified two Alport patients who had a homozygous mutation in intron 46 of COL4A4. The α5 and α6 (IV) chains were focally detected in the glomerular basement membrane of these patients. These findings indicate that although α5 and α6 (IV) chains are induced in the glomerular basement membrane in autosomal recessive Alport syndrome, their induction does not seem to play a major compensatory role.

COL4A6 is dispensable for autosomal recessive Alport syndrome

PubMed Central

Murata, Tomohiro; Katayama, Kan; Oohashi, Toshitaka; Jahnukainen, Timo; Yonezawa, Tomoko; Sado, Yoshikazu; Ishikawa, Eiji; Nomura, Shinsuke; Tryggvason, Karl; Ito, Masaaki

2016-01-01

Alport syndrome is caused by mutations in the genes encoding α3, α4, or α5 (IV) chains. Unlike X-linked Alport mice, α5 and α6 (IV) chains are detected in the glomerular basement membrane of autosomal recessive Alport mice, however, the significance of this finding remains to be investigated. We therefore generated mice lacking both α3 and α6 (IV) chains and compared their renal function and survival with Col4a3 knockout mice of 129 × 1/Sv background. No significant difference was observed in the renal function or survival of the two groups, or when the mice were backcrossed once to C57BL/6 background. However, the survival of backcrossed double knockout mice was significantly longer than that of the mice of 129 × 1/Sv background, which suggests that other modifier genes were involved in this phenomenon. In further studies we identified two Alport patients who had a homozygous mutation in intron 46 of COL4A4. The α5 and α6 (IV) chains were focally detected in the glomerular basement membrane of these patients. These findings indicate that although α5 and α6 (IV) chains are induced in the glomerular basement membrane in autosomal recessive Alport syndrome, their induction does not seem to play a major compensatory role. PMID:27377778

Hypothyroidism Compromises Hypothalamic Leptin Signaling in Mice

PubMed Central

Groba, Claudia; Mayerl, Steffen; van Mullem, Alies A.; Visser, Theo J.; Darras, Veerle M.; Habenicht, Andreas J.

2013-01-01

The impact of thyroid hormone (TH) on metabolism and energy expenditure is well established, but the role of TH in regulating nutritional sensing, particularly in the central nervous system, is only poorly defined. Here, we studied the consequences of hypothyroidism on leptin production as well as leptin sensing in congenital hypothyroid TRH receptor 1 knockout (Trhr1 ko) mice and euthyroid control animals. Hypothyroid mice exhibited decreased circulating leptin levels due to a decrease in fat mass and reduced leptin expression in white adipose tissue. In neurons of the hypothalamic arcuate nucleus, hypothyroid mice showed increased leptin receptor Ob-R expression and decreased suppressor of cytokine signaling 3 transcript levels. In order to monitor putative changes in central leptin sensing, we generated hypothyroid and leptin-deficient animals by crossing hypothyroid Trhr1 ko mice with the leptin-deficient ob/ob mice. Hypothyroid Trhr1/ob double knockout mice showed a blunted response to leptin treatment with respect to body weight and food intake and exhibited a decreased activation of phospho-signal transducer and activator of transcription 3 as well as a up-regulation of suppressor of cytokine signaling 3 upon leptin treatment, particularly in the arcuate nucleus. These data indicate alterations in the intracellular processing of the leptin signal under hypothyroid conditions and thereby unravel a novel mode of action by which TH affects energy metabolism. PMID:23518925

Genetic disruption of NRF2 promotes the development of necroinflammation and liver fibrosis in a mouse model of HFE-hereditary hemochromatosis.

PubMed

Duarte, Tiago L; Caldas, Carolina; Santos, Ana G; Silva-Gomes, Sandro; Santos-Gonçalves, Andreia; Martins, Maria João; Porto, Graça; Lopes, José Manuel

2017-04-01

In hereditary hemochromatosis, iron deposition in the liver parenchyma may lead to fibrosis, cirrhosis and hepatocellular carcinoma. Most cases are ascribed to a common mutation in the HFE gene, but the extent of clinical expression is greatly influenced by the combined action of yet unidentified genetic and/or environmental modifying factors. In mice, transcription factor NRF2 is a critical determinant of hepatocyte viability during exposure to acute dietary iron overload. We evaluated if the genetic disruption of Nrf2 would prompt the development of liver damage in Hfe -/- mice (an established model of human HFE-hemochromatosis). Wild-type, Nrf2 -/- , Hfe -/- and double knockout (Hfe/Nrf2 -/- ) female mice on C57BL/6 genetic background were sacrificed at the age of 6 (young), 12-18 (middle-aged) or 24 months (old) for evaluation of liver pathology. Despite the parenchymal iron accumulation, Hfe -/- mice presented no liver injury. The combination of iron overload (Hfe -/- ) and defective antioxidant defences (Nrf2 -/- ) increased the number of iron-related necroinflammatory lesions (sideronecrosis), possibly due to the accumulation of toxic oxidation products such as 4-hydroxy-2-nonenal-protein adducts. The engulfment of dead hepatocytes led to a gradual accumulation of iron within macrophages, featuring large aggregates. Myofibroblasts recruited towards the injury areas produced substantial amounts of collagen fibers involving the liver parenchyma of double-knockout animals with increased hepatic fibrosis in an age-dependent manner. The genetic disruption of Nrf2 promotes the transition from iron accumulation (siderosis) to liver injury in Hfe -/- mice, representing the first demonstration of spontaneous hepatic fibrosis in the long term in a mouse model of hereditary hemochromatosis displaying mildly elevated liver iron. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Skeletal mineralization deficits and impaired biogenesis and function of chondrocyte-derived matrix vesicles in Phospho1−/− and Phospho1/Pit1 double knockout mice

PubMed Central

Yadav, Manisha C.; Bottini, Massimo; Cory, Esther; Bhattacharya, Kunal; Kuss, Pia; Narisawa, Sonoko; Sah, Robert L.; Beck, Laurent; Fadeel, Bengt; Farquharson, Colin; Millán, José Luis

2016-01-01

We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis but that their chondrocyte- and osteoblast-derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1-mediated intravesicular generation of Pi and phosphate transporter-mediated influx of Pi. To test this hypothesis, we generated mice with col2a1-driven cre-mediated ablation of Slc20a1, hereafter referred to as Pit1, alone or in combination with a Phospho1 gene deletion. Pit1col2/col2 mice did not show any major phenotypic abnormalities, while severe skeletal deformities were observed in the [Phospho1−/−; Pit1col2/col2] double knockout mice that were more pronounced than those observed in the Phospho1−/− mice. Histological analysis of [Phospho1−/−; Pit1col2/col2] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1−/−; Pit1col2/col2] skeleton displayed significantly decreases in BV/TV%, trabecular number and bone mineral density, as well as decreased stiffness, decreased strength, and increased post-yield deflection compared to Phospho1−/− mice. Using atomic force microscopy we found that ~80% of [Phospho1−/−; Pit1col2/col2] MVs were devoid of mineral in comparison to ~50 % for the Phospho1−/− MVs and ~25% for the WT and Pit1col2/col2 MVs. We also found a significant decrease in the number of MVs produced by both Phospho1−/− and [Phospho1−/−; Pit1col2/col2] chondrocytes. These data support the involvement of PiT-1 in the initiation of skeletal mineralization and provide compelling evidence that PHOSPHO1 function is involved in MV biogenesis. PMID:26773408

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

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.

Genetic evidence supporting a critical role of endothelial caveolin-1 during the progression of atherosclerosis

PubMed Central

Fernández-Hernando, Carlos; Yu, Jun; Suárez, Yajaira; Rahner, Christoph; Dávalos, Alberto; Lasunción, Miguel A.; Sessa, William C.

2009-01-01

SUMMARY The accumulation of LDL-derived cholesterol in the artery wall is the initiating event that causes atherosclerosis. However, the mechanisms that lead to the initiation of atherosclerosis are still poorly understood. Here, by using endothelial cell-specific transgenesis of the caveolin-1 (Cav-1) gene in mice, we show the critical role of Cav-1 in promoting atherogenesis. Mice were generated lacking Cav-1 and apoE but expressing endothelial-specific Cav-1 in the double knockout background. Genetic ablation of Cav-1 on an apoE knockout background inhibits the progression of atherosclerosis while re-expression of Cav-1 in the endothelium promotes lesion expansion. Mechanistically, the loss of Cav-1 reduces LDL infiltration into the artery wall, promotes nitric oxide production and reduces the expression of leukocyte adhesion molecules, effects completely reversed in transgenic mice. In summary, this unique model provides physiological evidence supporting the important role of endothelial Cav-1 expression in regulating the entry of LDL into the vessel wall and the initiation of atherosclerosis. PMID:19583953

Differential cytokine expression in skin graft healing in inducible nitric oxide synthase knockout mice.

PubMed

Most, D; Efron, D T; Shi, H P; Tantry, U S; Barbul, A

2001-10-01

Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test. Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05). Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These data further delineate the modulatory effect of iNOS and nitric oxide in healing skin grafts.

Abrogated Freud-1/CC2D1A repression of 5-HT1A autoreceptors induces fluoxetine-resistant anxiety/depression-like behavior

PubMed Central

Vahid-Ansari, Faranak; Daigle, Mireille; Manzini, M. Chiara; Tanaka, Kenji F.; Hen, René; Geddes, Sean D.; Béïque, Jean-Claude; James, Jonathan; Merali, Zul; Albert, Paul R.

2017-01-01

Freud-1/CC2D1A represses the gene transcription of serotonin-1A (5-HT1A) autoreceptors, which negatively regulate 5-HT tone. To test the role of Freud-1 in vivo, we generated mice with adulthood conditional knockout of Freud-1 in 5-HT neurons (cF1ko). In cF1ko mice, 5-HT1A autoreceptor protein, binding and hypothermia response were increased, with reduced 5-HT content and neuronal activity in the dorsal raphe. The cF1ko mice displayed increased anxiety- and depression-like behavior that was resistant to chronic antidepressant (fluoxetine) treatment. Using conditional Freud-1/5-HT1A double knockout (cF1/1A dko) to disrupt both Freud-1 and 5-HT1A genes in 5-HT neurons, no increase in anxiety- or depression-like behaviour was seen upon knockout of Freud-1 on the 5-HT1A autoreceptor-negative background, rather a reduction in depression-like behaviour emerged. These studies implicate transcriptional dys-regulation of 5-HT1A autoreceptors by the repressor Freud-1 in anxiety and depression and provide a clinically relevant genetic model of antidepressant resistance. Targeting specific transcription factors like Freud-1 to restore transcriptional balance may augment response to antidepressant treatment. PMID:29101244

Structural and Functional Analysis of HIV-1 Coreceptors: Roles of Charged Residues and Posttranslational Modifications on Coreceptor Activity

DTIC Science & Technology

2000-01-01

to sites of inflammation. They may have additional functions. For example analysis of CXCR4 knockout mice show that CXCR4, which is chemotactic for... mice had similar phenotypes (195). Homozygous knockout of CXCR4 or SDF-1 results in embyonic lethality. Though CCR5 appears to be dispensable, other...chemokine receptors have vital functions. CXCR5 knockout mice have B-cell homing defects (118), and CXCR2 knockout mice overproduce B-cells and

Environment enrichment rescues the neurodegenerative phenotypes in presenilins-deficient mice.

PubMed

Dong, Suzhen; Li, Chunxia; Wu, Pu; Tsien, Joe Z; Hu, Yinghe

2007-07-01

Presenilin (PS) 1 and 2 conditional double knockout (cDKO) mice show progressive memory dysfunction and forebrain degeneration. Gene expression profiling results revealed a strong activation of immunity and inflammation responses in the brains of 10-month-old cDKO mice. As environmental enrichment (EE) has been shown to be able to improve memory and induce neurogenesis of the brain, we assessed the effects of EE on the memory performance and the neurodegeneration in cDKO mice. We found that EE effectively enhanced memory and partially rescued the forebrain atrophy of the cDKO mice. Our results suggest that immunity and inflammation could play important roles in the neurodegeneration of cDKO mice. Furthermore, the beneficial effects of EE may be associated with the inhibition of the expression of immunity and inflammation-related genes in the brain.

Changes in blood carnitine and acylcarnitine profiles of very long-chain acyl-CoA dehydrogenase-deficient mice subjected to stress.

PubMed

Spiekerkoetter, U; Tokunaga, C; Wendel, U; Mayatepek, E; Exil, V; Duran, M; Wijburg, F A; Wanders, R J A; Strauss, A W

2004-03-01

In humans with deficiency of the very long-chain acyl-CoA dehydrogenase (VLCAD), C14-C18 acylcarnitines accumulate. In this paper we have used the VLCAD knockout mouse as a model to study changes in blood carnitine and acylcarnitine profiles under stress. VLCAD knockout mice exhibit stress-induced hypoglycaemia and skeletal myopathy; symptoms resembling human VLCADD. To study the extent of biochemical derangement in response to different stressors, we determined blood carnitine and acylcarnitine profiles after exercise on a treadmill, fasting, or exposure to cold. Even in a nonstressed, well-fed state, knockout mice presented twofold higher C14-C18 acylcarnitines and a lower free carnitine of 72% as compared to wild-type littermates. After 1 h of intense exercise, the C14-C18 acylcarnitines in blood significantly increased, but free carnitine remained unchanged. After 8 h of fasting at 4 degrees C, the long-chain acylcarnitines were elevated 5-fold in knockout mice in comparison with concentrations in unstressed wild-type mice (P < 0.05), and four out of 12 knockout mice died. Free carnitine decreased to 44% as compared with unstressed wild-type mice. An increase in C14-C18 acylcarnitines and a decrease of free carnitine were also observed in fasted heterozygous and wild-type mice. Long-chain acylcarnitines in blood increase in knockout mice in response to different stressors and concentrations correlate with the clinical condition. A decrease in blood free carnitine in response to severe stress is observed in knockout mice but also in wild-type littermates. Monitoring blood acylcarnitine profiles in response to different stressors may allow systematic analysis of therapeutic interventions in VLCAD knockout mice.

Proper cytoskeletal architecture beneath the plasma membrane of red blood cells requires Ttll4

PubMed Central

Ijaz, Faryal; Hatanaka, Yasue; Hatanaka, Takahiro; Tsutsumi, Koji; Iwaki, Takayuki; Umemura, Kazuo; Ikegami, Koji; Setou, Mitsutoshi

2017-01-01

Mammalian red blood cells (RBCs) circulate through blood vessels, including capillaries, for tens of days under high mechanical stress. RBCs tolerate this mechanical stress while maintaining their shape because of their elastic membrane skeleton. This membrane skeleton consists of spectrin-actin lattices arranged as quasi-hexagonal units beneath the plasma membrane. In this study, we found that the organization of the RBC cytoskeleton requires tubulin tyrosine ligase–like 4 (Ttll4). RBCs from Ttll4-knockout mice showed larger average diameters in smear test. Based on the rate of hemolysis, Ttll4-knockout RBCs showed greater vulnerability to phenylhydrazine-induced oxidative stress than did wild-type RBCs. Ultrastructural analyses revealed the macromolecular aggregation of cytoskeletal components in RBCs of Ttll4-knockout mice. Immunoprecipitation using the anti-glutamylation antibody GT335 revealed nucleosome assembly protein 1 (NAP1) to be the sole target of TTLL4 in the RBCs, and NAP1 glutamylation was completely lost in Ttll4-knockout RBCs. In wild-type RBCs, the amount of glutamylated NAP1 in the membrane was nearly double that in the cytosol. Furthermore, the absence of TTLL4-dependent glutamylation of NAP1 weakened the binding of NAP1 to the RBC membrane. Taken together, these data demonstrate that Ttll4 is required for proper cytoskeletal organization in RBCs. PMID:27974641

Cholecystokinin Plays a Novel Protective Role in Diabetic Kidney Through Anti-inflammatory Actions on Macrophage

PubMed Central

Miyamoto, Satoshi; Shikata, Kenichi; Miyasaka, Kyoko; Okada, Shinichi; Sasaki, Motofumi; Kodera, Ryo; Hirota, Daisho; Kajitani, Nobuo; Takatsuka, Tetsuharu; Kataoka, Hitomi Usui; Nishishita, Shingo; Sato, Chikage; Funakoshi, Akihiro; Nishimori, Hisakazu; Uchida, Haruhito Adam; Ogawa, Daisuke; Makino, Hirofumi

2012-01-01

Inflammatory process is involved in the pathogenesis of diabetic nephropathy. In this article, we show that cholecystokinin (CCK) is expressed in the kidney and exerts renoprotective effects through its anti-inflammatory actions. DNA microarray showed that CCK was upregulated in the kidney of diabetic wild-type (WT) mice but not in diabetic intracellular adhesion molecule-1 knockout mice. We induced diabetes in CCK-1 receptor (CCK-1R) and CCK-2R double-knockout (CCK-1R−/−,-2R−/−) mice, and furthermore, we performed a bone marrow transplantation study using CCK-1R−/− mice to determine the role of CCK-1R on macrophages in the diabetic kidney. Diabetic CCK-1R−/−,-2R−/− mice revealed enhanced albuminuria and inflammation in the kidney compared with diabetic WT mice. In addition, diabetic WT mice with CCK-1R−/− bone marrow–derived cells developed more albuminuria than diabetic CCK-1R−/− mice with WT bone marrow–derived cells. Administration of sulfated cholecystokinin octapeptide (CCK-8S) ameliorated albuminuria, podocyte loss, expression of proinflammatory genes, and infiltration of macrophages in the kidneys of diabetic rats. Furthermore, CCK-8S inhibited both expression of tumor necrosis factor-α and chemotaxis in cultured THP-1 cells. These results suggest that CCK suppresses the activation of macrophage and expression of proinflammatory genes in diabetic kidney. Our findings may provide a novel strategy of therapy for the early stage of diabetic nephropathy. PMID:22357963

Distorted leukocyte migration, angiogenesis, wound repair and metastasis in Tspan8 and Tspan8/CD151 double knockout mice indicate complementary activities of Tspan8 and CD51.

PubMed

Zhao, Kun; Erb, Ulrike; Hackert, Thilo; Zöller, Margot; Yue, Shijing

2018-02-01

The tetraspanin Tspan8 supports via associated integrins and proteases tumor progression and angiogenesis. To shed light on its activities in non-transformed cells, we generated a Tspan8 knockout (ko) mouse, comparing leukocyte migration, angiogenesis, wound healing and tumor growth with wild type, CD151ko and Tspan8/CD151ko (dbko) mice. CD151ko mice were included as CD151 activities resemble that of Tspan8, and dbko mice to exclude mutual substitution. Tspan8ko and dbko mice show no pathological phenotype. However, delayed type hypersensitivity reactions are mitigated in Tspan8ko mice, angiogenesis is severely impaired in Tspan8ko, CD151ko and dbko mice, with Tspan8 mostly affecting lymphangiogenesis. Distinct contributions of CD151 and Tspan8 to skin wound healing rely on preferentially CD151 anchoring basal keratinocytes and Tspan8 promoting motility. Proliferation of wounded skin keratinocytes is not affected. Metastasis formation of a melanoma and a Tspan8-expressing pancreatic cancer line was impaired in Tspan8ko and dbko mice, pointing towards a contribution of host Tspan8 to tumor progression. In line with the importance of tetraspanins in exosome-mediated intercellular communication, defects became mitigated by Tspan8/CD151-competent serum exosomes, which offers a most promising therapeutic option for chronic wounds and arteriosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Oral aversion to dietary sugar, ethanol and glycerol correlates with alterations in specific hepatic metabolites in a mouse model of human citrin deficiency.

PubMed

Saheki, Takeyori; Inoue, Kanako; Ono, Hiromi; Fujimoto, Yuki; Furuie, Sumie; Yamamura, Ken-Ichi; Kuroda, Eishi; Ushikai, Miharu; Asakawa, Akihiro; Inui, Akio; Eto, Kazuhiro; Kadowaki, Takashi; Moriyama, Mitsuaki; Sinasac, David S; Yamamoto, Takashi; Furukawa, Tatsuhiko; Kobayashi, Keiko

2017-04-01

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

An inducible knockout mouse to model the cell-autonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias.

PubMed

Mirantes, Cristina; Eritja, Núria; Dosil, Maria Alba; Santacana, Maria; Pallares, Judit; Gatius, Sónia; Bergadà, Laura; Maiques, Oscar; Matias-Guiu, Xavier; Dolcet, Xavier

2013-05-01

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ER(T) under the control of a chicken actin promoter, we have generated a tamoxifen-inducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors.

An inducible knockout mouse to model the cell-autonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias

PubMed Central

Mirantes, Cristina; Eritja, Núria; Dosil, Maria Alba; Santacana, Maria; Pallares, Judit; Gatius, Sónia; Bergadà, Laura; Maiques, Oscar; Matias-Guiu, Xavier; Dolcet, Xavier

2013-01-01

SUMMARY PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ERT under the control of a chicken actin promoter, we have generated a tamoxifen-inducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors. PMID:23471917

New Advanced Technologies in Stem Cell Therapy

DTIC Science & Technology

2014-11-01

6-8 wks old utrophin/dystrophin double knockout (dKO) mice, a severe animal model of DMD, have an excess of ectopic fat , calcium deposits and...tissues in skeletal muscle alter the tissue environment and induce deregulation of muscle homeostasis; however, the cellular origin of muscle fat ...as a major contributor to ectopic fat cell, calcium deposits and fibrotic tissue formation within dystrophic muscle. In the current study, we propose

Studies of UCP2 transgenic and knockout mice reveal that liver UCP2 is not essential for the antiobesity effects of fish oil.

PubMed

Tsuboyama-Kasaoka, Nobuyo; Sano, Kayo; Shozawa, Chikako; Osaka, Toshimasa; Ezaki, Osamu

2008-03-01

Uncoupling protein 2 (UCP2) is a possible target molecule for energy dissipation. Many dietary fats, including safflower oil and lard, induce obesity in C57BL/6 mice, whereas fish oil does not. Fish oil increases UCP2 expression in hepatocytes and may enhance UCP2 activity by activating the UCP2 molecule or altering the lipid bilayer environment. To examine the role of liver UCP2 in obesity, we created transgenic mice that overexpressed human UCP2 in hepatocytes and examined whether UCP2 transgenic mice showed less obesity when fed a high-fat diet (safflower oil or lard). In addition, we examined whether fish oil had antiobesity effects in UCP2 knockout mice. UCP2 transgenic and wild-type mice fed a high-fat diet (safflower oil or lard) developed obesity to a similar degree. UCP2 knockout and wild-type mice fed fish oil had lower rates of obesity than mice fed safflower oil. Remarkably, safflower oil did not induce obesity in female UCP2 knockout mice, an unexpected phenotype for which we presently have no explanation. However, this unexpected effect was not observed in male UCP2 knockout mice or in UCP2 knockout mice fed a high-lard diet. These data indicate that liver UCP2 is not essential for fish oil-induced decreases in body fat.

Renal Dysfunction Induced by Kidney-Specific Gene Deletion of Hsd11b2 as a Primary Cause of Salt-Dependent Hypertension.

PubMed

Ueda, Kohei; Nishimoto, Mitsuhiro; Hirohama, Daigoro; Ayuzawa, Nobuhiro; Kawarazaki, Wakako; Watanabe, Atsushi; Shimosawa, Tatsuo; Loffing, Johannes; Zhang, Ming-Zhi; Marumo, Takeshi; Fujita, Toshiro

2017-07-01

Genome-wide analysis of renal sodium-transporting system has identified specific variations of Mendelian hypertensive disorders, including HSD11B2 gene variants in apparent mineralocorticoid excess. However, these genetic variations in extrarenal tissue can be involved in developing hypertension, as demonstrated in former studies using global and brain-specific Hsd11b2 knockout rodents. To re-examine the importance of renal dysfunction on developing hypertension, we generated kidney-specific Hsd11b2 knockout mice. The knockout mice exhibited systemic hypertension, which was abolished by reducing salt intake, suggesting its salt-dependency. In addition, we detected an increase in renal membrane expressions of cleaved epithelial sodium channel-α and T53-phosphorylated Na + -Cl - cotransporter in the knockout mice. Acute intraperitoneal administration of amiloride-induced natriuresis and increased urinary sodium/potassium ratio more in the knockout mice compared with those in the wild-type control mice. Chronic administration of amiloride and high-KCl diet significantly decreased mean blood pressure in the knockout mice, which was accompanied with the correction of hypokalemia and the resultant decrease in Na + -Cl - cotransporter phosphorylation. Accordingly, a Na + -Cl - cotransporter blocker hydrochlorothiazide significantly decreased mean blood pressure in the knockout mice. Chronic administration of mineralocorticoid receptor antagonist spironolactone significantly decreased mean blood pressure of the knockout mice along with downregulation of cleaved epithelial sodium channel-α and phosphorylated Na + -Cl - cotransporter expression in the knockout kidney. Our data suggest that kidney-specific deficiency of 11β-HSD2 leads to salt-dependent hypertension, which is attributed to mineralocorticoid receptor-epithelial sodium channel-Na + -Cl - cotransporter activation in the kidney, and provides evidence that renal dysfunction is essential for developing the phenotype of apparent mineralocorticoid excess. © 2017 American Heart Association, Inc.

Preaxial Polydactyly in Sost/Sostdc1 Double Knockouts

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

Yee, C M; Collette, N M; Loots, G G

2011-07-29

In the United States, {approx}5% are born with congenital birth defects due to abnormal function of cellular processes and interactions. Sclerosteosis, a rare autosomal recessive disease, causes hyperostosis of the axial and appendicular skeleton, and patients present radial deviation, digit syndactyly, nail dysplasia, and overall high bone mineral density. Sclerosteosis is due to a loss of function of sclerostin (Sost). Sost is a Wnt (abbrev.) antagonist; when mutated, nonfunctional Sost results in hyperactive osteoblast activity which leads to abnormal high bone mass. Previous studies have shown that Sost overexpression in transgenic mice causes reduced bone mineral density and a varietymore » of limb phenotypes ranging from lost, fused, and split phalanges. Consistent with clinical manifestations of Sclerosteosis, Sost knockout mice exhibit increased generalized bone mineral density and syndactyly of the digits. Sostdc1 is a paralog of Sost that has also been described as an antagonist of Wnt signaling, in developing tooth buds. Unlike Sost knockouts, Sostdc1 null mice do not display any limb abnormalities. To determine if Sost and Sostdc1 have redundant functions during limb patterning, we examined Sost; Sostdc1 mice determined that they exhibit a novel preaxial polydactyly phenotype with a low penetrance. LacZ staining, skeletal preparations, and in situ hybridization experiments were used to help characterize this novel phenotype and understand how this phenotype develops. We find Sost and Sostdc1 to have complementary expression patterns during limb development, and the loss of their expression alters the transcription of several key limb regulators, such as Fgf8, Shh and Grem.« less

Co-regulation of intragenic microRNA miR-153 and its host gene Ia-2 β: identification of miR-153 target genes with functions related to IA-2β in pancreas and brain.

PubMed

Mandemakers, W; Abuhatzira, L; Xu, H; Caromile, L A; Hébert, S S; Snellinx, A; Morais, V A; Matta, S; Cai, T; Notkins, A L; De Strooper, B

2013-07-01

We analysed the genomic organisation of miR-153, a microRNA embedded in genes that encode two of the major type 1 diabetes autoantigens, islet-associated protein (IA)-2 and IA-2β. We also identified miR-153 target genes that correlated with IA-2β localisation and function. A bioinformatics approach was used to identify miR-153's genomic organisation. To analyse the co-regulation of miR-153 and IA-2β, quantitative PCR analysis of miR-153 and Ia-2β (also known as Ptprn2) was performed after a glucose stimulation assay in MIN6B cells and isolated murine pancreatic islets, and also in wild-type Ia-2 (also known as Ptprn), Ia-2β single knockout and Ia-2/Ia-2β double knockout mouse brain and pancreatic islets. Bioinformatics identification of miR-153 target genes and validation via luciferase reporter assays, western blotting and quantitative PCR were also carried out. Two copies of miR-153, miR-153-1 and miR-153-2, are localised in intron 19 of Ia-2 and Ia-2β, respectively. In rodents, only miR-153-2 is conserved. We demonstrated that expression of miR-153-2 and Ia-2β in rodents is partially co-regulated as demonstrated by a strong reduction of miR-153 expression levels in Ia-2β knockout and Ia-2/Ia-2β double knockout mice. miR-153 levels were unaffected in Ia-2 knockout mice. In addition, glucose stimulation, which increases Ia-2 and Ia-2β expression, also significantly increased expression of miR-153. Several predicted targets of miR-153 were reduced after glucose stimulation in vitro, correlating with the increase in miR-153 levels. This study suggests the involvement of miR-153, IA-2β and miR-153 target genes in a regulatory network, which is potentially relevant to insulin and neurotransmitter release.

A novel CBL-Bflox/flox mouse model allows tissue-selective fully conditional CBL/CBL-B double-knockout: CD4-Cre mediated CBL/CBL-B deletion occurs in both T-cells and hematopoietic stem cells

PubMed Central

Goetz, Benjamin; An, Wei; Mohapatra, Bhopal; Zutshi, Neha; Iseka, Fany; Storck, Matthew D.; Meza, Jane; Sheinin, Yuri; Band, Vimla; Band, Hamid

2016-01-01

CBL-family ubiquitin ligases are critical negative regulators of tyrosine kinase signaling, with a clear redundancy between CBL and CBL-B evident in the immune cell and hematopoietic stem cell studies. Since CBL and CBL-B are negative regulators of immune cell activation, elimination of their function to boost immune cell activities could be beneficial in tumor immunotherapy. However, mutations of CBL are associated with human leukemias, pointing to tumor suppressor roles of CBL proteins; hence, it is critical to assess the tumor-intrinsic roles of CBL and CBL-B in cancers. This has not been possible since the only available whole-body CBL-B knockout mice exhibit constitutive tumor rejection. We engineered a new CBL-Bflox/flox mouse, combined this with an existing CBLflox/flox mouse to generate CBLflox/flox; CBL-Bflox/flox mice, and tested the tissue-specific concurrent deletion of CBL and CBL-B using the widely-used CD4-Cre transgenic allele to produce a T-cell-specific double knockout. Altered T-cell development, constitutive peripheral T-cell activation, and a lethal multi-organ immune infiltration phenotype largely resembling the previous Lck-Cre driven floxed-CBL deletion on a CBL-B knockout background establish the usefulness of the new model for tissue-specific CBL/CBL-B deletion. Unexpectedly, CD4-Cre-induced deletion in a small fraction of hematopoietic stem cells led to expansion of certain non-T-cell lineages, suggesting caution in the use of CD4-Cre for T-cell-restricted gene deletion. The establishment of a new model of concurrent tissue-selective CBL/CBL-B deletion should allow a clear assessment of the tumor-intrinsic roles of CBL/CBL-B in non-myeloid malignancies and help test the potential for CBL/CBL-B inactivation in immunotherapy of tumors. PMID:27276677

Fractionation of Spatial Memory in GRM2/3 (mGlu2/mGlu3) Double Knockout Mice Reveals a Role for Group II Metabotropic Glutamate Receptors at the Interface Between Arousal and Cognition

PubMed Central

Lyon, Louisa; Burnet, Philip WJ; Kew, James NC; Corti, Corrado; Rawlins, J Nicholas P; Lane, Tracy; De Filippis, Bianca; Harrison, Paul J; Bannerman, David M

2011-01-01

Group II metabotropic glutamate receptors (mGluR2 and mGluR3, encoded by GRM2 and GRM3) are implicated in hippocampal function and cognition, and in the pathophysiology and treatment of schizophrenia and other psychiatric disorders. However, pharmacological and behavioral studies with group II mGluR agonists and antagonists have produced complex results. Here, we studied hippocampus-dependent memory in GRM2/3 double knockout (GRM2/3−/−) mice in an iterative sequence of experiments. We found that they were impaired on appetitively motivated spatial reference and working memory tasks, and on a spatial novelty preference task that relies on animals' exploratory drive, but were unimpaired on aversively motivated spatial memory paradigms. GRM2/3−/− mice also performed normally on an appetitively motivated, non-spatial, visual discrimination task. These results likely reflect an interaction between GRM2/3 genotype and the arousal-inducing properties of the experimental paradigm. The deficit seen on appetitive and exploratory spatial memory tasks may be absent in aversive tasks because the latter induce higher levels of arousal, which rescue spatial learning. Consistent with an altered arousal–cognition relationship in GRM2/3−/− mice, injection stress worsened appetitively motivated, spatial working memory in wild-types, but enhanced performance in GRM2/3−/− mice. GRM2/3−/− mice were also hypoactive in response to amphetamine. This fractionation of hippocampus-dependent memory depending on the appetitive-aversive context is to our knowledge unique, and suggests a role for group II mGluRs at the interface of arousal and cognition. These arousal-dependent effects may explain apparently conflicting data from previous studies, and have translational relevance for the involvement of these receptors in schizophrenia and other disorders. PMID:21832989

Absence of SOCS3 in the cardiomyocyte increases mortality in a gp130 dependent manner accompanied by contractile dysfunction and ventricular arrhythmias

PubMed Central

Yajima, Toshitaka; Murofushi, Yoshiteru; Zhou, Hanbing; Park, Stanley; Housman, Jonathan; Zhong, Zhao-Hua; Nakamura, Michinari; Machida, Mitsuyo; Hwang, Kyung-Kuk; Gu, Yusu; Dalton, Nancy D.; Yajima, Tomoko; Yasukawa, Hideo; Peterson, Kirk L; Knowlton, Kirk U.

2011-01-01

Background Suppressor of cytokine signaling-3 (SOCS3) is a key negative-feedback regulator of gp130 receptor that provides crucial signaling for cardiac hypertrophy and survival; however, an in vivo role of SOCS3 regulation on cardiac gp130 signaling remains obscure. Methods and Results We generated cardiac-specific SOCS3 knockout (SOCS3 cKO) mice. These mice showed increased activation of gp130 downstream signaling targets (STAT3, ERK1/2, AKT and p38) from 15 weeks of age and developed cardiac dysfunction from around 25 weeks of age with signs of heart failure. Surprisingly, SOCS3 cKO failing hearts had minimal histological abnormalities with intact myofibril ultrastructure. In addition, Ca2+ transients were significantly increased in SOCS3 cKO failing hearts compared to wild-type (WT) hearts. We also found that Ser23/24 residues of troponin I were hypophosphorylated in SOCS3 cKO hearts before the manifestation of cardiac dysfunction. These data suggested the presence of abnormalities in myofilament Ca2+ sensitivity in SOCS3 cKO mice. In addition to the contractile dysfunction, we found various ventricular arrhythmias in SOCS3 cKO non-failing hearts accompanied by a sarcoplasmic reticulum Ca2+ overload. To determine the contribution of gp130 signaling to the cardiac phenotype that occurs with SOCS3 deficiency, we generated cardiac-specific gp130 and SOCS3 double knockout mice. Double KO mice lived significantly longer and had different histological abnormalities when compared to SOCS3 cKO mice; thus, demonstrating the importance of gp130 signaling in the SOCS3 cKO cardiac phenotype. Conclusions Our results demonstrate an important role of SOCS3 regulation on cardiac gp130 signaling in the pathogenesis of contractile dysfunction and ventricular arrhythmias. PMID:22082679

Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity.

PubMed

Groen, Annemiek; Romero, Marta Rodriguez; Kunne, Cindy; Hoosdally, Sarah J; Dixon, Peter H; Wooding, Carol; Williamson, Catherine; Seppen, Jurgen; Van den Oever, Karin; Mok, Kam S; Paulusma, Coen C; Linton, Kenneth J; Oude Elferink, Ronald P J

2011-11-01

Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4(-/-) mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts. To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts. Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1-CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice. ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1-CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

A mental retardation gene, motopsin/neurotrypsin/prss12, modulates hippocampal function and social interaction

PubMed Central

Mitsui, Shinichi; Osako, Yoji; Yokoi, Fumiaki; Dang, Mai T.; Yuri, Kazunari; Li, Yuqing; Yamaguchi, Nozomi

2010-01-01

Motopsin is a mosaic serine protease secreted from neuronal cells in various brain regions including the hippocampus. The loss of motopsin function causes nonsyndromic mental retardation in humans and impairs long-term memory formation in Drosophila. To understand motopsin’s function in the mammalian brain, motopsin knockout mice were generated. Motopsin knockout mice did not have significant deficit in memory formation, as was tested using in the Morris water maze, passive avoidance, and Y-maze tests. A social recognition test showed that the motopsin knockout mice had the ability to recognize two stimulator mice, suggesting normal social memory. In a social novelty test, motopsin knockout mice spent a longer time investigating a familiar mouse than wild-type mice did. In a resident-intruder test, motopsin knockout mice showed prolonged social interaction compared to wild-type mice. Consistent with the behavioral deficit, spine density was significantly decreased on apical dendrites, but not on basal dendrites, of hippocampal pyramidal neurons of motopsin knockout mice. In contrast, pyramidal neurons at the cingulate cortex showed normal spine density. Spatial learning and social interaction induced the phosphorylation of cAMP responsive element binding protein (CREB) in hippocampal neurons of wild-type mice, whereas the phosphorylation of CREB was markedly decreased in mutant mouse brains. Our results indicate that an extracellular protease, motopsin, preferentially affects social behaviors, and modulates the functions of hippocampal neurons. PMID:20092579

Distinct Roles of Opioid and Dopamine Systems in Lateral Hypothalamic Intracranial Self-Stimulation.

PubMed

Ide, Soichiro; Takahashi, Takehiro; Takamatsu, Yukio; Uhl, George R; Niki, Hiroaki; Sora, Ichiro; Ikeda, Kazutaka

2017-05-01

Opioid and dopamine systems play crucial roles in reward. Similarities and differences in the neural mechanisms of reward that are mediated by these 2 systems have remained largely unknown. Thus, in the present study, we investigated the differences in reward function in both µ-opioid receptor knockout mice and dopamine transporter knockout mice, important molecules in the opioid and dopamine systems. Mice were implanted with electrodes into the right lateral hypothalamus (l hour). Mice were then trained to put their muzzle into the hole in the head-dipping chamber for intracranial electrical stimulation, and the influences of gene knockout were assessed. Significant differences are observed between opioid and dopamine systems in reward function. µ-Opioid receptor knockout mice exhibited enhanced intracranial electrical stimulation, which induced dopamine release. They also exhibited greater motility under conditions of "despair" in both the tail suspension test and water wheel test. In contrast, dopamine transporter knockout mice maintained intracranial electrical stimulation responding even when more active efforts were required to obtain the reward. The absence of µ-opioid receptor or dopamine transporter did not lead to the absence of intracranial electrical stimulation responsiveness but rather differentially altered it. The present results in µ-opioid receptor knockout mice are consistent with the suppressive involvement of µ-opioid receptors in both positive incentive motivation associated with intracranial electrical stimulation and negative incentive motivation associated with depressive states. In contrast, the results in dopamine transporter knockout mice are consistent with the involvement of dopamine transporters in positive incentive motivation, especially its persistence. Differences in intracranial electrical stimulation in µ-opioid receptor and dopamine transporter knockout mice underscore the multidimensional nature of reward. © The Author 2016. Published by Oxford University Press on behalf of CINP.

Overexpression of the Type 1 Adenylyl Cyclase in the Forebrain Leads to Deficits of Behavioral Inhibition

PubMed Central

Cao, Hong; Saraf, Amit; Zweifel, Larry S.

2015-01-01

The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition. PMID:25568126

New insight into the role of the β3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout

PubMed Central

Ferguson, Carolyn; Hardy, Steven L; Werner, David F; Hileman, Stanley M; DeLorey, Timothy M; Homanics, Gregg E

2007-01-01

Background The β3 subunit of the γ-aminobutyric acid type A receptor (GABAA-R) has been reported to be important for palate formation, anesthetic action, and normal nervous system function. This subunit has also been implicated in the pathogenesis of Angelman syndrome and autism spectrum disorder. To further investigate involvement of this subunit, we previously produced mice with a global knockout of β3. However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model. To overcome many of these limitations, a mouse line with a conditionally inactivated β3 gene was engineered. Results Gene targeting and embryonic stem cell technologies were used to create mice in which exon 3 of the β3 subunit was flanked by loxP sites (i.e., floxed). Crossing the floxed β3 mice to a cre general deleter mouse line reproduced the phenotype of the previously described global knockout. Pan-neuronal knockout of β3 was achieved by crossing floxed β3 mice to Synapsin I-cre transgenic mice. Palate development was normal in pan-neuronal β3 knockouts but ~61% died as neonates. Survivors were overtly normal, fertile, and were less sensitive to etomidate. Forebrain selective knockout of β3 was achieved using α CamKII-cre transgenic mice. Palate development was normal in forebrain selective β3 knockout mice. These knockouts survived the neonatal period, but ~30% died between 15–25 days of age. Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese. Conclusion Conditional inactivation of the β3 gene revealed novel insight into the function of this GABAA-R subunit. The floxed β3 knockout mice described here will be very useful for conditional knockout studies to further investigate the role of the β3 subunit in development, ethanol and anesthetic action, normal physiology, and pathophysiologic processes. PMID:17927825

Reduced Extinction of Hippocampal-Dependent Memories in CPEB Knockout Mice

ERIC Educational Resources Information Center

Zearfoss, N. Ruth; Richter, Joel D.; Berger-Sweeney, Joanne

2006-01-01

CPEB is a sequence-specific RNA binding protein that regulates translation at synapses. In neurons of CPEB knockout mice, synaptic efficacy is reduced. Here, we have performed a battery of behavioral tests and find that relative to wild-type animals, CPEB knockout mice, although similar on many baseline behaviors, have reduced extinction of…

Senescence marker protein-30/superoxide dismutase 1 double knockout mice exhibit increased oxidative stress and hepatic steatosis

PubMed Central

Kondo, Yoshitaka; Masutomi, Hirofumi; Noda, Yoshihiro; Ozawa, Yusuke; Takahashi, Keita; Handa, Setsuko; Maruyama, Naoki; Shimizu, Takahiko; Ishigami, Akihito

2014-01-01

Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that converts superoxide anion radicals into hydrogen peroxide and molecular oxygen. The senescence marker protein-30 (SMP30) is a gluconolactonase that functions as an antioxidant protein in mammals due to its involvement in ascorbic acid (AA) biosynthesis. SMP30 also participates in Ca2+ efflux by activating the calmodulin-dependent Ca2+-pump. To reveal the role of oxidative stress in lipid metabolism defects occurring in non-alcoholic fatty liver disease pathogenesis, we generated SMP30/SOD1-double knockout (SMP30/SOD1-DKO) mice and investigated their survival curves, plasma and hepatic lipid profiles, amounts of hepatic oxidative stress, and hepatic protein levels expressed by genes related to lipid metabolism. While SMP30/SOD1-DKO pups had no growth retardation by 14 days of age, they did have low plasma and hepatic AA levels. Thereafter, 39% and 53% of male and female pups died by 15–24 and 89 days of age, respectively. Compared to wild type, SMP30-KO and SOD1-KO mice, by 14 days SMP30/SOD1-DKO mice exhibited: (1) higher plasma levels of triglyceride and aspartate aminotransferase; (2) severe accumulation of hepatic triglyceride and total cholesterol; (3) higher levels of superoxide anion radicals and thiobarbituric acid reactive substances in livers; and (4) decreased mRNA and protein levels of Apolipoprotein B (ApoB) in livers – ApoB is an essential component of VLDL secretion. These results suggest that high levels of oxidative stress due to concomitant deficiency of SMP30 and/or AA, and SOD1 cause abnormal plasma lipid metabolism, hepatic lipid accumulation and premature death resulting from impaired VLDL secretion. PMID:25003023

[Upregulation of P2X3 receptors in dorsal root ganglion of TRPV1 knockout female mice].

PubMed

Fang, Xiao; Shi, Xiao-Han; Huang, Li-Bin; Rong, Wei-Fang; Ma, Bei

2014-08-25

The study was aimed to investigate the changes in mechanical pain threshold in the condition of chronic inflammatory pain after transient receptor potential vanilloid 1 (TRPV1) gene was knockout. Hind-paw intraplantar injection of complete freund's adjuvant (CFA, 20 μL) produced peripheral inflammation in wild-type and TRPV1 knockout female mice. The mechanical pain thresholds were measured during the 8 days after injection and pre-injection by using Von-Frey hair. Nine days after injection, mice were killed and the differences of expression of c-Fos and P2X3 receptor in the dorsal root ganglia (DRG) and spinal cord dorsal horn were examined by Western blotting between the two groups. Compared with that in wild-type mice, the mechanical pain threshold was increased significantly in TRPV1 knockout mice (P < 0.05); 3 days after CFA injection, the baseline mechanical pain threshold in the TRPV1 knockout mice group was significantly higher than that in the wild-type mice group (P < 0.05); The result of Western blotting showed that the expression of c-Fos protein both in DRG and spinal cord dorsal horn of TRPV1 knockout mice group was decreased significantly compared with that in wild-type mice group (P < 0.01, P < 0.05), while the expression of P2X3 receptor in DRG of TRPV1 knockout mice group was increased significantly compared with that in wild-type mice group (P < 0.05). Our findings indicate that TRPV1 may influence the peripheral mechanical pain threshold by mediating the expression of c-Fos protein both in DRG and spinal cord dorsal horn and changing the expression of P2X3 receptor in DRG.

Earlier onset of motor deficits in mice with double mutations in Dyt1 and Sgce.

PubMed

Yokoi, Fumiaki; Yang, Guang; Li, Jindong; DeAndrade, Mark P; Zhou, Tong; Li, Yuqing

2010-10-01

DYT1 early-onset generalized torsion dystonia is an inherited movement disorder caused by mutations in DYT1 coding for torsinA with ∼30% penetrance. Most of the DYT1 dystonia patients exhibit symptoms during childhood and adolescence. On the other hand, DYT1 mutation carriers without symptoms during these periods mostly do not exhibit symptoms later in their life. Little is known about what controls the timing of the onset, a critical issue for DYT1 mutation carriers. DYT11 myoclonus-dystonia is caused by mutations in SGCE coding for ε-sarcoglycan. Two dystonia patients from a single family with double mutations in DYT1 and SGCE exhibited more severe symptoms. A recent study suggested that torsinA contributes to the quality control of ε-sarcoglycan. Here, we derived mice carrying mutations in both Dyt1 and Sgce and found that these double mutant mice showed earlier onset of motor deficits in beam-walking test. A novel monoclonal antibody against mouse ε-sarcoglycan was developed by using Sgce knock-out mice to avoid the immune tolerance. Western blot analysis suggested that functional deficits of torsinA and ε-sarcoglycan may independently cause motor deficits. Examining additional mutations in other dystonia genes may be beneficial to predict the onset in DYT1 mutation carriers.

Continuous intrathecal orexin delivery inhibits cataplexy in a murine model of narcolepsy.

PubMed

Kaushik, Mahesh K; Aritake, Kosuke; Imanishi, Aya; Kanbayashi, Takashi; Ichikawa, Tadashi; Shimizu, Tetsuo; Urade, Yoshihiro; Yanagisawa, Masashi

2018-06-05

Narcolepsy-cataplexy is a chronic neurological disorder caused by loss of orexin (hypocretin)-producing neurons, associated with excessive daytime sleepiness, sleep attacks, cataplexy, sleep paralysis, hypnagogic hallucinations, and fragmentation of nighttime sleep. Currently, human narcolepsy is treated by providing symptomatic therapies, which can be associated with an array of side effects. Although peripherally administered orexin does not efficiently penetrate the blood-brain barrier, centrally delivered orexin can effectively alleviate narcoleptic symptoms in animal models. Chronic intrathecal drug infusion through an implantable pump is a clinically available strategy to treat a number of neurological diseases. Here we demonstrate that the narcoleptic symptoms of orexin knockout mice can be reversed by lumbar-level intrathecal orexin delivery. Orexin was delivered via a chronically implanted intrathecal catheter at the upper lumbar level. The computed tomographic scan confirmed that intrathecally administered contrast agent rapidly moved from the spinal cord to the brain. Intrathecally delivered orexin was detected in the brain by radioimmunoassay at levels comparable to endogenous orexin levels. Cataplexy and sleep-onset REM sleep were significantly decreased in orexin knockout mice during and long after slow infusion of orexin (1 nmol/1 µL/h). Sleep/wake states remained unchanged both quantitatively as well as qualitatively. Intrathecal orexin failed to induce any changes in double orexin receptor-1 and -2 knockout mice. This study supports the concept of intrathecal orexin delivery as a potential therapy for narcolepsy-cataplexy to improve the well-being of patients.

The chemical chaperones tauroursodeoxycholic and 4-phenylbutyric acid accelerate thyroid hormone activation and energy expenditure

PubMed Central

da-Silva, Wagner S.; Ribich, Scott; e Drigo, Rafael Arrojo; Castillo, Melany; Patty, Mary-Elizabeth; Bianco, Antonio C.

2011-01-01

Exposure of cell lines endogenously expressing the thyroid hormone activating enzyme type 2 deiodinase (D2) to the chemical chaperones tauroursodeoxycholic acid (TUDCA) or 4-phenylbutiric acid (4-PBA) increases D2 expression, activity and T3 production. In brown adipocytes, TUDCA or 4-PBA induced T3-dependent genes and oxygen consumption (~2-fold), an effect partially lost in D2 knockout cells. In wild type, but not in D2 knockout mice, administration of TUDCA lowered the respiratory quotient, doubled brown adipose tissue D2 activity and normalized the glucose intolerance associated with high fat feeding. Thus, D2 plays a critical role in the metabolic effects of chemical chaperones. PMID:21237159

The dense core vesicle protein IA-2, but not IA-2β, is required for active avoidance learning.

PubMed

Carmona, G N; Nishimura, T; Schindler, C W; Panlilio, L V; Notkins, A L

2014-06-06

The islet-antigens IA-2 and IA-2β are major autoantigens in type-1 diabetes and transmembrane proteins in dense core vesicles (DCV). Recently we showed that deletion of both IA-2 and IA-2β alters the secretion of hormones and neurotransmitters and impairs behavior and learning. The present study was designed to evaluate the contribution to learning of each of these genes by using single knockout (SKO) and double knockout (DKO) mice in an active avoidance test. After 5 days of training, wild-type (WT) mice showed 60-70% active avoidance responses, whereas the DKO mice showed only 10-15% active avoidance responses. The degree of active avoidance responses in the IA-2 SKO mice was similar to that of the DKO mice, but in contrast, the IA-2β SKO mice behaved like WT mice showing 60-70% active avoidance responses. Molecular studies revealed a marked decrease in the phosphorylation of the cAMP response element-binding protein (CREB) and Ca(2+)/calmodulin-dependent protein kinase II (CAMKII) in the striatum and hippocampus of the IA-2 SKO and DKO mice, but not in the IA-2β SKO mice. To evaluate the role of CREB and CAMKII in the SKO and DKO mice, GBR-12909, which selectively blocks the dopamine uptake transporter and increases CREB and CAMKII phosphorylation, was administered. GBR-12909 restored the phosphorylation of CREB and CAMKII and increased active avoidance learning in the DKO and IA-2 SKO to near the normal levels found in the WT and IA-2β SKO mice. We conclude that in the absence of the DCV protein IA-2, active avoidance learning is impaired. Published by Elsevier Ltd.

Object recognition impairment in Fmr1 knockout mice is reversed by amphetamine: involvement of dopamine in the medial prefrontal cortex.

PubMed

Ventura, R; Pascucci, T; Catania, M V; Musumeci, S A; Puglisi-Allegra, S

2004-09-01

Fragile X syndrome is an X-linked form of mental retardation including, among others, symptoms such as stereotypic behaviour, hyperactivity, hyperarousal, and cognitive deficits. We hypothesized that hyperactivity and/or compromised attentional, cognitive functions may lead to impaired performance in cognitive tasks in Fmr1 knockout mice, the most widely used animal model of fragile X syndrome, and suggested that psychostimulant treatment may improve performance by acting on one or both components. Since hyperactivity and cognitive functions have been suggested to depend on striatal and prefrontal cortex dopaminergic dysfunction, we assessed whether amphetamine produced beneficial, positive effects by acting on dopaminergic corticostriatal systems. Our results show that Fmr1 knockout mice are not able to discriminate between a familiar object and a novel one in the object recognition test, thus showing a clear-cut cognitive impairment that, to date, has been difficult to demonstrate in other cognitive tasks. Amphetamine improved performance of Fmr1 knockout mice, leading to enhanced ability to discriminate novel versus familiar objects, without significantly affecting locomotor activity. In agreement with behavioural data, amphetamine produced a greater increase in dopamine release in the prefrontal cortex of Fmr1 knockout compared with the wild-type mice, while a weak striatal dopaminergic response was observed in Fmr1 knockout mice. Our data support the view that the psychostimulant ameliorates performance in Fmr1 knockout mice by improving merely cognitive functions through its action on prefrontal cortical dopamine, irrespective of its action on motor hyperactivity. These results indicate that prefrontal cortical dopamine plays a major role in cognitive impairments characterizing Fmr1 knockout mice, thus pointing to an important aetiological factor in the fragile X syndrome.

Morphological characterization of the AlphaA- and AlphaB-crystallin double knockout mouse lens

PubMed Central

Boyle, Daniel L; Takemoto, Larry; Brady, James P; Wawrousek, Eric F

2003-01-01

Background One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alphaA- and alphaB-crystallin genes (alphaA/BKO). Methods Lenses from 129SvEvTac mice and alphaA/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. Results Equatorial and axial (sagittal) dimensions of lenses for alphaA/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alphaA/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alphaA/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. Conclusion These results indicated that both alphaA- and alphaB-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation. PMID:12546709

Haploinsufficiency in the PPAR{alpha} and LDL receptor genes leads to gender- and age-specific obesity and hyperinsulinemia

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

Sugiyama, Eiko; Tanaka, Naoki; Nakajima, Tamie

2006-11-17

When preparing peroxisome proliferator-activated receptor (PPAR){alpha}:low-density lipoprotein receptor (LDLR) (-/-) double knockout mice, we unexpectedly found a unique gender- and age-specific obesity in the F1 generation, PPAR{alpha} (+/-):LDLR (+/-), even in mice fed standard chow. Body weights of the male heterozygous mice increased up to about 60 g at 75 weeks of age, then decreased by about 30 g at 100 weeks of age. More than 95% of the heterozygous mice between 35- and 75-week-olds were overweight. Of interest, the obese heterozygous mice also exhibited hyperinsulinemia correlating with moderate insulin resistance. Hepatic gene expression of LDLR was lower than expectedmore » in the heterozygous mice, particularly at 50 and 75 weeks of age. In contrast, the hepatic expression of PPAR{alpha} was higher than expected in obese heterozygous mice, but decreased in non-obese older heterozygous mice. Modulated expression of these genes may be partially associated with the onset of the hyperinsulinemia.« less

Genetic manipulation of the ApoF/Stat2 locus supports an important role for type I interferon signaling in atherosclerosis.

PubMed

Lagor, William R; Fields, David W; Bauer, Robert C; Crawford, Alison; Abt, Michael C; Artis, David; Wherry, E John; Rader, Daniel J

2014-03-01

Apolipoprotein F (ApoF) is a sialoglycoprotein that is a component of the HDL and LDL fractions of human serum. We sought to test the hypothesis that ApoF plays an important role in atherosclerosis in mice by modulating lipoprotein function. Atherosclerosis was assessed in male low density lipoprotein receptor knockout (Ldlr KO) and ApoF/Ldlr double knockout (DKO) mice fed a Western diet for 16 weeks. ApoF/Ldlr DKO mice showed a 39% reduction in lesional area by en face analysis of aortas (p < 0.05), despite no significant differences in plasma lipid parameters. ApoF KO mice had reduced expression of Interferon alpha (IFNα) responsive genes in liver and spleen, as well as impaired macrophage activation. Interferon alpha induced gene 27 like 2a (Ifi27l2a), Oligoadenylate synthetases 2 and 3 (Oas2 and Oas3) were significantly reduced in the ApoF KO mice relative to wild type controls. These effects were attributable to hypomorphic expression of Stat2 in the ApoF KO mice, a critical gene in the Type I IFN pathway that is situated just 425 base pairs downstream of ApoF. These studies implicate STAT2 as a potentially important player in atherosclerosis, and support the growing evidence that the Type I IFN pathway may contribute to this complex disease. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Deficiency of eNOS exacerbates early-stage NAFLD pathogenesis by changing the fat distribution.

PubMed

Nozaki, Yuichi; Fujita, Koji; Wada, Koichiro; Yoneda, Masato; Shinohara, Yoshiyasu; Imajo, Kento; Ogawa, Yuji; Kessoku, Takaomi; Nakamuta, Makoto; Saito, Satoru; Masaki, Naohiko; Nagashima, Yoji; Terauchi, Yasuo; Nakajima, Atsushi

2015-12-17

Although many factors and molecules that are closely associated with non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) have been reported, the role of endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) in the pathogenesis of NAFLD/NASH remains unclear. We therefore investigated the role of eNOS-derived NO in NAFLD pathogenesis using systemic eNOS-knockout mice fed a high-fat diet. eNOS-knockout and wild-type mice were fed a basal diet or a high-fat diet for 12 weeks. Lipid accumulation and inflammation were evaluated in the liver, and various factors that are closely associated with NAFLD/NASH and hepatic tissue blood flow were analyzed. Lipid accumulation and inflammation were more extensive in the liver and lipid accumulation was less extensive in the visceral fat tissue in eNOS-knockout mice, compared with wild-type mice, after 12 weeks of being fed a high-fat diet. While systemic insulin resistance was comparable between the eNOS-knockout and wild-type mice fed a high-fat diet, hepatic tissue blood flow was significantly suppressed in the eNOS-knockout mice, compared with the wild-type mice, in mice fed a high-fat diet. The microsomal triglyceride transfer protein activity was down-regulated in eNOS-knockout mice, compared with wild-type mice, in mice fed a high-fat diet. A deficiency of eNOS-derived NO may exacerbate the early-stage of NASH pathogenesis by changing the fat distribution in a mouse model via the regulation of hepatic tissue blood flow.

Heat shock factor-1 knockout enhances cholesterol 7α-hydroxylase (CYP7A1) and multidrug transporter (MDR1) gene expressions to attenuate atherosclerosis

PubMed Central

Krishnamurthy, Karthikeyan; Glaser, Shannon; Alpini, Gianfranco D.; Cardounel, Arturo J.; Liu, Zhenguo; Ilangovan, Govindasamy

2016-01-01

Aims Stress response, in terms of activation of stress factors, is known to cause obesity and coronary heart disease such as atherosclerosis in human. However, the underlying mechanism(s) of these pathways are not known. Here, we investigated the effect of heat shock factor-1 (HSF-1) on atherosclerosis. Methods and results HSF-1 and low-density lipoprotein receptor (LDLr) double knockout (HSF-1−/−/LDLr−/−) and LDLr knockout (LDLr−/−) mice were fed with atherogenic western diet (WD) for 12 weeks. WD-induced weight gain and atherosclerotic lesion in aortic arch and carotid regions were reduced in HSF-1−/−/LDLr−/− mice, compared with LDLr−/− mice. Also, repression of PPAR-γ2 and AMPKα expression in adipose tissue, low hepatic steatosis, and lessened plasma adiponectins and lipoproteins were observed. In HSF-1−/−/LDLr−/− liver, higher cholesterol 7α-hydroxylase (CYP7A1) and multidrug transporter [MDR1/P-glycoprotein (P-gp)] gene expressions were observed, consistent with higher bile acid transport and larger hepatic bile ducts. Luciferase reporter gene assays with wild-type CYP7A1 and MDR1 promoters showed lesser luminescence than with mutant promoters (HSF-1 binding site deleted), indicating that HSF-1 binding is repressive of CYP7A1 and MDR1 gene expressions. Conclusion HSF-1 ablation not only eliminates heat shock response, but it also transcriptionally up-regulates CYP7A1 and MDR1/P-gp axis in WD-diet fed HSF-1−/−/LDLr−/− mice to reduce atherosclerosis. PMID:27131506

A Phex Mutation in a Murine Model of X-linked Hypophosphatemia Alters Phosphate Responsiveness of Bone Cells

PubMed Central

Ichikawa, Shoji; Austin, Anthony M.; Gray, Amie K.; Econs, Michael J.

2011-01-01

Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH – high dose phosphate and calcitriol – further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (PhexK496X) and hyperphosphatemic tumoral calcinosis (Galnt3 -/-), and Galnt3/Phex double mutant mice. Phex mutant mice had not only increased Fgf23 expression, but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by up-regulating Fgf23 expression as much as 24 fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for “normal” phosphate levels. PMID:22006791

A Phex mutation in a murine model of X-linked hypophosphatemia alters phosphate responsiveness of bone cells.

PubMed

Ichikawa, Shoji; Austin, Anthony M; Gray, Amie K; Econs, Michael J

2012-02-01

Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH--high-dose phosphate and calcitriol--further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (Phex(K496X)) and hyperphosphatemic tumoral calcinosis (Galnt3(-/-)), and Galnt3/Phex double-mutant mice. Phex mutant mice had not only increased Fgf23 expression but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double-mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double-mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by upregulating Fgf23 expression as much as 24-fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for "normal" phosphate levels.

Knockout of the Na,K-ATPase α2-isoform in cardiac myocytes delays pressure overload-induced cardiac dysfunction

PubMed Central

Rindler, Tara N.; Lasko, Valerie M.; Nieman, Michelle L.; Okada, Motoi; Lorenz, John N.

2013-01-01

The α2-isoform of the Na,K-ATPase (α2) is the minor isoform of the Na,K-ATPase expressed in the cardiovascular system and is thought to play a critical role in the regulation of cardiovascular hemodynamics. However, the organ system/cell type expressing α2 that is required for this regulation has not been fully defined. The present study uses a heart-specific knockout of α2 to further define the tissue-specific role of α2 in the regulation of cardiovascular hemodynamics. To accomplish this, we developed a mouse model using the Cre/loxP system to generate a tissue-specific knockout of α2 in the heart using β-myosin heavy chain Cre. We have achieved a 90% knockout of α2 expression in the heart of the knockout mice. Interestingly, the heart-specific knockout mice exhibit normal basal cardiac function and systolic blood pressure, and in addition, these mice develop ACTH-induced hypertension in response to ACTH treatment similar to control mice. Surprisingly, the heart-specific knockout mice display delayed onset of cardiac dysfunction compared with control mice in response to pressure overload induced by transverse aortic constriction; however, the heart-specific knockout mice deteriorated to control levels by 9 wk post-transverse aortic constriction. These results suggest that heart expression of α2 does not play a role in the regulation of basal cardiovascular function or blood pressure; however, heart expression of α2 plays a role in the hypertrophic response to pressure overload. This study further emphasizes that the tissue localization of α2 determines its unique roles in the regulation of cardiovascular function. PMID:23436327

Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice.

PubMed

Walentiny, D Matthew; Vann, Robert E; Wiley, Jenny L

2015-06-01

A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184). Copyright © 2015 Elsevier Ltd. All rights reserved.

Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes.

PubMed Central

Weinstock, P H; Bisgaier, C L; Aalto-Setälä, K; Radner, H; Ramakrishnan, R; Levak-Frank, S; Essenburg, A D; Zechner, R; Breslow, J L

1995-01-01

Lipoprotein lipase (LPL)-deficient mice have been created by gene targeting in embryonic stem cells. At birth, homozygous knockout pups have threefold higher triglycerides and sevenfold higher VLDL cholesterol levels than controls. When permitted to suckle, LPL-deficient mice become pale, then cyanotic, and finally die at approximately 18 h of age. Before death, triglyceride levels are severely elevated (15,087 +/- 3,805 vs 188 +/- 71 mg/dl in controls). Capillaries in tissues of homozygous knockout mice are engorged with chylomicrons. This is especially significant in the lung where marginated chylomicrons prevent red cell contact with the endothelium, a phenomenon which is presumably the cause of cyanosis and death in these mice. Homozygous knockout mice also have diminished adipose tissue stores as well as decreased intracellular fat droplets. By crossbreeding with transgenic mice expressing human LPL driven by a muscle-specific promoter, mouse lines were generated that express LPL exclusively in muscle but not in any other tissue. This tissue-specific LPL expression rescued the LPL knockout mice and normalized their lipoprotein pattern. This supports the contention that hypertriglyceridemia caused the death of these mice and that LPL expression in a single tissue was sufficient for rescue. Heterozygous LPL knockout mice survive to adulthood and have mild hypertriglyceridemia, with 1.5-2-fold elevated triglyceride levels compared with controls in both the fed and fasted states on chow, Western-type, or 10% sucrose diets. In vivo turnover studies revealed that heterozygous knockout mice had impaired VLDL clearance (fractional catabolic rate) but no increase in transport rate. In summary, total LPL deficiency in the mouse prevents triglyceride removal from plasma, causing death in the neonatal period, and expression of LPL in a single tissue alleviates this problem. Furthermore, half-normal levels of LPL cause a decrease in VLDL fractional catabolic rate and mild hypertriglyceridemia, implying that partial LPL deficiency has physiological consequences. Images PMID:8675619

Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.

PubMed

Hutchens, Steven; Liu, Chunyi; Jursa, Thomas; Shawlot, William; Chaffee, Beth K; Yin, Weiling; Gore, Andrea C; Aschner, Michael; Smith, Donald R; Mukhopadhyay, Somshuvra

2017-06-09

Manganese is an essential metal that becomes toxic at elevated levels. Loss-of-function mutations in SLC30A10, a cell-surface-localized manganese efflux transporter, cause a heritable manganese metabolism disorder resulting in elevated manganese levels and parkinsonian-like movement deficits. The underlying disease mechanisms are unclear; therefore, treatment is challenging. To understand the consequences of loss of SLC30A10 function at the organism level, we generated Slc30a10 knock-out mice. During early development, knock-outs were indistinguishable from controls. Surprisingly, however, after weaning and compared with controls, knock-out mice failed to gain weight, were smaller, and died prematurely (by ∼6-8 weeks of age). At 6 weeks, manganese levels in the brain, blood, and liver of the knock-outs were ∼20-60-fold higher than controls. Unexpectedly, histological analyses revealed that the brain and liver of the knock-outs were largely unaffected, but their thyroid exhibited extensive alterations. Because hypothyroidism leads to growth defects and premature death in mice, we assayed for changes in thyroid and pituitary hormones. At 6 weeks and compared with controls, the knock-outs had markedly reduced thyroxine levels (∼50-80%) and profoundly increased thyroid-stimulating hormone levels (∼800-1000-fold), indicating that Slc30a10 knock-out mice develop hypothyroidism. Importantly, a low-manganese diet produced lower tissue manganese levels in the knock-outs and rescued the phenotype, suggesting that manganese toxicity was the underlying cause. Our unanticipated discovery highlights the importance of determining the role of thyroid dysfunction in the onset and progression of manganese-induced disease and identifies Slc30a10 knock-out mice as a new model for studying thyroid biology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

LXRα Regulates Hepatic ChREBPα Activity and Lipogenesis upon Glucose, but Not Fructose Feeding in Mice

PubMed Central

Nørgaard, Rikke C.; Bindesbøll, Christian; Lucas, Christin; Dalen, Knut Tomas; Itkonen, Harri M.; Matthews, Jason; Nebb, Hilde I.; Grønning-Wang, Line M.

2017-01-01

Liver X receptors (LXRα/β) and carbohydrate response element-binding proteins (ChREBPα/β) are key players in the transcriptional control of hepatic de novo lipogenesis. LXRα/β double knockout (LXRα−/−/β−/−) mice have reduced feeding-induced nuclear O-linked N-acetylglucosamine (O-GlcNAc) signaling, ChREBPα activity, and lipogenic gene expression in livers, suggesting important roles for LXRs in linking hepatic glucose utilization to lipid synthesis. However, the role of LXRs in fructose-induced ChREBP activation and lipogenesis is currently unknown. In this study, we studied the effects of high fructose or high glucose feeding on hepatic carbohydrate metabolism and lipogenic gene expression in livers from fasted (24 h) and fasted-refed (12 h) wild type and LXRα knockout (LXRα−/−) mice. Hepatic lipogenic gene expression was reduced in glucose fed, but not fructose fed LXRα−/− mice. This was associated with lower expression of liver pyruvate-kinase (L-pk) and Chrebpβ, indicating reduced ChREBPα activity in glucose fed, but not fructose fed mice. Interestingly, ChREBP binding to the L-pk promoter was increased in fructose fed LXRα−/− mice, concomitant with increased glucose-6-phosphatase (G6pc) expression and O-GlcNAc modified LXRβ, suggesting a role for LXRβ in regulating ChREBPα activity upon fructose feeding. In conclusion, we propose that LXRα is an important regulator of hepatic lipogenesis and ChREBPα activity upon glucose, but not fructose feeding in mice. PMID:28661453

The TLR3/TICAM-1 signal constitutively controls spontaneous polyposis through suppression of c-Myc in Apc Min/+ mice.

PubMed

Ono, Junya; Shime, Hiroaki; Takaki, Hiromi; Takashima, Ken; Funami, Kenji; Yoshida, Sumito; Takeda, Yohei; Matsumoto, Misako; Kasahara, Masanori; Seya, Tsukasa

2017-10-17

Intestinal tumorigenesis is promoted by myeloid differentiation primary response gene 88 (MyD88) activation in response to the components of microbiota in Apc Min/+ mice. Microbiota also contains double-stranded RNA (dsRNA), a ligand for TLR3, which activates the toll-like receptor adaptor molecule 1 (TICAM-1, also known as TRIF) pathway. We established Apc Min/+ Ticam1 -/- mice and their survival was compared to survival of Apc Min/+ Myd88 -/- and wild-type (WT) mice. The properties of polyps were investigated using immunofluorescence staining and RT-PCR analysis. We demonstrate that TICAM-1 is essential for suppression of polyp formation in Apc Min/+ mice. TICAM-1 knockout resulted in shorter survival of mice compared to WT mice or mice with knockout of MyD88 in the Apc Min/+ background. Polyps were more frequently formed in the distal intestine of Apc Min/+ Ticam1 -/- mice than in Apc Min/+ mice. Infiltration of immune cells such as CD11b + and CD8α + cells into the polyps was detected histologically. CD11b and CD8α mRNAs were increased in polyps of Apc Min/+ Ticam1 -/- mice compared to Apc Min/+ mice. Gene expression of inducible nitric oxide synthase (iNOS), interferon (IFN)-γ, CXCL9 and IL-12p40 was increased in polyps of Apc Min/+ Ticam1 -/- mice. mRNA and protein expression of c-Myc, a critical transcription factor for inflammation-associated polyposis, were increased in polyps of Apc Min/+ Ticam1 -/- mice. A Lactobacillus strain producing dsRNA was detected in feces of Apc Min/+ mice. These results imply that the TLR3/TICAM-1 pathway inhibits polyposis through suppression of c-Myc expression and supports long survival in Apc Min/+ mice.

MONOAMINE OXIDASE: From Genes to Behavior

PubMed Central

Shih, J. C.; Chen, K.; Ridd, M. J.

2010-01-01

Cloning of MAO (monoamine oxidase) A and B has demonstrated unequivocally that these enzymes are made up of different polypeptides, and our understanding of MAO structure, regulation, and function has been significantly advanced by studies using their cDNA. MAO A and B genes are located on the X-chromosome (Xp11.23) and comprise 15 exons with identical intron-exon organization, which suggests that they are derived from the same ancestral gene. MAO A and B knockout mice exhibit distinct differences in neurotransmitter metabolism and behavior. MAO A knock-out mice have elevated brain levels of serotonin, norephinephrine, and dopamine and manifest aggressive behavior similar to human males with a deletion of MAO A. In contrast, MAO B knock-out mice do not exhibit aggression and only levels of phenylethylamine are increased. Mice lacking MAO B are resistant to the Parkinsongenic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine. Both MAO A and B knock-out mice show increased reactivity to stress. These knock-out mice are valuable models for investigating the role of monoamines in psychoses and neurodegenerative and stress-related disorders. PMID:10202537

Thymosin Beta-4 Induces Mouse Hair Growth

PubMed Central

Hou, Fang; Zhang, Zhipeng; Nuo, Mingtu; Guo, Xudong; Liu, Dongjun

2015-01-01

Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts. PMID:26083021

Thymosin Beta-4 Induces Mouse Hair Growth.

PubMed

Gao, Xiaoyu; Liang, Hao; Hou, Fang; Zhang, Zhipeng; Nuo, Mingtu; Guo, Xudong; Liu, Dongjun

2015-01-01

Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.

PubMed

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

2018-01-01

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

Dcdc2 knockout mice display exacerbated developmental disruptions following knockdown of Dcx

PubMed Central

Wang, Yu; Yin, Xiuyin; Rosen, Glenn; Gabel, Lisa; Guadiana, Sarah M.; Sarkisian, Matthew R; Galaburda, Albert M.; LoTurco, Joseph J.

2011-01-01

The dyslexia-associated gene DCDC2 is a member of the DCX family of genes known to play roles in neurogenesis, neuronal migration and differentiation. Here we report the first phenotypic analysis of a Dcdc2 knockout mouse. Comparisons between Dcdc2 knockout mice and wild type littermates revealed no significant differences in neuronal migration, neocortical lamination, neuronal cilliogenesis or dendritic differentiation. Considering previous studies showing genetic interactions and potential functional redundancy among members of the DCX family, we tested whether decreasing Dcx expression by RNAi would differentially impair neurodevelopment in Dcdc2 knockouts and wild type mice. Consistent with this hypothesis, we found that deficits in neuronal migration, and dendritic growth caused by RNAi of Dcx were more severe in Dcdc2 knockouts than in wild type mice with the same transfection. These results indicate that Dcdc2 is not required for neurogenesis, neuronal migration or differentiation in mice, but may have partial functional redundancy with Dcx. PMID:21689730

Abrogation of both short and long forms of latent transforming growth factor-β binding protein-1 causes defective cardiovascular development and is perinatally lethal.

PubMed

Horiguchi, Masahito; Todorovic, Vesna; Hadjiolova, Krassimira; Weiskirchen, Ralf; Rifkin, Daniel B

2015-04-01

Latent transforming growth factor-β binding protein-1 (LTBP-1) is an extracellular protein that is structurally similar to fibrillin and has an important role in controlling transforming growth factor-β (TGF-β) signaling by storing the cytokine in the extracellular matrix and by being involved in the conversion of the latent growth factor to its active form. LTBP-1 is found as both short (LTBP-1S) and long (LTBP-1L) forms, which are derived through the use of separate promoters. There is controversy regarding the importance of LTBP-1L, as Ltbp1L knockout mice showed multiple cardiovascular defects but the complete null mice did not. Here, we describe a third line of Ltbp1 knockout mice generated utilizing a conditional knockout strategy that ablated expression of both L and S forms of LTBP-1. These mice show severe developmental cardiovascular abnormalities and die perinatally; thus these animals display a phenotype similar to previously reported Ltbp1L knockout mice. We reinvestigated the other "complete" knockout line and found that these mice express a splice variant of LTBP-1L and, therefore, are not complete Ltbp1 knockouts. Our results clarify the phenotypes of Ltbp1 null mice and re-emphasize the importance of LTBP-1 in vivo. Copyright © 2015. Published by Elsevier B.V.

Olivocochlear neuron central anatomy is normal in alpha 9 knockout mice.

PubMed

Brown, M Christian; Vetter, Douglas E

2009-03-01

Olivocochlear (OC) neurons were studied in a transgenic mouse with deletion of the alpha 9 nicotinic acetylcholine receptor subunit. In this alpha 9 knockout mouse, the peripheral effects of OC stimulation are lacking and the peripheral terminals of OC neurons under outer hair cells have abnormal morphology. To account for this mouse's apparently normal hearing, it has been proposed to have central compensation via collateral branches to the cochlear nucleus. We tested this idea by staining OC neurons for acetylcholinesterase and examining their morphology in knockout mice, wild-type mice of the same background strain, and CBA/CaJ mice. Knockout mice had normal OC systems in terms of numbers of OC neurons, dendritic patterns, and numbers of branches to the cochlear nucleus. The branch terminations were mainly to edge regions and to a lesser extent the core of the cochlear nucleus, and were similar among the strains in terms of the distribution and staining density. These data demonstrate that there are no obvious changes in the central morphology of the OC neurons in alpha 9 knockout mice and make less attractive the idea that there is central compensation for deletion of the peripheral receptor in these mice.

Behavioral characterization of mice deficient in the phosphodiesterase-10A (PDE10A) enzyme on a C57/Bl6N congenic background.

PubMed

Siuciak, Judith A; McCarthy, Sheryl A; Chapin, Douglas S; Martin, Ashley N; Harms, John F; Schmidt, Christopher J

2008-02-01

The phenotype of genetically modified animals is strongly influenced by both the genetic background of the animal as well as environmental factors. We have previously reported the behavioral and neurochemical characterization of PDE10A knockout mice maintained on a DBA1LacJ (PDE10A(DBA)) genetic background. The aim of the present studies was to assess the behavioral and neurochemical phenotype of PDE10A knockout mice on an alternative congenic C57BL/6N (PDE10A(C57)) genetic background. Consistent with our previous results, PDE10A(C57) knockout mice showed a decrease in exploratory locomotor activity and a delay in the acquisition of conditioned avoidance responding. Also consistent with previous studies, the elimination of PDE10A did not alter basal levels of striatal cGMP or cAMP or affect behavior in several other well-characterized behavioral assays. PDE10A(C57) knockout mice showed a blunted response to MK-801, although to a lesser degree than previously observed in the PDE10A(DBA) knockout mice, and no differences were observed following a PCP challenge. PDE10A(C57) knockout mice showed a significant change in striatal dopamine turnover, which was accompanied by an enhanced locomotor response to AMPH, These studies demonstrate that while many of the behavioral effects of the PDE10A gene deletion appear to be independent of genetic background, the impact of the deletion on behavior can vary in magnitude. Furthermore, the effects on the dopaminergic system appear to be background-dependent, with significant effects observed only in knockout mice on the C57BL6N genetic background.

Methionine Sulfoxide Reductase A Knockout Mice Show Progressive Hearing Loss and Sensitivity to Acoustic Trauma.

PubMed

Alqudah, Safa; Chertoff, Mark; Durham, Dianne; Moskovitz, Jackob; Staecker, Hinrich; Peppi, Marcello

2018-06-21

Methionine sulfoxide reductases (MsrA and MsrB) protect the biological activity of proteins from oxidative modifications to methionine residues and are important for protecting against the pathological effects of neurodegenerative diseases. In the current study, we characterized the auditory phenotype of the MsrA knockout mouse. Young MsrA knockout mice showed small high-frequency threshold elevations for auditory brainstem response and distortion product otoacoustic emission compared to those of wild-type mice, which progressively worsened in older MsrA knockout mice. MsrA knockout mice showed an increased sensitivity to noise at young and older ages, suggesting that MsrA is part of a mechanism that protects the cochlea from acoustic damage. MsrA mRNA in the cochlea was increased following acoustic stimulation. Finally, expression of mRNA MsrB1 was compromised at 6 months old, but not in younger MsrA knockout mice (compared to controls). The identification of MsrA in the cochlea as a protective mediator from both early onset hearing loss and acoustic trauma expands our understanding of the pathways that may induce protection from acoustic trauma and foster further studies on how to prevent the damaging effect of noise exposure through Msr-based therapy. © 2018 S. Karger AG, Basel.

Hepatic changes in metabolic gene expression in old ghrelin and ghrelin receptor knockout mice

USDA-ARS?s Scientific Manuscript database

Ghrelin knockout (GKO) and ghrelin receptor (growth hormone secretagogue receptor) knockout (GHSRKO) mice exhibit enhanced insulin sensitivity, but the mechanism is unclear. Insulin sensitivity declines with age and is inversely associated with accumulation of lipid in liver, a key glucoregulatory ...

Discrete change in volatile anesthetic sensitivity in mice with inactivated tandem pore potassium ion channel TRESK.

PubMed

Chae, Yun Jeong; Zhang, Jianan; Au, Paul; Sabbadini, Marta; Xie, Guo-Xi; Yost, C Spencer

2010-12-01

We investigated the role of tandem pore potassium ion channel (K2P) TRESK in neurobehavioral function and volatile anesthetic sensitivity in genetically modified mice. Exon III of the mouse TRESK gene locus was deleted by homologous recombination using a targeting vector. The genotype of bred mice (wild type, knockout, or heterozygote) was determined using polymerase chain reaction. Morphologic and behavioral evaluations of TRESK knockout mice were compared with wild-type littermates. Sensitivity of bred mice to isoflurane, halothane, sevoflurane, and desflurane were studied by determining the minimum alveolar concentration preventing movement to tail clamping in 50% of each genotype. With the exception of decreased number of inactive periods and increased thermal pain sensitivity (20% decrease in latency with hot plate test), TRESK knockout mice had healthy development and behavior. TRESK knockout mice showed a statistically significant 8% increase in isoflurane minimum alveolar concentration compared with wild-type littermates. Sensitivity to other volatile anesthetics was not significantly different. Spontaneous mortality of TRESK knockout mice after initial anesthesia testing was nearly threefold higher than that of wild-type littermates. TRESK alone is not critical for baseline central nervous system function but may contribute to the action of volatile anesthetics. The inhomogeneous change in anesthetic sensitivity corroborates findings in other K2P knockout mice and supports the theory that the mechanism of volatile anesthetic action involves multiple targets. Although it was not shown in this study, a compensatory effect by other K2P channels may also contribute to these observations.

Circulating levels of IGF-1 directly regulate bone growth and density

PubMed Central

Yakar, Shoshana; Rosen, Clifford J.; Beamer, Wesley G.; Ackert-Bicknell, Cheryl L.; Wu, Yiping; Liu, Jun-Li; Ooi, Guck T.; Setser, Jennifer; Frystyk, Jan; Boisclair, Yves R.; LeRoith, Derek

2002-01-01

IGF-1 is a growth-promoting polypeptide that is essential for normal growth and development. In serum, the majority of the IGFs exist in a 150-kDa complex including the IGF molecule, IGF binding protein 3 (IGFBP-3), and the acid labile subunit (ALS). This complex prolongs the half-life of serum IGFs and facilitates their endocrine actions. Liver IGF-1–deficient (LID) mice and ALS knockout (ALSKO) mice exhibited relatively normal growth and development, despite having 75% and 65% reductions in serum IGF-1 levels, respectively. Double gene disrupted mice were generated by crossing LID+ALSKO mice. These mice exhibited further reductions in serum IGF-1 levels and a significant reduction in linear growth. The proximal growth plates of the tibiae of LID+ALSKO mice were smaller in total height as well as in the height of the proliferative and hypertrophic zones of chondrocytes. There was also a 10% decrease in bone mineral density and a greater than 35% decrease in periosteal circumference and cortical thickness in these mice. IGF-1 treatment for 4 weeks restored the total height of the proximal growth plate of the tibia. Thus, the double gene disruption LID+ALSKO mouse model demonstrates that a threshold concentration of circulating IGF-1 is necessary for normal bone growth and suggests that IGF-1, IGFBP-3, and ALS play a prominent role in the pathophysiology of osteoporosis. PMID:12235108

CD34 EXPRESSION BY HAIR FOLLICLE STEM CELLS IS REQUIRED FOR SKIN TUMOR DEVELOPMENT IN MICE

EPA Science Inventory

We used knockout mice to show that a cell surface protein called CD34 is required for skin tumor formation in mice. Wild type mice treated with 7-12-Dimethylbenz(a)anthracene (DMBA) and a tumor promoter developed papillomas. When we treated CD34 knockout (KO) mice the same way, n...

Reduced extinction of hippocampal-dependent memories in CPEB knockout mice.

PubMed

Berger-Sweeney, Joanne; Zearfoss, N Ruth; Richter, Joel D

2006-01-01

CPEB is a sequence-specific RNA binding protein that regulates translation at synapses. In neurons of CPEB knockout mice, synaptic efficacy is reduced. Here, we have performed a battery of behavioral tests and find that relative to wild-type animals, CPEB knockout mice, although similar on many baseline behaviors, have reduced extinction of memories on two hippocampal-dependent tasks. A corresponding microarray analysis reveals that about 0.14% of hippocampal genes have an altered expression in the CPEB knockout mouse. These data suggest that CPEB-dependent local protein synthesis may be an important cellular mechanism underlying extinction of hippocampal-dependent memories.

Enhanced serotonin response in the hippocampus of Galphaz protein knock-out mice.

PubMed

Oleskevich, Sharon; Leck, Kwong-Joo; Matthaei, Klaus; Hendry, Ian A

2005-06-21

The serotonin-1A [5-hydroxytryptamine 1A (5HT1A)] receptor is important for emotional and homeostatic processes in the central nervous system. In the hippocampus, the 5HT1A receptor couples to inhibitory Gi/o proteins to decrease pyramidal cell excitability. Here we investigate the 5HT1A receptor in a mouse deficient in the alpha-subunit of Gz protein (Galphaz knock-out). Behavioural tests showed heightened anxiety and depression-like behaviour in the Galphaz knock-out mice. Whole-cell recording in CA1 pyramidal neurons showed a significantly greater 5HT1A receptor-mediated potassium current in Galphaz knock-out mice. The effect was independent of 5HT4 receptors as the slow after-hyperpolarization was unaffected and a slow depolarization was absent in the Galphaz knock-out mice. Other receptors linked to Gi/o proteins [gamma-aminobutyric acid type B receptor (GABAB), adenosine A1 and muscarinic acetylcholine receptors] were not affected in Galphaz knock-out mice. These results suggest that the 5HT1A receptor may be linked to Galphaz protein, as reported previously in cell culture but shown here in an intact neural network.

Protection of the Villus Epithelial Cells of the Small Intestine from Rotavirus Infection Does Not Require Immunoglobulin A

PubMed Central

O'Neal, Christine M.; Harriman, Gregory R.; Conner, Margaret E.

2000-01-01

Immunoglobulin A (IgA) is the primary immune response induced in the intestine by rotavirus infection, but vaccination with virus-like particles induces predominantly IgG, not IgA. To definitively assess the role of IgA in protection from rotavirus infection, IgA knockout mice, which are devoid of serum and secretory IgA, were infected and then rechallenged with murine rotavirus at either 6 weeks or 10 months. Following primary rotavirus infection, IgA knockout mice cleared virus as effectively as IgA normal control mice. Rotavirus-infected IgA knockout mice produced no serum or fecal IgA but did have high levels of antirotavirus serum IgG and IgM and fecal IgG, whereas IgA normal control mice made both serum IgA and IgG and fecal IgA. Both IgA normal and IgA knockout mice were totally protected from rotavirus challenge at 42 days. Ten months following a primary infection, both IgA normal and knockout mice still had high levels of serum and fecal antirotavirus antibody and were totally protected from rotavirus challenge. To determine if compensatory mechanisms other than IgG were responsible for protection from rotavirus infection in IgA knockout mice, mice were depleted of CD4+ T cells or CD8+ T cells. No changes in the level of protection were seen in depleted mice. These data show that fecal or systemic IgA is not essential for protection from rotavirus infection and suggest that in the absence of IgA, IgG may play a significant role in protection from mucosal pathogens. PMID:10756022

Differential effects of thiopurine methyltransferase (TPMT) and multidrug resistance-associated protein gene 4 (MRP4) on mercaptopurine toxicity.

PubMed

Liu, Chengcheng; Janke, Laura J; Yang, Jun J; Evans, William E; Schuetz, John D; Relling, Mary V

2017-08-01

Mercaptopurine plays a pivotal role in treatment of acute lymphoblastic leukemia (ALL) and autoimmune diseases, and inter-individual variability in mercaptopurine tolerance can influence treatment outcome. Thiopurine methyltransferase (TPMT) and multi-drug resistant Protein 4 (MRP4) have both been associated with mercaptopurine toxicity in clinical studies, but their relative contributions remain unclear. We studied the metabolism of and tolerance to mercaptopurine in murine knockout models of Tpmt, Mrp4, and both genes simultaneously. Upon mercaptopurine treatment, Tpmt -/- Mrp4 -/- mice had the highest concentration of bone marrow thioguanine nucleotides (8.5 pmol/5 × 10 6 cells, P = 7.8 × 10 -4 compared with 2.7 pmol/5 × 10 6 cells in wild-types), followed by those with Mrp4 or Tpmt deficiency alone (6.1 and 4.3 pmol/5 × 10 6 cells, respectively). Mrp4-deficient mice accumulated higher concentrations of methylmercaptopurine metabolites compared with wild-type (76.5 vs. 23.2 pmol/5 × 10 6 cells, P = 0.027). Mice exposed to a clinically relevant mercaptopurine dosing regimen displayed differences in toxicity and survival among the genotypes. The double knock-out of both genes experienced greater toxicity and shorter survival compared to the single knockout of either Tpmt (P = 1.7 × 10 -6 ) or Mrp4 (P = 7.4 × 10 -10 ). We showed that both Tpmt and Mrp4 influence mercaptopurine disposition and toxicity.

Knockout Mice for Dyslexia Susceptibility Gene Homologs KIAA0319 and KIAA0319L have Unaffected Neuronal Migration but Display Abnormal Auditory Processing

PubMed Central

Guidi, Luiz G; Mattley, Jane; Martinez-Garay, Isabel; Monaco, Anthony P; Linden, Jennifer F; Velayos-Baeza, Antonio

2017-01-01

Abstract Developmental dyslexia is a neurodevelopmental disorder that affects reading ability caused by genetic and non-genetic factors. Amongst the susceptibility genes identified to date, KIAA0319 is a prime candidate. RNA-interference experiments in rats suggested its involvement in cortical migration but we could not confirm these findings in Kiaa0319-mutant mice. Given its homologous gene Kiaa0319L (AU040320) has also been proposed to play a role in neuronal migration, we interrogated whether absence of AU040320 alone or together with KIAA0319 affects migration in the developing brain. Analyses of AU040320 and double Kiaa0319;AU040320 knockouts (dKO) revealed no evidence for impaired cortical lamination, neuronal migration, neurogenesis or other anatomical abnormalities. However, dKO mice displayed an auditory deficit in a behavioral gap-in-noise detection task. In addition, recordings of click-evoked auditory brainstem responses revealed suprathreshold deficits in wave III amplitude in AU040320-KO mice, and more general deficits in dKOs. These findings suggest that absence of AU040320 disrupts firing and/or synchrony of activity in the auditory brainstem, while loss of both proteins might affect both peripheral and central auditory function. Overall, these results stand against the proposed role of KIAA0319 and AU040320 in neuronal migration and outline their relationship with deficits in the auditory system. PMID:29045729

Acute heat-evoked temperature sensation is impaired but not abolished in mice lacking TRPV1 and TRPV3 channels.

PubMed

Marics, Irène; Malapert, Pascale; Reynders, Ana; Gaillard, Stéphane; Moqrich, Aziz

2014-01-01

The discovery of heat-sensitive Transient Receptor Potential Vanilloid ion channels (ThermoTRPVs) greatly advanced our molecular understanding of acute and injury-evoked heat temperature sensation. ThermoTRPV channels are activated by partially overlapping temperatures ranging from warm to supra-threshold noxious heat. TRPV1 is activated by noxious heat temperature whereas TRPV3 can be activated by warm as well as noxious heat temperatures. Loss-of-function studies in single TRPV1 and TRPV3 knock-out mice have shown that heat temperature sensation is not completely abolished suggesting functional redundancies among these two channels and highlighting the need of a detailed analysis of TRPV1::TRPV3 double knock-out mice (V1V3dKO) which is hampered by the close proximity of the loci expressing the two channels. Here we describe the generation of a novel mouse model in which trpv1 and trpv3 genes have been inactivated using bacterial artificial chromosome (BAC)-based homologous recombination in embryonic stem cells. In these mice, using classical thermosensory tests such hot plate, tail flick and the thermotaxis gradient paradigms, we confirm that TRPV1 is the master channel for sensing noxious heat temperatures and identify a cooperative role of TRPV1 and TRPV3 for sensing a well-defined window of acute moderate heat temperature. Using the dynamic hot plate assay, we unravel an intriguing and unexpected pronounced escape behavior in TRPV1 knock-out mice that was attenuated in the V1V3dKO. Together, and in agreement with the temperature activation overlap between TRPV1 and TRPV3 channels, our data provide in vivo evidence of a cooperative role between skin-derived TRPV3 and primary sensory neurons-enriched TRPV1 in modulation of moderate and noxious heat temperature sensation and suggest that other mechanisms are required for heat temperature sensation.

Acute Heat-Evoked Temperature Sensation Is Impaired but Not Abolished in Mice Lacking TRPV1 and TRPV3 Channels

PubMed Central

Reynders, Ana; Gaillard, Stéphane; Moqrich, Aziz

2014-01-01

The discovery of heat-sensitive Transient Receptor Potential Vanilloid ion channels (ThermoTRPVs) greatly advanced our molecular understanding of acute and injury-evoked heat temperature sensation. ThermoTRPV channels are activated by partially overlapping temperatures ranging from warm to supra-threshold noxious heat. TRPV1 is activated by noxious heat temperature whereas TRPV3 can be activated by warm as well as noxious heat temperatures. Loss-of-function studies in single TRPV1 and TRPV3 knock-out mice have shown that heat temperature sensation is not completely abolished suggesting functional redundancies among these two channels and highlighting the need of a detailed analysis of TRPV1::TRPV3 double knock-out mice (V1V3dKO) which is hampered by the close proximity of the loci expressing the two channels. Here we describe the generation of a novel mouse model in which trpv1 and trpv3 genes have been inactivated using bacterial artificial chromosome (BAC)-based homologous recombination in embryonic stem cells. In these mice, using classical thermosensory tests such hot plate, tail flick and the thermotaxis gradient paradigms, we confirm that TRPV1 is the master channel for sensing noxious heat temperatures and identify a cooperative role of TRPV1 and TRPV3 for sensing a well-defined window of acute moderate heat temperature. Using the dynamic hot plate assay, we unravel an intriguing and unexpected pronounced escape behavior in TRPV1 knock-out mice that was attenuated in the V1V3dKO. Together, and in agreement with the temperature activation overlap between TRPV1 and TRPV3 channels, our data provide in vivo evidence of a cooperative role between skin-derived TRPV3 and primary sensory neurons-enriched TRPV1 in modulation of moderate and noxious heat temperature sensation and suggest that other mechanisms are required for heat temperature sensation. PMID:24925072

Loss of Urinary Macromolecules in Mice Causes Interstitial and Intratubular Renal Calcification Dependent on the Underlying Conditions

NASA Astrophysics Data System (ADS)

Wu, Xue-Ru; Lieske, John C.; Evan, Andrew P.; Sommer, Andre J.; Liaw, Lucy; Mo, Lan

2008-09-01

Urinary protein macromolecules have long been thought to play a role in influencing the various phases of urolithiasis including nucleation, growth, aggregation of mineral crystals and their subsequent adhesion to the renal epithelial cells. However, compelling evidence regarding their precise role was lacking, due partly to the fact that most prior studies were done in vitro and results were highly variable depending on the experimental conditions. The advent of genetic engineering technology has made it possible to study urinary protein macromolecules within an in vivo biological system. Indeed, recent studies have begun to shed light on the net effects of loss of one or more macromolecules on the earliest steps of urolithiasis. This paper focuses on the in vivo consequences of inactivating Tamm-Horsfall protein and/or osteopontin, two major urinary glycoproteins, using the knockout approach. The renal phenotypes of both single and double knockout mice under spontaneous or hyperoxaluric conditions will be described. The functional significance of the urinary macromolecules as critical defense factors against renal calcification will also be discussed.

Genome Editing in Mice Using TALE Nucleases.

PubMed

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

2016-01-01

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

A minor role of WNK3 in regulating phosphorylation of renal NKCC2 and NCC co-transporters in vivo.

PubMed

Oi, Katsuyuki; Sohara, Eisei; Rai, Tatemitsu; Misawa, Moko; Chiga, Motoko; Alessi, Dario R; Sasaki, Sei; Uchida, Shinichi

2012-02-15

Mutations in WNK1 and WNK4 kinase genes have been shown to cause a human hereditary hypertensive disease, pseudohypoaldosteronism type II (PHAII). We previously discovered that WNK kinases phosphorylate and activate OSR1/SPAK kinases that regulate renal SLC12A family transporters such as NKCC2 and NCC, and clarified that the constitutive activation of this cascade causes PHAII. WNK3, another member of the WNK kinase family, was reported to be a strong activator of NCC/NKCC2 when assayed in Xenopus oocytes, suggesting that WNK3 also plays a major role in regulating blood pressure and sodium reabsorption in the kidney. However, it remains to be determined whether WNK3 is in fact involved in the regulation of these transporters in vivo. To clarify this issue, we generated and analyzed WNK3 knockout mice. Surprisingly, phosphorylation and expression of OSR1, SPAK, NKCC2 and NCC did not decrease in knockout mouse kidney under normal and low-salt diets. Similarly, expression of epithelial Na channel and Na/H exchanger 3 were not affected in knockout mice. Na(+) and K(+) excretion in urine in WNK3 knockout mice was not affected under different salt diets. Blood pressure in WNK3 knockout mice was not lower under normal diet. However, lower blood pressure was observed in WNK3 knockout mice fed low-salt diet. WNK4 and WNK1 expression was slightly elevated in the knockout mice under low-salt diet, suggesting compensation for WNK3 knockout by these WNKs. Thus, WNK3 may have some role in the WNK-OSR1/SPAK-NCC/NKCC2 signal cascade in the kidney, but its contribution to total WNK kinase activity may be minimal.

Role of melanopsin in circadian responses to light.

PubMed

Ruby, Norman F; Brennan, Thomas J; Xie, Xinmin; Cao, Vinh; Franken, Paul; Heller, H Craig; O'Hara, Bruce F

2002-12-13

Melanopsin has been proposed as an important photoreceptive molecule for the mammalian circadian system. Its importance in this role was tested in melanopsin knockout mice. These mice entrained to a light/dark cycle, phase-shifted after a light pulse, and increased circadian period when light intensity increased. Induction of the immediate-early gene c-fos was observed after a nighttime light pulse in both wild-type and knockout mice. However, the magnitude of these behavioral responses in knockout mice was 40% lower than in wild-type mice. Although melanopsin is not essential for the circadian clock to receive photic input, it contributes significantly to the magnitude of photic responses.

Increased Contextual Fear Conditioning in iNOS Knockout Mice: Additional Evidence for the Involvement of Nitric Oxide in Stress-Related Disorders and Contribution of the Endocannabinoid System

PubMed Central

Gomes, Felipe V.; Silva, Andréia L.; Uliana, Daniela L.; Camargo, Laura H. A.; Guimarães, Francisco S.; Cunha, Fernando Q.; Joca, Sâmia R. L.; Resstel, Leonardo B. M.

2015-01-01

Background: Inducible or neuronal nitric oxide synthase gene deletion increases or decreases anxiety-like behavior in mice, respectively. Since nitric oxide and endocannabinoids interact to modulate defensive behavior, the former effect could involve a compensatory increase in basal brain nitric oxide synthase activity and/or changes in the endocannabinoid system. Thus, we investigated the expression and extinction of contextual fear conditioning of inducible nitric oxide knockout mice and possible involvement of endocannabinoids in these responses. Methods: We evaluated the effects of a preferential neuronal nitric oxide synthase inhibitor, 7-nitroindazol, nitric oxide synthase activity, and mRNA changes of nitrergic and endocannabinoid systems components in the medial prefrontal cortex and hippocampus of wild-type and knockout mice. The effects of URB597, an inhibitor of the fatty acid amide hydrolase enzyme, which metabolizes the endocannabinoid anandamide, WIN55,212-2, a nonselective cannabinoid agonist, and AM281, a selective CB1 antagonist, on contextual fear conditioning were also evaluated. Results: Contextual fear conditioning expression was similar in wild-type and knockout mice, but the latter presented extinction deficits and increased basal nitric oxide synthase activity in the medial prefrontal cortex. 7-Nitroindazol decreased fear expression and facilitated extinction in wild-type and knockout mice. URB597 decreased fear expression in wild-type and facilitated extinction in knockout mice, whereas WIN55,212-2 and AM281 increased it in wild-type mice. Nonconditioned knockout mice showed changes in the mRNA expression of nitrergic and endocannabinoid system components in the medial prefrontal cortex and hippocampus that were modified by fear conditioning. Conclusion: These data reinforce the involvement of the nitric oxide and endocannabinoids (anandamide) in stress-related disorders and point to a deregulation of the endocannabinoid system in situations where nitric oxide signaling is increased. PMID:25618404

Increased Contextual Fear Conditioning in iNOS Knockout Mice: Additional Evidence for the Involvement of Nitric Oxide in Stress-Related Disorders and Contribution of the Endocannabinoid System.

PubMed

Lisboa, Sabrina F; Gomes, Felipe V; Silva, Andréia L; Uliana, Daniela L; Camargo, Laura H A; Guimarães, Francisco S; Cunha, Fernando Q; Joca, Sâmia R L; Resstel, Leonardo B M

2015-01-24

Inducible or neuronal nitric oxide synthase gene deletion increases or decreases anxiety-like behavior in mice, respectively. Since nitric oxide and endocannabinoids interact to modulate defensive behavior, the former effect could involve a compensatory increase in basal brain nitric oxide synthase activity and/or changes in the endocannabinoid system. Thus, we investigated the expression and extinction of contextual fear conditioning of inducible nitric oxide knockout mice and possible involvement of endocannabinoids in these responses. We evaluated the effects of a preferential neuronal nitric oxide synthase inhibitor, 7-nitroindazol, nitric oxide synthase activity, and mRNA changes of nitrergic and endocannabinoid systems components in the medial prefrontal cortex and hippocampus of wild-type and knockout mice. The effects of URB597, an inhibitor of the fatty acid amide hydrolase enzyme, which metabolizes the endocannabinoid anandamide, WIN55,212-2, a nonselective cannabinoid agonist, and AM281, a selective CB1 antagonist, on contextual fear conditioning were also evaluated. Contextual fear conditioning expression was similar in wild-type and knockout mice, but the latter presented extinction deficits and increased basal nitric oxide synthase activity in the medial prefrontal cortex. 7-Nitroindazol decreased fear expression and facilitated extinction in wild-type and knockout mice. URB597 decreased fear expression in wild-type and facilitated extinction in knockout mice, whereas WIN55,212-2 and AM281 increased it in wild-type mice. Nonconditioned knockout mice showed changes in the mRNA expression of nitrergic and endocannabinoid system components in the medial prefrontal cortex and hippocampus that were modified by fear conditioning. These data reinforce the involvement of the nitric oxide and endocannabinoids (anandamide) in stress-related disorders and point to a deregulation of the endocannabinoid system in situations where nitric oxide signaling is increased. © The Author 2015. Published by Oxford University Press on behalf of CINP.

Decreased consumption of sweet fluids in mu opioid receptor knockout mice: a microstructural analysis of licking behavior

PubMed Central

Ostlund, Sean B.; Kosheleff, Alisa; Maidment, Nigel T.; Murphy, Niall P.

2013-01-01

Summary Rationale Evidence suggests that the palatability of food (i.e., the hedonic impact produced by its sensory features) can promote feeding and may underlie compulsive eating, leading to obesity. Pharmacological studies implicate opioid transmission in the hedonic control of feeding, though these studies often rely on agents lacking specificity for particular opioid receptors. Objectives Here, we investigated the role of mu opioid receptors (MORs) specifically in determining hedonic responses to palatable sweet stimuli. Methods In Experiment 1, licking microstructure when consuming sucrose solution (2 to 20 %) was compared in MOR knockout and wildtype mice as a function of sucrose concentration and level of food deprivation. In Experiment 2, a similar examination was conducted using the palatable but calorie-free stimulus sucralose (0.001 to 1%), allowing study of licking behavior independent of homeostatic variables. Results In Experiment 1, MOR knockout mice exhibited several alterations in sucrose licking. Although wildtype mice exhibited a two-fold increase in the burst length when food deprived, relative to the nondeprived test, this aspect of sucrose licking was generally insensitive to manipulations of food deprivation for MOR knockout mice. Furthermore, during concentration testing, their rate of sucrose licking was less than half that of wildtype mice. During sucralose testing (Experiment 2), MOR knockout mice licked at approximately half the wildtype rate, providing more direct evidence that MOR knockout mice were impaired in processing stimulus palatability. Conclusions These results suggest that transmission through MORs mediates hedonic responses to palatable stimuli, and therefore likely contributes to normal and pathological eating. PMID:23568577

Glutaminyl Cyclase Knock-out Mice Exhibit Slight Hypothyroidism but No Hypogonadism

PubMed Central

Schilling, Stephan; Kohlmann, Stephanie; Bäuscher, Christoph; Sedlmeier, Reinhard; Koch, Birgit; Eichentopf, Rico; Becker, Andreas; Cynis, Holger; Hoffmann, Torsten; Berg, Sabine; Freyse, Ernst-Joachim; von Hörsten, Stephan; Rossner, Steffen; Graubner, Sigrid; Demuth, Hans-Ulrich

2011-01-01

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms, like spontaneous cyclization or conversion by isoQC. The different effects of QC depletion on the hypothalamic pituitary thyroid and gonadal axes might indicate slightly different modes of substrate conversion of both enzymes. The absence of significant abnormalities in QC knock-out mice suggests the presence of a therapeutic window for suppression of QC activity in current drug development. PMID:21330373

Vasoactive intestinal peptide-null mice demonstrate enhanced sweet taste preference, dysglycemia, and reduced taste bud leptin receptor expression.

PubMed

Martin, Bronwen; Shin, Yu-Kyong; White, Caitlin M; Ji, Sunggoan; Kim, Wook; Carlson, Olga D; Napora, Joshua K; Chadwick, Wayne; Chapter, Megan; Waschek, James A; Mattson, Mark P; Maudsley, Stuart; Egan, Josephine M

2010-05-01

It is becoming apparent that there is a strong link between taste perception and energy homeostasis. Recent evidence implicates gut-related hormones in taste perception, including glucagon-like peptide 1 and vasoactive intestinal peptide (VIP). We used VIP knockout mice to investigate VIP's specific role in taste perception and connection to energy regulation. Body weight, food intake, and plasma levels of multiple energy-regulating hormones were measured and pancreatic morphology was determined. In addition, the immunocytochemical profile of taste cells and gustatory behavior were examined in wild-type and VIP knockout mice. VIP knockout mice demonstrate elevated plasma glucose, insulin, and leptin levels, with no islet beta-cell number/topography alteration. VIP and its receptors (VPAC1, VPAC2) were identified in type II taste cells of the taste bud, and VIP knockout mice exhibit enhanced taste preference to sweet tastants. VIP knockout mouse taste cells show a significant decrease in leptin receptor expression and elevated expression of glucagon-like peptide 1, which may explain sweet taste preference of VIP knockout mice. This study suggests that the tongue can play a direct role in modulating energy intake to correct peripheral glycemic imbalances. In this way, we could view the tongue as a sensory mechanism that is bidirectionally regulated and thus forms a bridge between available foodstuffs and the intricate hormonal balance in the animal itself.

Lithium ameliorates open-field and elevated plus maze behaviors, and brain phospho-glycogen synthase kinase 3-beta expression in fragile X syndrome model mice.

PubMed

Chen, Xi; Sun, Weiwen; Pan, Ying; Yang, Quan; Cao, Kaiyi; Zhang, Jin; Zhang, Yizhi; Chen, Mincong; Chen, Feidi; Huang, Yueling; Dai, Lijun; Chen, Shengqiang

2013-10-01

To investigate whether lithium modifies open-field and elevated plus maze behavior, and brain phospho-glycogen synthase kinase 3 (P-GSK3beta) expression in Fmr1 knockout mice. One hundred and eighty FVB mice, including knockout and wild type, with an age of 30 days were used. An open-field and elevated plus maze was utilized to test behavior, while western blot was used to measure the P-GSK3beta expression. Six groups were formed: control (saline), lithium chloride 30, 60, 90, 120, and 200 mg/kg. The experiments were carried out in the Institute of Neuroscience, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China between January and June 2012. Lithium significantly decreased total distance, crossing, central area time, and center entry in the open-field test (p<0.05), and significantly reduced open-arm tracking, open-arm entry, and open-arm time in the elevated plus maze (p<0.05) in knockout mice. In wild type mice, significant changes were observed in both behavior tests in some treatment groups. Lithium ameliorated P-GSK3beta expression in the hippocampus of all the treatment groups in knockout mice (p<0.05). However, lithium did not modify either GSK3beta expression in tissues of knockout mice, or P-GSK3beta or GSK3beta expression in tissues of wild type mice. Lithium ameliorated open-field and elevated plus maze behaviors of Fmr1 knockout mice. This effect may be related to its enhancement of P-GSK3beta expression. Our findings suggest that lithium might have a therapeutic effect in fragile X syndrome.

Myeloid-derived suppressor cells in murine AIDS inhibit B-cell responses in part via soluble mediators including reactive oxygen and nitrogen species, and TGF-β

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

Rastad, Jessica L.

2016-12-15

Monocytic myeloid-derived suppressor cells (M-MDSCs) were increased during LP-BM5 retroviral infection, and were capable of suppressing not only T-cell, but also B-cell responses. In addition to previously demonstrating iNOS- and VISTA-dependent M-MDSC mechanisms, in this paper, we detail how M-MDSCs utilized soluble mediators, including the reactive oxygen and nitrogen species superoxide, peroxynitrite, and nitric oxide, and TGF-β, to suppress B cells in a predominantly contact-independent manner. Suppression was independent of cysteine-depletion and hydrogen peroxide production. When two major mechanisms of suppression (iNOS and VISTA) were eliminated in double knockout mice, M-MDSCs from LP-BM5-infected mice were able to compensate using other,more » soluble mechanisms in order to maintain suppression of B cells. The IL-10 producing regulatory B-cell compartment was among the targets of M-MDSC-mediated suppression. -- Highlights: •LP-BM5-expanded M-MDSCs utilized soluble mediators nitric oxide, superoxide, peroxynitrite, and TGF-β to suppress B cells. •When two major mechanisms of suppression were eliminated through knockouts, M-MDSCs maintained suppression. •M-MDSCs from LP-BM5-infected mice decreased proliferation of IL-10 producing regulatory B cells.« less

Antiatherosclerotic and renoprotective effects of ebselen in the diabetic apolipoprotein E/GPx1-double knockout mouse.

PubMed

Chew, Phyllis; Yuen, Derek Y C; Stefanovic, Nada; Pete, Josefa; Coughlan, Melinda T; Jandeleit-Dahm, Karin A; Thomas, Merlin C; Rosenfeldt, Franklin; Cooper, Mark E; de Haan, Judy B

2010-12-01

To investigate the effect of the GPx1-mimetic ebselen on diabetes-associated atherosclerosis and renal injury in a model of increased oxidative stress. The study was performed using diabetic apolipoprotein E/GPx1 (ApoE(-/-)GPx1(-/-))-double knockout (dKO) mice, a model combining hyperlipidemia and hyperglycemia with increased oxidative stress. Mice were randomized into two groups, one injected with streptozotocin, the other with vehicle, at 8 weeks of age. Groups were further randomized to receive either ebselen or no treatment for 20 weeks. Ebselen reduced diabetes-associated atherosclerosis in most aortic regions, with the exception of the aortic sinus, and protected dKO mice from renal structural and functional injury. The protective effects of ebselen were associated with a reduction in oxidative stress (hydroperoxides in plasma, 8-isoprostane in urine, nitrotyrosine in the kidney, and 4-hydroxynonenal in the aorta) as well as a reduction in VEGF, CTGF, VCAM-1, MCP-1, and Nox2 after 10 weeks of diabetes in the dKO aorta. Ebselen also significantly reduced the expression of proteins implicated in fibrosis and inflammation in the kidney as well as reducing related key intracellular signaling pathways. Ebselen has an antiatherosclerotic and renoprotective effect in a model of accelerated diabetic complications in the setting of enhanced oxidative stress. Our data suggest that ebselen effectively repletes the lack of GPx1, and indicate that ebselen may be an effective therapeutic for the treatment of diabetes-related atherosclerosis and nephropathy. Furthermore, this study highlights the feasibility of addressing two diabetic complications with one treatment regimen through the unifying approach of targeted antioxidant therapy.

Transgenic and gene knockout mice in gastric cancer research

PubMed Central

Jiang, Yannan; Yu, Yingyan

2017-01-01

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

Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation

PubMed Central

Dykes, Iain M.; Tempest, Lynne; Lee, Su-In; Turner, Eric E.

2011-01-01

The combinatorial expression of transcription factors frequently marks cellular identity in the nervous system, yet how these factors interact to determine specific neuronal phenotypes is not well understood. Sensory neurons of the trigeminal (TG) and dorsal root ganglia (DRG) co-express the homeodomain transcription factors Brn3a and Islet1, and past work has revealed partially overlapping programs of gene expression downstream of these factors. Here we examine sensory development in Brn3a/Islet1 double knockout mice (DKO mice). Sensory neurogenesis and the formation of the TG and DRG occur in DKO embryos, but the DRG are dorsally displaced, and the peripheral projections of the ganglia are markedly disturbed. Sensory neurons in DKO embryos show a profound loss of all early markers of sensory subtypes, including the Ntrk neurotrophin receptors, and the runt-family transcription factors Runx1 and Runx3. Examination of global gene expression in the E12.5 DRG of single and double mutant embryos shows that Brn3a and Islet1 are together required for nearly all aspects of sensory-specific gene expression, including several newly identified sensory markers. On a majority of targets Brn3a and Islet1 exhibit negative epistasis, in which the effects of the individual knockout alleles are less than additive in the DKO. Smaller subsets of targets exhibit positive epistasis, or are regulated exclusively by one factor. Brn3a/Islet1 double mutants also fail to developmentally repress neurogenic bHLH genes, and in vivo chromatin immunoprecipitation shows that Islet1 binds to a known Brn3a -regulated enhancer in the neurod4 gene, suggesting a mechanism of interaction between these genes. PMID:21734270

P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP

PubMed Central

Cockayne, Debra A; Dunn, Philip M; Zhong, Yu; Rong, Weifang; Hamilton, Sara G; Knight, Gillian E; Ruan, Huai-Zhen; Ma, Bei; Yip, Ping; Nunn, Philip; McMahon, Stephen B; Burnstock, Geoffrey; Ford, Anthony PDW

2005-01-01

Extracellular ATP plays a role in nociceptive signalling and sensory regulation of visceral function through ionotropic receptors variably composed of P2X2 and P2X3 subunits. P2X2 and P2X3 subunits can form homomultimeric P2X2, homomultimeric P2X3, or heteromultimeric P2X2/3 receptors. However, the relative contribution of these receptor subtypes to afferent functions of ATP in vivo is poorly understood. Here we describe null mutant mice lacking the P2X2 receptor subunit (P2X2−/−) and double mutant mice lacking both P2X2 and P2X3 subunits (P2X2/P2X3Dbl−/−), and compare these with previously characterized P2X3−/− mice. In patch-clamp studies, nodose, coeliac and superior cervical ganglia (SCG) neurones from wild-type mice responded to ATP with sustained inward currents, while dorsal root ganglia (DRG) neurones gave predominantly transient currents. Sensory neurones from P2X2−/− mice responded to ATP with only transient inward currents, while sympathetic neurones had barely detectable responses. Neurones from P2X2/P2X3Dbl−/− mice had minimal to no response to ATP. These data indicate that P2X receptors on sensory and sympathetic ganglion neurones involve almost exclusively P2X2 and P2X3 subunits. P2X2−/− and P2X2/P2X3Dbl−/− mice had reduced pain-related behaviours in response to intraplantar injection of formalin. Significantly, P2X3−/−, P2X2−/−, and P2X2/P2X3Dbl−/− mice had reduced urinary bladder reflexes and decreased pelvic afferent nerve activity in response to bladder distension. No deficits in a wide variety of CNS behavioural tests were observed in P2X2−/− mice. Taken together, these data extend our findings for P2X3−/− mice, and reveal an important contribution of heteromeric P2X2/3 receptors to nociceptive responses and mechanosensory transduction within the urinary bladder. PMID:15961431

Genetic cathepsin B deficiency reduces beta-amyloid in transgenic mice expressing human wild-type amyloid precursor protein.

PubMed

Hook, Vivian Y H; Kindy, Mark; Reinheckel, Thomas; Peters, Christoph; Hook, Gregory

2009-08-21

Neurotoxic beta-amyloid (Abeta) peptides participate in Alzheimer's disease (AD); therefore, reduction of Abeta generated from APP may provide a therapeutic approach for AD. Gene knockout studies in transgenic mice producing human Abeta may identify targets for reducing Abeta. This study shows that knockout of the cathepsin B gene in mice expressing human wild-type APP (hAPPwt) results in substantial decreases in brain Abeta40 and Abeta42 by 67% and decreases in levels of the C-terminal beta-secretase fragment (CTFbeta) derived from APP. In contrast, knockout of cathepsin B in mice expressing hAPP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Abeta. The difference in reduction of Abeta in hAPPwt mice, but not in hAPPSwe/Ind mice, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express hAPPwt, these data validate cathepsin B as a target for development of inhibitors to lower Abeta in AD.

Alterations of amino acids and monoamine metabolism in male Fmr1 knockout mice: a putative animal model of the human fragile X mental retardation syndrome.

PubMed

Gruss, M; Braun, K

2001-01-01

The Fragile X syndrome, a common form of mental retardation in humans, is caused by silencing the fragile X mental retardation (FMR1) gene leading to the absence of the encoded fragile X mental retardation protein 1 (FMRP). We describe morphological and behavioral abnormalities for both affected humans and Fmr1 knockout mice, a putative animal model for the human Fragile X syndrome. The aim of the present study was to identify possible neurochemical abnormalities in Fmr1 knockout mice, with particular focus on neurotransmission. Significant region-specific differences of basal neurotransmitter and metabolite levels were found between wildtype and Fmr1 knockout animals, predominantly in juveniles (post-natal days 28 to 31). Adults (postnatal days 209 to 221) showed only few abnormalities as compared with the wildtype. In juvenile knockout mice, aspartate and taurine were especially increased in cortical regions, striatum, hippocampus, cerebellum, and brainstem. In addition, juveniles showed an altered balance between excitatory and inhibitory amino acids in the caudal cortex, hippocampus, and brainstem. We detected very few differences in monoamine turnover in both age stages. The results presented here provide the first evidence that lack of FMRP expression in FMRP knockout mice is accompanied by age-dependent, region-specific alterations in neurotransmission.

Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out

PubMed Central

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

2012-01-01

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

Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice.

PubMed

Seki, Ekihiro; Tsutsui, Hiroko; Tsuji, Noriko M; Hayashi, Nobuki; Adachi, Keishi; Nakano, Hiroki; Futatsugi-Yumikura, Shizue; Takeuchi, Osamu; Hoshino, Katsuaki; Akira, Shizuo; Fujimoto, Jiro; Nakanishi, Kenji

2002-10-01

Listeria monocytogenes (LM), a facultative intracellular Gram-positive bacterium, often causes lethal infection of the host. In this study we investigated the molecular mechanism underlying LM eradication in the early phase of infection. Upon infection with LM, both IL-12 and IL-18 were produced, and then they synergistically induced IFN-gamma production, leading to normal LM clearance in the host. IFN-gamma knockout (KO) mice were highly susceptible to LM infection. IL-12/IL-18 double knockout mice were also highly susceptible. Their susceptibility was less than that of IFN-gamma KO mice, but more than that of single IL-12 or IL-18 KO mice. Mice deficient in myeloid differentiation factor 88 (MyD88), an essential adaptor molecule used by signal transduction pathways of all members of the Toll-like receptor (TLR) family, showed an inability to produce IL-12 and IFN-gamma following LM infection and were most susceptible to LM. Furthermore, MyD88-deficient, but not IFN-gamma-deficient, Kupffer cells could not produce TNF-alpha in response to LM in vitro, indicating the importance of MyD88-dependent TNF-alpha production for host defense. As TLR2 KO, but not TLR4 KO, mice showed partial impairment in their capacity to produce IL-12, IFN-gamma, and TNF-alpha, TLR2 activation partly contributed to the induction of IL-12-mediated IFN-gamma production. These results indicated a critical role for TLRs/MyD88-dependent IL-12/TNF-alpha production and for IL-12- and IL-18-mediated IFN-gamma production in early phase clearance of LM.

A Role for Calmodulin-Stimulated Adenylyl Cyclases in Cocaine Sensitization

PubMed Central

DiRocco, Derek P.; Scheiner, Zachary S.; Sindreu, Carlos Balet; Chan, Guy C-K; Storm, Daniel R.

2009-01-01

Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca2+/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that while AC1 and AC8 single knockout mice (AC1−/− and AC8−/−) exhibit Ca2+-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knockout (DKO) mice do not. Furthermore, DKO mice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization following chronic cocaine treatment. Because of the known role for the ERK/MAP kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated extracellular signal-regulated kinase (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase positive (ChAT+) interneurons in DKO mice relative to wild-type (WT) controls. Following acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice. We demonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581, and cAMP response element-binding protein (pCREB) at Ser133 following acute cocaine treatment. Our results demonstrate that the Ca2+-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs. PMID:19244515

Scavenger Receptor Class B Type 1 Deletion Led to Coronary Atherosclerosis and Ischemic Heart Disease in Low-density Lipoprotein Receptor Knockout Mice on Modified Western-type Diet

PubMed Central

Liao, Jiawei; Guo, Xin; Wang, Mengyu; Dong, Chengyan; Gao, Mingming; Wang, Huan; Kayoumu, Abudurexiti; Shen, Qiang; Wang, Yuhui; Wang, Fan; Liu, George

2017-01-01

Aim: Atherosclerosis-prone apolipoprotein E (apoE) or low-density lipoprotein receptor (LDL-R) knockout (KO) mice are generally resistant to developing coronary atherosclerosis (CA) and ischemic heart disease (IHD). However, studies have demonstrated the occurrence of spontaneous CA and IHD in scavenger receptor class B type 1 (SR-BI)/apoE double KO (dKO) mice, which suggests that SR-BI could be a potential target for the prevention and therapy of CA and IHD. This possibility was later investigated in SR-BI/LDL-R dKO mice, but no signs of CA or IHD was identified when mice were fed a normal western-type diet. Here we explored whether SR-BI deletion could result in CA and IHD in LDL-R KO mice when fed a modified western-type diet containing higher (0.5%) cholesterol. Methods: Cardiac functions were detected by electrocardiography, single photon emission computed tomography (SPECT), echocardiography (Echo) and 2,3,5-triphenyltetrazolium chloride staining. CA was visualized by hematoxylin-eosin staining. Results: After 12 weeks on the modified diet, SR-BI/LDL-R dKO mice developed cardiac ischemia/infarction, together with systolic dysfunction and left ventricular dilatation. CA was most severe at the aortic sinus level to an extent that no dKO mice survived to 20 weeks on the modified diet. None of control mice, however, developed CA or IHD. Conclusions: SR-BI deletion led to CA and IHD in LDL-R KO mice when fed the modified western-type diet. We established SR-BI/LDL-R dKO mice as a diet-induced murine model of human IHD and developed detection methods, using a combination of SPECT and Echo, for effective in vivo evaluation of cardiac functions. PMID:27373983

Genetic background can result in a marked or minimal effect of gene knockout (GPR55 and CB2 receptor) in experimental autoimmune encephalomyelitis models of multiple sclerosis.

PubMed

Sisay, Sofia; Pryce, Gareth; Jackson, Samuel J; Tanner, Carolyn; Ross, Ruth A; Michael, Gregory J; Selwood, David L; Giovannoni, Gavin; Baker, David

2013-01-01

Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim)) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen)) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim) mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

PubMed Central

Jackson, Samuel J.; Tanner, Carolyn; Ross, Ruth A.; Michael, Gregory J.; Selwood, David L.; Giovannoni, Gavin; Baker, David

2013-01-01

Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 tm1Zim) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 Dgen) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility. PMID:24130809

Biopolymer encapsulated live influenza virus as a universal CD8+ T cell vaccine against influenza virus.

PubMed

Boesteanu, Alina C; Babu, Nadarajan S; Wheatley, Margaret; Papazoglou, Elisabeth S; Katsikis, Peter D

2010-12-16

Current influenza virus vaccines primarily elicit antibodies and can be rendered ineffective by antigenic drift and shift. Vaccines that elicit CD8+ T cell responses targeting less variable proteins may function as universal vaccines that have broad reactivity against different influenza virus strains. To generate such a universal vaccine, we encapsulated live influenza virus in a biopolymer and delivered it to mice subcutaneously. This vaccine was safe, induced potent CD8+ T cell immunity and protected mice against heterosubtypic lethal challenge. Safety of subcutaneous (SQ) vaccination was tested in Rag-/-γc-/- double knockout mice which we show cannot control intranasal infection. Biopolymer encapsulation of live influenza virus could be used to develop universal CD8+ T cell vaccines against heterosubtypic and pandemic strains. Copyright © 2010 Elsevier Ltd. All rights reserved.

IL-6-Type Cytokine Signaling in Adipocytes Induces Intestinal GLP-1 Secretion.

PubMed

Wueest, Stephan; Laesser, Céline I; Böni-Schnetzler, Marianne; Item, Flurin; Lucchini, Fabrizio C; Borsigova, Marcela; Müller, Werner; Donath, Marc Y; Konrad, Daniel

2018-01-01

We recently showed that interleukin (IL)-6-type cytokine signaling in adipocytes induces free fatty acid release from visceral adipocytes, thereby promoting obesity-induced hepatic insulin resistance and steatosis. In addition, IL-6-type cytokines may increase the release of leptin from adipocytes and by those means induce glucagon-like peptide 1 (GLP-1) secretion. We thus hypothesized that IL-6-type cytokine signaling in adipocytes may regulate insulin secretion. To this end, mice with adipocyte-specific knockout of gp130, the signal transducer protein of IL-6, were fed a high-fat diet for 12 weeks. Compared with control littermates, knockout mice showed impaired glucose tolerance and circulating leptin, GLP-1, and insulin levels were reduced. In line, leptin release from isolated adipocytes was reduced, and intestinal proprotein convertase subtilisin/kexin type 1 ( Pcsk1 ) expression, the gene encoding PC1/3, which controls GLP-1 production, was decreased in knockout mice. Importantly, treatment with the GLP-1 receptor antagonist exendin 9-39 abolished the observed difference in glucose tolerance between control and knockout mice. Ex vivo, supernatant collected from isolated adipocytes of gp130 knockout mice blunted Pcsk1 expression and GLP-1 release from GLUTag cells. In contrast, glucose- and GLP-1-stimulated insulin secretion was not affected in islets of knockout mice. In conclusion, adipocyte-specific IL-6 signaling induces intestinal GLP-1 release to enhance insulin secretion, thereby counteracting insulin resistance in obesity. © 2017 by the American Diabetes Association.

Proteome analysis of a hepatocyte-specific BIRC5 (survivin)-knockout mouse model during liver regeneration.

PubMed

Bracht, Thilo; Hagemann, Sascha; Loscha, Marius; Megger, Dominik A; Padden, Juliet; Eisenacher, Martin; Kuhlmann, Katja; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara

2014-06-06

The Baculoviral IAP repeat-containing protein 5 (BIRC5), also known as inhibitor of apoptosis protein survivin, is a member of the chromosomal passenger complex and a key player in mitosis. To investigate the function of BIRC5 in liver regeneration, we analyzed a hepatocyte-specific BIRC5-knockout mouse model using a quantitative label-free proteomics approach. Here, we present the analyses of the proteome changes in hepatocyte-specific BIRC5-knockout mice compared to wildtype mice, as well as proteome changes during liver regeneration induced by partial hepatectomy in wildtype mice and mice lacking hepatic BIRC5, respectively. The BIRC5-knockout mice showed an extensive overexpression of proteins related to cellular maintenance, organization and protein synthesis. Key regulators of cell growth, transcription and translation MTOR and STAT1/STAT2 were found to be overexpressed. During liver regeneration proteome changes representing a response to the mitotic stimulus were detected in wildtype mice. Mainly proteins corresponding to proliferation, cell cycle and cytokinesis were up-regulated. The hepatocyte-specific BIRC5-knockout mice showed impaired liver regeneration, which had severe consequences on the proteome level. However, several proteins with function in mitosis were found to be up-regulated upon the proliferative stimulus. Our results show that the E3 ubiquitin-protein ligase UHRF1 is strongly up-regulated during liver regeneration independently of BIRC5.

Role of light and the circadian clock in the rhythmic oscillation of intraocular pressure: Studies in VPAC2 receptor and PACAP deficient mice.

PubMed

Fahrenkrug, Jan; Georg, Birgitte; Hannibal, Jens; Jørgensen, Henrik Løvendahl

2018-04-01

The intraocular pressure of mice displays a daily rhythmicity being highest during the dark period. The present study was performed to elucidate the role of the circadian clock and light in the diurnal and the circadian variations in intraocular pressure in mice, by using animals with disrupted clock function (VPAC2 receptor knockout mice) or impaired light information to the clock (PACAP knockout mice). In wildtype mice, intraocular pressure measured under light/dark conditions showed a statistically significant 24 h sinusoidal rhythm with nadir during the light phase and peak during the dark phase. After transfer of the wildtype mice into constant darkness, the intraocular pressure increased, but the rhythmic changes in intraocular pressure continued with a pattern identical to that obtained during the light/dark cycle. The intraocular pressure in VPAC2 receptor deficient mice during light/dark conditions also showed a sinusoidal pattern with significant changes as a function of a 24 h cycle. However, transfer of the VPAC2 receptor knockout mice into constant darkness completely abolished the rhythmic changes in intraocular pressure. The intraocular pressure in PACAP deficient mice oscillated significantly during both 24 h light and darkness and during constant darkness. During LD conditions, the amplitude of PACAP deficient was significantly lower compared to wildtype mice, resulting in higher daytime and lower nighttime values. In conclusion, by studying the VPAC2 receptor knockout mouse which lacks circadian control and the PACAP knockout mouse which displays impaired light signaling, we provided evidence that the daily intraocular pressure rhythms are primarily generated by the circadian master clock and to a lesser extent by environmental light and darkness. Copyright © 2018 Elsevier Ltd. All rights reserved.

Increase of Total Nephron Albumin Filtration and Reabsorption in Diabetic Nephropathy.

PubMed

Mori, Keita P; Yokoi, Hideki; Kasahara, Masato; Imamaki, Hirotaka; Ishii, Akira; Kuwabara, Takashige; Koga, Kenichi; Kato, Yukiko; Toda, Naohiro; Ohno, Shoko; Kuwahara, Koichiro; Endo, Tomomi; Nakao, Kazuwa; Yanagita, Motoko; Mukoyama, Masashi; Mori, Kiyoshi

2017-01-01

The amount of albumin filtered through the glomeruli and reabsorbed at the proximal tubules in normal and in diabetic kidneys is debated. The megalin/cubilin complex mediates protein reabsorption, but genetic knockout of megalin is perinatally lethal. To overcome current technical problems, we generated a drug-inducible megalin-knockout mouse line, megalin(lox/lox);Ndrg1-CreER T2 (iMegKO), in which megalin expression can be shut off at any time by administration of tamoxifen (Tam). Tam administration in adult iMegKO mice decreased the expression of renal megalin protein by 92% compared with that in wild-type C57BL/6J mice and almost completely abrogated renal reabsorption of intravenously injected retinol-binding protein. Furthermore, urinary albumin excretion increased to 175 μg/d (0.46 mg albumin/mg creatinine) in Tam-treated iMegKO mice, suggesting that this was the amount of total nephron albumin filtration. By comparing Tam-treated, streptozotocin-induced diabetic iMegKO mice with Tam-treated nondiabetic iMegKO mice, we estimated that the development of diabetes led to a 1.9-fold increase in total nephron albumin filtration, a 1.8-fold increase in reabsorption, and a significant reduction in reabsorption efficiency (86% efficiency versus 96% efficiency in nondiabetic mice). Insulin treatment normalized these abnormalities. Akita;iMegKO mice, another model of type 1 diabetes, showed equivalent results. Finally, nondiabetic iMegKO mice had a glomerular sieving coefficient of albumin of 1.7×10 -5 , which approximately doubled in diabetic iMegKO mice. This study reveals actual values and changes of albumin filtration and reabsorption in early diabetic nephropathy in mice, bringing new insights to our understanding of renal albumin dynamics associated with the hyperfiltration status of diabetic nephropathy. Copyright © 2016 by the American Society of Nephrology.

Neuron-specific (pro)renin receptor knockout prevents the development of salt-sensitive hypertension

PubMed Central

Li, Wencheng; Peng, Hua; Mehaffey, Eamonn P.; Kimball, Christie D.; Grobe, Justin L.; van Gool, Jeanette M.G.; Sullivan, Michelle N.; Earley, Scott; Danser, A.H. Jan; Ichihara, Atsuhiro; Feng, Yumei

2013-01-01

The (pro)renin receptor, which binds both renin and prorenin, is a newly discovered component of the renin angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, non-proteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate salt induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. (Pro)renin receptor expression, detected by immunostaining and RT-PCR, was significantly decreased in the brains of knockout compared with wide-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild type mice. This hypertensive response was abolished in (pro)renin receptor knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate salt increased (pro)renin receptor expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in (pro)renin receptor knockout mice. (Pro)renin receptor knockout in neurons prevented the development of Deoxycorticosterone acetate salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, non-proteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate salt-induced hypertension, possibly through diminished angiotensin II formation. PMID:24246383

Role of Aquaporin-4 in Airspace-to-Capillary Water Permeability in Intact Mouse Lung Measured by a Novel Gravimetric Method

PubMed Central

Song, Yuanlin; Ma, Tonghui; Matthay, Michael A.; Verkman, A.S.

2000-01-01

The mammalian peripheral lung contains at least three aquaporin (AQP) water channels: AQP1 in microvascular endothelia, AQP4 in airway epithelia, and AQP5 in alveolar epithelia. In this study, we determined the role of AQP4 in airspace-to-capillary water transport by comparing water permeability in wild-type mice and transgenic null mice lacking AQP1, AQP4, or AQP1/AQP4 together. An apparatus was constructed to measure lung weight continuously during pulmonary artery perfusion of isolated mouse lungs. Osmotically induced water flux (Jv) between the airspace and capillary compartments was measured from the kinetics of lung weight change in saline-filled lungs in response to changes in perfusate osmolality. Jv in wild-type mice varied linearly with osmotic gradient size (4.4 × 10−5 cm3 s−1 mOsm−1) and was symmetric, independent of perfusate osmolyte size, weakly temperature dependent, and decreased 11-fold by AQP1 deletion. Transcapillary osmotic water permeability was greatly reduced by AQP1 deletion, as measured by the same method except that the airspace saline was replaced by an inert perfluorocarbon. Hydrostatically induced lung edema was characterized by lung weight changes in response to changes in pulmonary arterial inflow or pulmonary venous outflow pressure. At 5 cm H2O outflow pressure, the filtration coefficient was 4.7 cm3 s−1 mOsm−1 and reduced 1.4-fold by AQP1 deletion. To study the role of AQP4 in lung water transport, AQP1/AQP4 double knockout mice were generated by crossbreeding of AQP1 and AQP4 null mice. Jv were (cm3 s−1 mOsm−1 × 10−5, SEM, n = 7–12 mice): 3.8 ± 0.4 (wild type), 0.35 ± 0.02 (AQP1 null), 3.7 ± 0.4 (AQP4 null), and 0.25 ± 0.01 (AQP1/AQP4 null). The significant reduction in P f in AQP1 vs. AQP1/AQP4 null mice was confirmed by an independent pleural surface fluorescence method showing a 1.6 ± 0.2-fold (SEM, five mice) reduced P f in the AQP1/AQP4 double knockout mice vs. AQP1 null mice. These results establish a simple gravimetric method to quantify osmosis and filtration in intact mouse lung and provide direct evidence for a contribution of the distal airways to airspace-to-capillary water transport. PMID:10613915

Genetic characterization of p27(kip1) and stathmin in controlling cell proliferation in vivo.

PubMed

Berton, Stefania; Pellizzari, Ilenia; Fabris, Linda; D'Andrea, Sara; Segatto, Ilenia; Canzonieri, Vincenzo; Marconi, Daniela; Schiappacassi, Monica; Benevol, Sara; Gattei, Valter; Colombatti, Alfonso; Belletti, Barbara; Baldassarre, Gustavo

2014-01-01

The CDK inhibitor p27(kip1) is a critical regulator of cell cycle progression, but the mechanisms by which p27(kip1) controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner. To get more insights into the in vivo significance of this interaction, we generated p27(kip1) and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27(kip1) null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

Genetic characterization of p27kip1 and stathmin in controlling cell proliferation in vivo

PubMed Central

Berton, Stefania; Pellizzari, Ilenia; Fabris, Linda; D'Andrea, Sara; Segatto, Ilenia; Canzonieri, Vincenzo; Marconi, Daniela; Schiappacassi, Monica; Benevol, Sara; Gattei, Valter; Colombatti, Alfonso; Belletti, Barbara; Baldassarre, Gustavo

2014-01-01

The CDK inhibitor p27kip1 is a critical regulator of cell cycle progression, but the mechanisms by which p27kip1 controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27kip1 binding partner. To get more insights into the in vivo significance of this interaction, we generated p27kip1 and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27kip1 null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27kip1 null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27kip1 to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression. PMID:25486569

Impaired fear extinction learning in adult heterozygous BDNF knock-out mice.

PubMed

Psotta, Laura; Lessmann, Volkmar; Endres, Thomas

2013-07-01

Brain-derived neurotrophic factor (BDNF) is a crucial regulator of neuroplasticity, which underlies learning and memory processes in different brain areas. To investigate the role of BDNF in the extinction of amygdala-dependent cued fear memories, we analyzed fear extinction learning in heterozygous BDNF knock-out mice, which possess a reduction of endogenous BDNF protein levels to ~50% of wild-type animals. Since BDNF expression has been shown to decline with aging of animals, we tested the performance in extinction learning of these mice at 2 months (young adults) and 7 months (older adults) of age. The present study shows that older adult heterozygous BDNF knock-out mice, which have a chronic 50% lack of BDNF, also possess a deficit in the acquisition of extinction memory, while extinction learning remains unaffected in young adult heterozygous BDNF knock-out mice. This deficit in extinction learning is accompanied by a reduction of BDNF protein in the hippocampus, amygdala and the prefrontal cortex. Copyright © 2013 Elsevier Inc. All rights reserved.

Physiological roles of A1 and A2A adenosine receptors in regulating heart rate, body temperature, and locomotion as revealed using knockout mice and caffeine

PubMed Central

Yang, Jiang-Ning; Chen, Jiang-Fan; Fredholm, Bertil B.

2009-01-01

Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O2C) were studied in awake mice lacking one or both of the adenosine A1 or A2A receptors (A1R or A2AR, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A1R knockout (A1RKO) mice but lower in A2ARKO mice and intermediate in A1-A2AR double KO mice. A single dose of an unselective β-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A1Rs had little effect on Temp, whereas deletion of A2ARs increased it in females and decreased it in males. A1-A2ARKO mice had lower Temp than WT mice. LA was unaltered in A1RKO mice and lower in A2ARKO and A1-A2ARKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A2AR. Caffeine ingestion also increased LA in an A2AR-dependent manner in male mice. Caffeine ingestion significantly increased O2C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A1R or A2AR blockade. Selective A2B antagonism had little or no effect. Thus A1R and A2AR influence HR, Temp, LA, and O2C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A2AR plays an important role in the modulation of O2C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A1R and A2AR. PMID:19218506

GENETIC CATHEPSIN B DEFICIENCY REDUCES β-AMYLOID IN TRANSGENIC MICE EXPRESSING HUMAN WILD-TYPE AMYLOID PRECURSOR PROTEIN

PubMed Central

Hook, Vivian Y. H.; Kindy, Mark; Reinheckel, Thomas; Peters, Christoph; Hook, Gregory

2009-01-01

Neurotoxic β-amyloid (Aβ) peptides participate in Alzheimer’s disease (AD); therefore, reduction of Aβ generated from APP may provide a therapeutic approach for AD. Gene knockout studies in transgenic mice producing human Aβ may identify targets for reducing Aβ. This study shows that knockout of the cathepsin B gene in mice expressing human wild-type APP (hAPPwt) results in substantial decrease of Aβ40 and Aβ42 by 67% in brain, and decreases levels of the C-terminal β-secretase fragment (CTFβ) derived from APP. In contrast, knockout of cathepsin B in mice expressing hAPP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Aβ. The difference in reduction of Aβ in hAPPwt mice, but not in hAPPSwe/Ind mice, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express hAPPwt, these data validate cathepsin B as a target for development of inhibitors to lower Aβ in AD. PMID:19501042

Degenerative phenotypes caused by the combined deficiency of murine HIP1 and HIP1r are rescued by human HIP1.

PubMed

Bradley, Sarah V; Hyun, Teresa S; Oravecz-Wilson, Katherine I; Li, Lina; Waldorff, Erik I; Ermilov, Alexander N; Goldstein, Steven A; Zhang, Claire X; Drubin, David G; Varela, Kate; Parlow, Al; Dlugosz, Andrzej A; Ross, Theodora S

2007-06-01

The members of the huntingtin-interacting protein-1 (HIP1) family, HIP1 and HIP1-related (HIP1r), are multi-domain proteins that interact with inositol lipids, clathrin and actin. HIP1 is over-expressed in a variety of cancers and both HIP1 and HIP1r prolong the half-life of multiple growth factor receptors. To better understand the physiological importance of the HIP1 family in vivo, we have analyzed a large cohort of double Hip1/Hip1r knockout (DKO) mice. All DKO mice were dwarfed, afflicted with severe vertebral defects and died in early adulthood. These phenotypes were not observed during early adulthood in the single Hip1 or Hip1r knockouts, indicating that HIP1 and HIP1r compensate for one another. Despite the ability of HIP1 and HIP1r to modulate growth factor receptor levels when over-expressed, studies herein using DKO fibroblasts indicate that the HIP1 family is not necessary for endocytosis but is necessary for the maintenance of diverse adult tissues in vivo. To test if human HIP1 can function similar to mouse HIP1, transgenic mice with 'ubiquitous' expression of the human HIP1 cDNA were generated and crossed with DKO mice. Strikingly, the compound human HIP1 transgenic DKO mice were completely free from dwarfism and spinal defects. This successful rescue demonstrates that the human HIP1 protein shares some interchangeable functions with both HIP1 and HIP1r in vivo. In addition, we conclude that the degenerative phenotypes seen in the DKO mice are due mainly to HIP1 and HIP1r protein deficiency rather than altered expression of neighboring genes or disrupted intronic elements.

Importance of GluA1 Subunit-Containing AMPA Glutamate Receptors for Morphine State-Dependency

PubMed Central

Aitta-aho, Teemu; Möykkynen, Tommi P.; Panhelainen, Anne E.; Vekovischeva, Olga Yu.; Bäckström, Pia; Korpi, Esa R.

2012-01-01

In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days) using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test); in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards), in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity. PMID:22675452

Genetic loss of diazepam binding inhibitor in mice impairs social interest.

PubMed

Ujjainwala, A L; Courtney, C D; Rhoads, S G; Rhodes, J S; Christian, C A

2018-06-01

Neuropsychiatric disorders in which reduced social interest is a common symptom, such as autism, depression, and anxiety, are frequently associated with genetic mutations affecting γ-aminobutyric acid (GABA)ergic transmission. Benzodiazepine treatment, acting via GABA type-A receptors, improves social interaction in male mouse models with autism-like features. The protein diazepam binding inhibitor (DBI) can act as an endogenous benzodiazepine, but a role for DBI in social behavior has not been described. Here, we investigated the role of DBI in the social interest and recognition behavior of mice. The responses of DBI wild-type and knockout male and female mice to ovariectomized female wild-type mice (a neutral social stimulus) were evaluated in a habituation/dishabituation task. Both male and female knockout mice exhibited reduced social interest, and DBI knockout mice lacked the sex difference in social interest levels observed in wild-type mice, in which males showed higher social interest levels than females. The ability to discriminate between familiar and novel stimulus mice (social recognition) was not impaired in DBI-deficient mice of either sex. DBI knockouts could learn a rotarod motor task, and could discriminate between social and nonsocial odors. Both sexes of DBI knockout mice showed increased repetitive grooming behavior, but not in a manner that would account for the decrease in social investigation time. Genetic loss of DBI did not alter seminal vesicle weight, indicating that the social interest phenotype of males lacking DBI is not due to reduced circulating testosterone. Together, these studies show a novel role of DBI in driving social interest and motivation. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Skeletal muscle-specific HMG-CoA reductase knockout mice exhibit rhabdomyolysis: A model for statin-induced myopathy.

PubMed

Osaki, Yoshinori; Nakagawa, Yoshimi; Miyahara, Shoko; Iwasaki, Hitoshi; Ishii, Akiko; Matsuzaka, Takashi; Kobayashi, Kazuto; Yatoh, Shigeru; Takahashi, Akimitsu; Yahagi, Naoya; Suzuki, Hiroaki; Sone, Hirohito; Ohashi, Ken; Ishibashi, Shun; Yamada, Nobuhiro; Shimano, Hitoshi

2015-10-23

HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonic acid (MVA); this is the rate-limiting enzyme of the mevalonate pathway that synthesizes cholesterol. Statins, HMGCR inhibitors, are widely used as cholesterol-reducing drugs. However, statin-induced myopathy is the most adverse side effect of statins. To eludicate the mechanisms underlying statin the myotoxicity and HMGCR function in the skeletal muscle, we developed the skeletal muscle-specific HMGCR knockout mice. Knockout mice exhibited postnatal myopathy with elevated serum creatine kinase levels and necrosis. Myopathy in knockout mice was completely rescued by the oral administration of MVA. These results suggest that skeletal muscle toxicity caused by statins is dependent on the deficiencies of HMGCR enzyme activity and downstream metabolites of the mevalonate pathway in skeletal muscles rather than the liver or other organs. Copyright © 2015 Elsevier Inc. All rights reserved.

Dietary phosphate restriction normalizes biochemical and skeletal abnormalities in a murine model of tumoral calcinosis.

PubMed

Ichikawa, Shoji; Austin, Anthony M; Gray, Amie K; Allen, Matthew R; Econs, Michael J

2011-12-01

Mutations in the GALNT3 gene cause tumoral calcinosis characterized by ectopic calcifications due to persistent hyperphosphatemia. We recently developed Galnt3 knockout mice in a mixed background, which had hyperphosphatemia with increased bone mineral density (BMD) and infertility in males. To test the effect of dietary phosphate intake on their phenotype, Galnt3 knockout mice were generated in the C57BL/6J strain and fed various phosphate diets: 0.1% (low), 0.3% (low normal), 0.6% (normal), and 1.65% (high). Sera were analyzed for calcium, phosphorus, alkaline phosphatase, creatinine, blood urine nitrogen, 1,25-dihydroxyvitamin D, osteocalcin, tartrate-resistant acid phosphatase 5b, and fibroblast growth factor 23 (Fgf23). Femurs were evaluated by dual-energy x-ray absorptiometry, dynamic histomorphometry, and/or microcomputed tomography. Galnt3 knockout mice in C57BL/6J had the same biochemical phenotype observed in our previous study: hyperphosphatemia, inappropriately normal 1,25-dihydroxyvitamin D level, decreased alkaline phosphatase activity, and low intact Fgf23 concentration but high Fgf23 fragments. Skeletal analyses of their femurs revealed significantly high BMD with increased cortical bone area and trabecular bone volume. On all four phosphate diets, Galnt3 knockout mice had consistently higher phosphorus levels and lower alkaline phosphatase and intact Fgf23 concentrations than littermate controls. The low-phosphate diet normalized serum phosphorus, alkaline phosphatase, and areal BMD but failed to correct male infertility in Galnt3 knockout mice. The high-phosphate diet did not increase serum phosphorus concentration in either mutant or control mice due to a compensatory increase in circulating intact Fgf23 levels. In conclusion, dietary phosphate restriction normalizes biochemical and skeletal phenotypes of Galnt3 knockout mice and, thus, can be an effective therapy for tumoral calcinosis.

Dietary Phosphate Restriction Normalizes Biochemical and Skeletal Abnormalities in a Murine Model of Tumoral Calcinosis

PubMed Central

Austin, Anthony M.; Gray, Amie K.; Allen, Matthew R.; Econs, Michael J.

2011-01-01

Mutations in the GALNT3 gene cause tumoral calcinosis characterized by ectopic calcifications due to persistent hyperphosphatemia. We recently developed Galnt3 knockout mice in a mixed background, which had hyperphosphatemia with increased bone mineral density (BMD) and infertility in males. To test the effect of dietary phosphate intake on their phenotype, Galnt3 knockout mice were generated in the C57BL/6J strain and fed various phosphate diets: 0.1% (low), 0.3% (low normal), 0.6% (normal), and 1.65% (high). Sera were analyzed for calcium, phosphorus, alkaline phosphatase, creatinine, blood urine nitrogen, 1,25-dihydroxyvitamin D, osteocalcin, tartrate-resistant acid phosphatase 5b, and fibroblast growth factor 23 (Fgf23). Femurs were evaluated by dual-energy x-ray absorptiometry, dynamic histomorphometry, and/or microcomputed tomography. Galnt3 knockout mice in C57BL/6J had the same biochemical phenotype observed in our previous study: hyperphosphatemia, inappropriately normal 1,25-dihydroxyvitamin D level, decreased alkaline phosphatase activity, and low intact Fgf23 concentration but high Fgf23 fragments. Skeletal analyses of their femurs revealed significantly high BMD with increased cortical bone area and trabecular bone volume. On all four phosphate diets, Galnt3 knockout mice had consistently higher phosphorus levels and lower alkaline phosphatase and intact Fgf23 concentrations than littermate controls. The low-phosphate diet normalized serum phosphorus, alkaline phosphatase, and areal BMD but failed to correct male infertility in Galnt3 knockout mice. The high-phosphate diet did not increase serum phosphorus concentration in either mutant or control mice due to a compensatory increase in circulating intact Fgf23 levels. In conclusion, dietary phosphate restriction normalizes biochemical and skeletal phenotypes of Galnt3 knockout mice and, thus, can be an effective therapy for tumoral calcinosis. PMID:22009723

Age- and region-specific imbalances of basal amino acids and monoamine metabolism in limbic regions of female Fmr1 knock-out mice.

PubMed

Gruss, Michael; Braun, Katharina

2004-07-01

The Fragile X syndrome, a common form of mental retardation in humans, originates from the loss of expression of the Fragile X mental retardation gene leading to the absence of the encoded Fragile X mental retardation protein 1 (FMRP). A broad pattern of morphological and behavioral abnormalities is well described for affected humans as well as Fmr1 knock-out mice, a transgenic animal model for the human Fragile X syndrome. In the present study, we examined neurochemical differences between female Fmr1 knock-out and wildtype mice with particular focus on neurotransmission. Significant age- and region-specific differences of basal tissue neurotransmitter and metabolite levels measured by high performance liquid chromatography were found. Those differences were more numerous in juvenile animals (postnatal day (PND) 28-31) compared to adults (postnatal day 209-221). In juvenile female knock-out mice, especially aspartate and taurine were increased in cortical regions, striatum, cerebellum, and brainstem. Furthermore, compared to the wildtype animals, the juvenile knock-out mice displayed an increased level of neuronal inhibition in the hippocampus and brainstem reflected by decreased ratios of (aspartate + glutamate)/(taurine + GABA), as well as an increased dopamine (DA) turnover in cortical regions, striatum, and hippocampus. These results provide the first evidence that the lack of FMRP expression in female Fmr1 knock-out mice is accompanied by age-dependent, region-specific alterations in brain amino acids, and monoamine turnover, which might be related to the reported synaptical and behavioural alterations in these animals.

Microarray expression profiling identifies genes with altered expression in HDL-deficient mice

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

Callow, Matthew J.; Dudoit, Sandrine; Gong, Elaine L.

2000-05-05

Based on the assumption that severe alterations in the expression of genes known to be involved in HDL metabolism may affect the expression of other genes we screened an array of over 5000 mouse expressed sequence tags (ESTs) for altered gene expression in the livers of two lines of mice with dramatic decreases in HDL plasma concentrations. Labeled cDNA from livers of apolipoprotein AI (apo AI) knockout mice, Scavenger Receptor BI (SR-BI) transgenic mice and control mice were co-hybridized to microarrays. Two-sample t-statistics were used to identify genes with altered expression levels in the knockout or transgenic mice compared withmore » the control mice. In the SR-BI group we found 9 array elements representing at least 5 genes to be significantly altered on the basis of an adjusted p value of less than 0.05. In the apo AI knockout group 8 array elements representing 4 genes were altered compared with the control group (p < 0.05). Several of the genes identified in the SR-BI transgenic suggest altered sterol metabolism and oxidative processes. These studies illustrate the use of multiple-testing methods for the identification of genes with altered expression in replicated microarray experiments of apo AI knockout and SR-BI transgenic mice.« less

OAS and PKR are not Required for the Anti-viral Effect of Ad:IFN-γ Against Acute HSV-1 in Primary Trigeminal Ganglia Cultures

PubMed Central

Austin, Bobbie Ann; Halford, William; Silverman, Robert H.; Williams, Bryan R. G.; Carr, Daniel J. J.

2005-01-01

Three interferon-gamma (IFNG) induced anti-viral pathways have been reported. Involved anti-viral proteins include: Mx, RNaseL/2'-5'-OAS, and PKR. Involvement of OAS and PKR in IFNG-induced anti-HSV-1 pathways has not been previously reported, but IFNG induces OAS and PKR when other viruses invade the nervous system. The aim of the current study was to determine whether the absence of intact OAS and PKR anti-viral pathways affect the anti-viral activity of IFNG during acute HSV-1 infection within trigeminal ganglia (TG). To investigate this, primary TG cultures were established using TGs removed from C57BL/6 (Wt), RNase L knockout, and RNase L/PKR double knockout mice. Each dissociated TG was transduced with an adenoviral vector containing an IFNG transgene or vector alone. Viral titers following HSV-1 infection of primary TG cell cultures were determined. Significant differences in viral titer for Ad:Null versus Ad:IFNG tranduced TGs were found in each genotype. However, the effectiveness of Ad:IFNG was not reduced in the absence of both OAS and PKR pathways or OAS alone. Recombinant IFNG also exhibited anti-HSV-1 activity. The effectiveness of the IFNG transgene was lost in primary TG cells from IFNG receptor knockout mice. The data suggest that novel anti-HSV-1 mechanisms are induced by IFNG. PMID:16704298

Cholecystokinin levels in prohormone convertase 2 knock-out mouse brain regions reveal a complex phenotype of region-specific alterations.

PubMed

Beinfeld, Margery C; Blum, Alissa; Vishnuvardhan, Daesety; Fanous, Sanya; Marchand, James E

2005-11-18

Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA.

Effects of blueberries in prevention of atherosclerosis in apoe knockout mice

USDA-ARS?s Scientific Manuscript database

ApoE knockout (ApoE-/-) mice were fed AIN-93G diet (CD) or CD formulated to contain 1% freeze-dried whole wild blueberries (CD1% BB). Mice were sacrificed after 20 weeks on the specified diet. Atherosclerotic lesions in aortic sinus were determined by staining cryosections (10 µm) with Oil Red O. Th...

Toll-like receptor 4 knockout ameliorates neuroinflammation due to lung-brain interaction in mechanically ventilated mice.

PubMed

Chen, Ting; Chen, Chang; Zhang, Zongze; Zou, Yufeng; Peng, Mian; Wang, Yanlin

2016-08-01

Toll-like receptor 4 (TLR4) is a crucial receptor in the innate immune system, and increasing evidence supports its role in inflammation, stress, and tissue injury, including injury to the lung and brain. We aimed to investigate the effects of TLR4 on neuroinflammation due to the lung-brain interaction in mechanically ventilated mice. Male wild-type (WT) C57BL/6 and TLR4 knockout (TLR4 KO) mice were divided into three groups: (1) control group (C): spontaneous breathing; (2) anesthesia group (A): spontaneous breathing under anesthesia; and (3) mechanical ventilation group (MV): 6h of MV under anesthesia. The behavioral responses of mice were tested with fear conditioning tests. The histological changes in the lung and brain were assessed using hematoxylin-eosin (HE) staining. The level of TLR4 mRNA in tissue was measured using reverse transcription-polymerase chain reaction (RT-PCR). The levels of inflammatory cytokines were measured with an enzyme-linked immunosorbent assay (ELISA). Microgliosis, astrocytosis, and the TLR4 immunoreactivity in the hippocampus were measured by double immunofluorescence. MV mice exhibited impaired cognition, and this impairment was less severe in TLR4 KO mice than in WT mice. In WT mice, MV increased TLR4 mRNA expression in the lung and brain. MV induced mild lung injury, which was prevented in TLR4 KO mice. MV mice exhibited increased levels of inflammatory cytokines, increased microglia and astrocyte activation. Microgliosis was alleviated in TLR4 KO mice. MV mice exhibited increased TLR4 immunoreactivity, which was expressed in microglia and astrocytes. These results demonstrate that TLR4 is involved in neuroinflammation due to the lung-brain interaction and that TLR4 KO ameliorates neuroinflammation due to lung-brain interaction after prolonged MV. In addition, Administration of a TLR4 antagonist (100μg/mice) to WT mice also significantly attenuated neuroinflammation of lung-brain interaction due to prolonged MV. TLR4 antagonism may be a new and novel approach for the treatment and management of neuroinflammation in long-term mechanically ventilated patients. Copyright © 2016 Elsevier Inc. All rights reserved.

CB2 Cannabinoid Receptor Knockout in Mice Impairs Contextual Long-Term Memory and Enhances Spatial Working Memory

PubMed Central

Li, Yong; Kim, Jimok

2016-01-01

Neurocognitive effects of cannabinoids have been extensively studied with a focus on CB1 cannabinoid receptors because CB1 receptors have been considered the major cannabinoid receptor in the nervous system. However, recent discoveries of CB2 cannabinoid receptors in the brain demand accurate determination of whether and how CB2 receptors are involved in the cognitive effects of cannabinoids. CB2 cannabinoid receptors are primarily involved in immune functions, but also implicated in psychiatric disorders such as schizophrenia and depression. Here, we examined the effects of CB2 receptor knockout in mice on memory to determine the roles of CB2 receptors in modulating cognitive function. Behavioral assays revealed that hippocampus-dependent, long-term contextual fear memory was impaired whereas hippocampus-independent, cued fear memory was normal in CB2 receptor knockout mice. These mice also displayed enhanced spatial working memory when tested in a Y-maze. Motor activity and anxiety of CB2 receptor knockout mice were intact when assessed in an open field arena and an elevated zero maze. In contrast to the knockout of CB2 receptors, acute blockade of CB2 receptors by AM603 in C57BL/6J mice had no effect on memory, motor activity, or anxiety. Our results suggest that CB2 cannabinoid receptors play diverse roles in regulating memory depending on memory types and/or brain areas. PMID:26819779

Ontogeny of brain and blood serotonin levels in 5-HT receptor knockout mice: potential relevance to the neurobiology of autism.

PubMed

Janusonis, Skirmantas; Anderson, George M; Shifrovich, Ilya; Rakic, Pasko

2006-11-01

The most consistent neurochemical finding in autism has been elevated group mean levels of blood platelet 5-hydroxytryptamine (5-HT, serotonin). The origin and significance of this platelet hyperserotonemia remain poorly understood. The 5-HT(1A) receptor plays important roles in the developing brain and is also expressed in the gut, the main source of platelet 5-HT. Post-natal tissue levels of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) and tryptophan were examined in the brain, duodenum and blood of 5-HT(1A) receptor-knockout and wild-type mice. At 3 days after birth, the knockout mice had lower mean brain 5-HT levels and normal mean platelet 5-HT levels. Also, at 3 days after birth, the mean tryptophan levels in the brain, duodenum and blood of the knockout mice were around 30% lower than those of the wild-type mice. By 2 weeks after birth, the mean brain 5-HT levels of the knockout mice normalized, but their mean platelet 5-HT levels became 24% higher than normal. The possible causes of these dynamic shifts were explored by examining correlations between central and peripheral levels of 5-HT, 5-HIAA and tryptophan. The results are discussed in relation to the possible role of 5-HT in the ontogeny of autism.

IL-23 is critical in the induction but not in the effector phase of experimental autoimmune encephalomyelitis.

PubMed

Thakker, Paresh; Leach, Michael W; Kuang, Wen; Benoit, Stephen E; Leonard, John P; Marusic, Suzana

2007-02-15

Experimental autoimmune encephalomyelitis (EAE), a T cell-mediated inflammatory disease of the CNS, is a rodent model of human multiple sclerosis. IL-23 is one of the critical cytokines in EAE development and is currently believed to be involved in the maintenance of encephalitogenic responses during the tissue damage effector phase of the disease. In this study, we show that encephalitogenic T cells from myelin oligodendrocyte glycopeptide (MOG)-immunized wild-type (WT) mice caused indistinguishable disease when adoptively transferred to WT or IL-23-deficient (p19 knockout (KO)) recipient mice, demonstrating that once encephalitogenic cells have been generated, EAE can develop in the complete absence of IL-23. Furthermore, IL-12/23 double-deficient (p35/p19 double KO) recipient mice developed EAE that was indistinguishable from WT recipients, indicating that IL-12 did not compensate for IL-23 deficiency during the effector phase of EAE. In contrast, MOG-specific T cells from p19KO mice induced EAE with delayed onset and much lower severity when transferred to WT recipient mice as compared with the EAE that was induced by cells from WT controls. MOG-specific T cells from p19KO mice were highly deficient in the production of IFN-gamma, IL-17A, and TNF, indicating that IL-23 plays a critical role in development of encephalitogenic T cells and facilitates the development of T cells toward both Th1 and Th17 pathways.

Crimean-Congo Hemorrhagic Fever Virus Subunit Vaccines Induce High Levels of Neutralizing Antibodies But No Protection in STAT1 Knockout Mice.

PubMed

Kortekaas, Jeroen; Vloet, Rianka P M; McAuley, Alexander J; Shen, Xiaoli; Bosch, Berend Jan; de Vries, Laura; Moormann, Rob J M; Bente, Dennis A

2015-12-01

Crimean-Congo hemorrhagic fever virus is a tick-borne bunyavirus of the Nairovirus genus that causes hemorrhagic fever in humans with high case fatality. Here, we report the development of subunit vaccines and their efficacy in signal transducer and activator of transcription 1 (STAT1) knockout mice. Ectodomains of the structural glycoproteins Gn and Gc were produced using a Drosophila insect cell-based expression system. A single vaccination of STAT129 mice with adjuvanted Gn or Gc ectodomains induced neutralizing antibody responses, which were boosted by a second vaccination. Despite these antibody responses, mice were not protected from a CCHFV challenge infection. These results suggest that neutralizing antibodies against CCHFV do not correlate with protection of STAT1 knockout mice.

Tenascin-C Deficiency in Apo E−/− Mouse Increases Eotaxin Levels: Implications for Atherosclerosis

PubMed Central

Wang, Lai; Shah, Prediman K.; Wang, Wei; Song, Lei; Yang, Mingjie; Sharifi, Behrooz G.

2013-01-01

Aim To investigate the potential role of inflammatory cytokines in apo E−/− mouse in response to deletion of Tenascin-C (TNC) gene. Methods and results We used antibody array and ELISA to compare the profile of circulating inflammatory cytokines in apo E−/− mice and apo E−/− TNC−/− double knockout mice. In addition, tissue culture studies were performed to investigate the activity of cells from each mouse genotype in vitro. Cytokine array analysis and subsequent ELISA showed that circulating eotaxin levels were selectively and markedly increased in response to TNC gene deletion in apo E−/− mice. In addition, considerable variation was noted in the circulating level of eotaxin among the control apo E−/− mouse group. Inbreeding of apo E−/− mice with high or low levels of plasma eotaxin showed that the level of eotaxin per se determines the extent of atherosclerosis in this mouse genotype. While endothelial cells from apo E−/− mice had low level of eotaxin expression, cells derived from apo E−/−TNC−/− mice expressed a high level of eotaxin. Transient transfection of eotaxin promoter-reporter constructs revealed that eotaxin expression is regulated at the transcriptional level by TNC. Histochemical analysis of aortic sections revealed the massive accumulation of mast cells in the adventitia of double KO mice lesions whereas no such accumulation was detected in the control group. Plasma from the apo E−/−TNC−/− mice markedly stimulated mast cell migration whereas plasma from the apo E−/− mice had no such effect. Conclusion These observations support the emerging hypothesis that TNC expression controls eotaxin level in apo E−/− mice and that this chemokine plays a key role in the development of atherosclerosis. PMID:23433402

Rectal Delivery of a DNAzyme That Specifically Blocks the Transcription Factor GATA3 and Reduces Colitis in Mice.

PubMed

Popp, Vanessa; Gerlach, Katharina; Mott, Stefanie; Turowska, Agnieszka; Garn, Holger; Atreya, Raja; Lehr, Hans-Anton; Ho, I-Cheng; Renz, Harald; Weigmann, Benno; Neurath, Markus F

2017-01-01

GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice. We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n = 61) or ulcerative colitis (UC, n = 74) or from patients without inflammatory bowel diseases (n = 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated and analyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by in vivo imaging (endoscopy) of mice. Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA, compared with mice given a control DNAzyme. Mice given hgd40 did not develop colitis after administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid; lamina propria cells from these mice expressed lower levels of IL6, IL9, and IL13 than cells from mice given the control DNAzyme. Mini-endoscopic images revealed that hgd40 and anti-TNF reduced colon inflammation over 3 days; hgd40 reduced colitis in TNFR double-knockout mice. Levels of GATA3 are increased in patients with UC and correlate with production of inflammatory cytokines in mice and humans. A DNAzyme that prevents expression of GATA3 reduces colitis in mice, independently of TNF, and reduces levels of cytokines in the colon. This DNAzyme might be developed for treatment of patients with UC. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Severe Intestinal Inflammation in the Small Intestine of Mice Induced by Controllable Deletion of Claudin-7.

PubMed

Li, Wen-Jing; Xu, Chang; Wang, Kun; Li, Teng-Yan; Wang, Xiao-Nan; Yang, Hui; Xing, Tiaosi; Li, Wen-Xia; Chen, Yan-Hua; Gao, Hong; Ding, Lei

2018-05-01

As a potential tumor suppressor gene, Claudin-7 (Cldn7), which is a component of tight junctions, may play an important role in colorectal cancer occurrence and development. To generate a knockout mouse model of inducible conditional Cldn7 in the intestine and analyze the phenotype of the mice after induction with tamoxifen. We constructed Cldn7-flox transgenic mice and crossed them with Villin-CreERT2 mice. The Cldn7 inducible conditional knockout mice appeared normal and were well developed at birth. We induced Cldn7 gene deletion by injecting different dosages of tamoxifen into the mice and then conducted a further phenotypic analysis. After induction for 5 days in succession at a dose of 200 µl tamoxifen in sunflower oil at 10 mg/ml per mouse every time, the mice appeared dehydrated, had a lower temperature, and displayed inactivity or death. The results of hematoxylin-eosin staining showed that the intestines of the Cldn7 inducible conditional knockout mice had severe intestinal defects that included epithelial cell sloughing, necrosis, inflammation and hyperplasia. Owing to the death of ICKO mice, we adjusted the dose of tamoxifen to a dose of 100 µl in sunflower oil at 10 mg/ml per mouse (aged more than 8 weeks old) every 4 days. And we could induce atypical hyperplasia and adenoma in the intestine. Immunofluorescent staining indicated that the intestinal epithelial structure was destroyed. Electron microscopy experimental analysis indicated that the intercellular gap along the basolateral membrane of Cldn7 inducible conditional knockout mice in the intestine was increased and that contact between the cells and matrix was loosened. We generated a model of intestinal Cldn7 inducible conditional knockout mice. Intestinal Cldn7 deletion induced by tamoxifen initiated inflammation and hyperplasia in mice.

Cathepsin K knockout alleviates aging-induced cardiac dysfunction

PubMed Central

Hua, Yinan; Robinson, Timothy J; Cao, Yongtao; Shi, Guo-Ping; Ren, Jun; Nair, Sreejayan

2015-01-01

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis. PMID:25692548

Glutathione peroxidase-2 and selenium decreased inflammation and tumors in a mouse model of inflammation-associated carcinogenesis whereas sulforaphane effects differed with selenium supply

PubMed Central

Krehl, Susanne; Loewinger, Maria; Florian, Simone; Kipp, Anna P.; Banning, Antje; Wessjohann, Ludger A.; Brauer, Martin N.; Iori, Renato; Esworthy, Robert S.; Chu, Fong-Fong; Brigelius-Flohé, Regina

2012-01-01

Chronic inflammation and selenium deficiency are considered as risk factors for colon cancer. The protective effect of selenium might be mediated by specific selenoproteins, such as glutathione peroxidases (GPx). GPx-1 and -2 double knockout, but not single knockout mice, spontaneously develop ileocolitis and intestinal cancer. Since GPx2 is induced by the chemopreventive sulforaphane (SFN) via the nuclear factor E2-related factor 2 (Nrf2)/Keap1 system, the susceptibility of GPx2-KO and wild-type (WT) mice to azoxymethane and dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis was tested under different selenium states and SFN applications. WT and GPx2-KO mice were grown on a selenium-poor, -adequate or -supranutritional diet. SFN application started either 1 week before (SFN4) or along with (SFN3) a single AOM application followed by DSS treatment for 1 week. Mice were assessed 3 weeks after AOM for colitis and Nrf2 target gene expression and after 12 weeks for tumorigenesis. NAD(P)H:quinone oxidoreductases, thioredoxin reductases and glutathione-S-transferases were upregulated in the ileum and/or colon by SFN, as was GPx2 in WT mice. Inflammation scores were more severe in GPx2-KO mice and highest in selenium-poor groups. Inflammation was enhanced by SFN4 in both genotypes under selenium restriction but decreased in selenium adequacy. Total tumor numbers were higher in GPx2-KO mice but diminished by increasing selenium in both genotypes. SFN3 reduced inflammation and tumor multiplicity in both Se-adequate genotypes. Tumor size was smaller in Se-poor GPx2-KO mice. It is concluded that GPx2, although supporting tumor growth, inhibits inflammation-mediated tumorigenesis, but the protective effect of selenium does not strictly depend on GPx2 expression. Similarly, SFN requires selenium but not GPx2 for being protective. PMID:22180572

Glutathione peroxidase-2 and selenium decreased inflammation and tumors in a mouse model of inflammation-associated carcinogenesis whereas sulforaphane effects differed with selenium supply.

PubMed

Krehl, Susanne; Loewinger, Maria; Florian, Simone; Kipp, Anna P; Banning, Antje; Wessjohann, Ludger A; Brauer, Martin N; Iori, Renato; Esworthy, Robert S; Chu, Fong-Fong; Brigelius-Flohé, Regina

2012-03-01

Chronic inflammation and selenium deficiency are considered as risk factors for colon cancer. The protective effect of selenium might be mediated by specific selenoproteins, such as glutathione peroxidases (GPx). GPx-1 and -2 double knockout, but not single knockout mice, spontaneously develop ileocolitis and intestinal cancer. Since GPx2 is induced by the chemopreventive sulforaphane (SFN) via the nuclear factor E2-related factor 2 (Nrf2)/Keap1 system, the susceptibility of GPx2-KO and wild-type (WT) mice to azoxymethane and dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis was tested under different selenium states and SFN applications. WT and GPx2-KO mice were grown on a selenium-poor, -adequate or -supranutritional diet. SFN application started either 1 week before (SFN4) or along with (SFN3) a single AOM application followed by DSS treatment for 1 week. Mice were assessed 3 weeks after AOM for colitis and Nrf2 target gene expression and after 12 weeks for tumorigenesis. NAD(P)H:quinone oxidoreductases, thioredoxin reductases and glutathione-S-transferases were upregulated in the ileum and/or colon by SFN, as was GPx2 in WT mice. Inflammation scores were more severe in GPx2-KO mice and highest in selenium-poor groups. Inflammation was enhanced by SFN4 in both genotypes under selenium restriction but decreased in selenium adequacy. Total tumor numbers were higher in GPx2-KO mice but diminished by increasing selenium in both genotypes. SFN3 reduced inflammation and tumor multiplicity in both Se-adequate genotypes. Tumor size was smaller in Se-poor GPx2-KO mice. It is concluded that GPx2, although supporting tumor growth, inhibits inflammation-mediated tumorigenesis, but the protective effect of selenium does not strictly depend on GPx2 expression. Similarly, SFN requires selenium but not GPx2 for being protective.

CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation.

PubMed

Chang, Chung-Hsing; Kuo, Che-Jung; Ito, Takamichi; Su, Yu-Ya; Jiang, Si-Tse; Chiu, Min-Hsi; Lin, Yi-Hsiung; Nist, Andrea; Mernberger, Marco; Stiewe, Thorsten; Ito, Shosuke; Wakamatsu, Kazumasa; Hsueh, Yi-An; Shieh, Sheau-Yann; Snir-Alkalay, Irit; Ben-Neriah, Yinon

2017-09-19

Casein kinase 1α (CK1α), a component of the β-catenin destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1α (encoded by Csnk1a1 ) in skin physiology, we crossed mice harboring floxed Csnk1a1 with mice expressing K14-Cre-ER T2 to generate mice in which tamoxifen induces the deletion of Csnk1a1 exclusively in keratinocytes [single-knockout (SKO) mice]. As expected, CK1α loss was accompanied by β-catenin and p53 stabilization, with the preferential induction of p53 target genes, but phenotypically most striking was hyperpigmentation of the skin, importantly without tumorigenesis, for at least 9 mo after Csnk1a1 ablation. The number of epidermal melanocytes and eumelanin levels were dramatically increased in SKO mice. To clarify the putative role of p53 in epidermal hyperpigmentation, we established K14-Cre-ER T2 CK1α/p53 double-knockout (DKO) mice and found that coablation failed to induce epidermal hyperpigmentation, demonstrating that it was p53-dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit ligand (KitL). SKO mice treated with ACK2 (a Kit-neutralizing antibody) or imatinib (a Kit inhibitor) abrogated the CK1α ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway. Pro-opiomelanocortin (POMC), a precursor of α-melanocyte-stimulating hormone (α-MSH), was not activated in the CK1α ablation-induced hyperpigmentation, which is in contrast to the mechanism of p53-dependent UV tanning. Nevertheless, acute sunburn effects were successfully prevented in the hyperpigmented skin of SKO mice. CK1α inhibition induces skin-protective eumelanin but no carcinogenic pheomelanin and may therefore constitute an effective strategy for safely increasing eumelanin via UV-independent pathways, protecting against acute sunburn.

CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation

PubMed Central

Chang, Chung-Hsing; Kuo, Che-Jung; Ito, Takamichi; Su, Yu-Ya; Jiang, Si-Tse; Chiu, Min-Hsi; Lin, Yi-Hsiung; Nist, Andrea; Mernberger, Marco; Stiewe, Thorsten; Ito, Shosuke; Wakamatsu, Kazumasa; Hsueh, Yi-An; Shieh, Sheau-Yann; Snir-Alkalay, Irit; Ben-Neriah, Yinon

2017-01-01

Casein kinase 1α (CK1α), a component of the β-catenin destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1α (encoded by Csnk1a1) in skin physiology, we crossed mice harboring floxed Csnk1a1 with mice expressing K14–Cre–ERT2 to generate mice in which tamoxifen induces the deletion of Csnk1a1 exclusively in keratinocytes [single-knockout (SKO) mice]. As expected, CK1α loss was accompanied by β-catenin and p53 stabilization, with the preferential induction of p53 target genes, but phenotypically most striking was hyperpigmentation of the skin, importantly without tumorigenesis, for at least 9 mo after Csnk1a1 ablation. The number of epidermal melanocytes and eumelanin levels were dramatically increased in SKO mice. To clarify the putative role of p53 in epidermal hyperpigmentation, we established K14–Cre–ERT2 CK1α/p53 double-knockout (DKO) mice and found that coablation failed to induce epidermal hyperpigmentation, demonstrating that it was p53-dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit ligand (KitL). SKO mice treated with ACK2 (a Kit-neutralizing antibody) or imatinib (a Kit inhibitor) abrogated the CK1α ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway. Pro-opiomelanocortin (POMC), a precursor of α-melanocyte–stimulating hormone (α-MSH), was not activated in the CK1α ablation-induced hyperpigmentation, which is in contrast to the mechanism of p53-dependent UV tanning. Nevertheless, acute sunburn effects were successfully prevented in the hyperpigmented skin of SKO mice. CK1α inhibition induces skin-protective eumelanin but no carcinogenic pheomelanin and may therefore constitute an effective strategy for safely increasing eumelanin via UV-independent pathways, protecting against acute sunburn. PMID:28878021

Kv4.2 Knockout Mice Have Hippocampal-Dependent Learning and Memory Deficits

ERIC Educational Resources Information Center

Lugo, Joaquin N.; Brewster, Amy L.; Spencer, Corinne M.; Anderson, Anne E.

2012-01-01

Kv4.2 channels contribute to the transient, outward K[superscript +] current (A-type current) in hippocampal dendrites, and modulation of this current substantially alters dendritic excitability. Using Kv4.2 knockout (KO) mice, we examined the role of Kv4.2 in hippocampal-dependent learning and memory. We found that Kv4.2 KO mice showed a deficit…

LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors

PubMed Central

2012-01-01

Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation. We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis. Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes. PMID:22647713

Mild deficits in mice lacking pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) performing on memory tasks.

PubMed

Sauvage, M; Brabet, P; Holsboer, F; Bockaert, J; Steckler, T

2000-12-08

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor subtype 1 (PAC1) have been suggested to play a role in the modulation of learning and memory. However, behavioral evidence for altered mnemonic function due to altered PAC1 activity is missing. Therefore, the role of PAC1 in learning and memory was studied in mouse mutants lacking this receptor (PAC1 knock-out mice), tested in water maze two-choice spatial discrimination, one-trial contextual and cued fear conditioning, and multiple-session contextual discrimination. Water maze spatial discrimination was unaffected in PAC1 mutants, while a mild deficit was observed in multiple session contextual discrimination in PAC1 knock-out mice. Furthermore, PAC1 knock-out mice were able to learn the association between context and shock in one-trial contextual conditioning, but showed faster return to baseline than wild-type mice. Thus, the effects of PAC1 knock-out on modulating performance in these tasks were subtle and suggest that PAC1 only plays a limited role in learning and memory.

Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides.

PubMed

Pan, Xuefang; De Aragão, Camila De Britto Pará; Velasco-Martin, Juan P; Priestman, David A; Wu, Harry Y; Takahashi, Kohta; Yamaguchi, Kazunori; Sturiale, Luisella; Garozzo, Domenico; Platt, Frances M; Lamarche-Vane, Nathalie; Morales, Carlos R; Miyagi, Taeko; Pshezhetsky, Alexey V

2017-08-01

Gangliosides (sialylated glycolipids) play an essential role in the CNS by regulating recognition and signaling in neurons. Metabolic blocks in processing and catabolism of gangliosides result in the development of severe neurologic disorders, including gangliosidoses manifesting with neurodegeneration and neuroinflammation. We demonstrate that 2 mammalian enzymes, neuraminidases 3 and 4, play important roles in catabolic processing of brain gangliosides by cleaving terminal sialic acid residues in their glycan chains. In neuraminidase 3 and 4 double-knockout mice, G M3 ganglioside is stored in microglia, vascular pericytes, and neurons, causing micro- and astrogliosis, neuroinflammation, accumulation of lipofuscin bodies, and memory loss, whereas their cortical and hippocampal neurons have lower rate of neuritogenesis in vitro Double-knockout mice also have reduced levels of G M1 ganglioside and myelin in neuronal axons. Furthermore, neuraminidase 3 deficiency drastically increased storage of G M2 in the brain tissues of an asymptomatic mouse model of Tay-Sachs disease, a severe human gangliosidosis, indicating that this enzyme is responsible for the metabolic bypass of β-hexosaminidase A deficiency. Together, our results provide the first in vivo evidence that neuraminidases 3 and 4 have important roles in CNS function by catabolizing gangliosides and preventing their storage in lipofuscin bodies.-Pan, X., De Britto Pará De Aragão, C., Velasco-Martin, J. P., Priestman, D. A., Wu, H. Y., Takahashi, K., Yamaguchi, K., Sturiale, L., Garozzo, D., Platt, F. M., Lamarche-Vane, N., Morales, C. R., Miyagi, T., Pshezhetsky, A. V. Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides. © FASEB.

Changes in the expression of neurotransmitter receptors in Parkin and DJ-1 knockout mice--A quantitative multireceptor study.

PubMed

Cremer, J N; Amunts, K; Schleicher, A; Palomero-Gallagher, N; Piel, M; Rösch, F; Zilles, K

2015-12-17

Parkinson's disease (PD) is a well-characterized neurological disorder with regard to its neuropathological and symptomatic appearance. At the genetic level, mutations of particular genes, e.g. Parkin and DJ-1, were found in human hereditary PD with early onset. Neurotransmitter receptors constitute decisive elements in neural signal transduction. Furthermore, since they are often altered in neurological and psychiatric diseases, receptors have been successful targets for pharmacological agents. However, the consequences of PD-associated gene mutations on the expression of transmitter receptors are largely unknown. Therefore, we studied the expression of 16 different receptor binding sites of the neurotransmitters glutamate, GABA, acetylcholine, adrenaline, serotonin, dopamine and adenosine by means of quantitative receptor autoradiography in Parkin and DJ-1 knockout mice. These knockout mice exhibit electrophysiological and behavioral deficits, but do not show the typical dopaminergic cell loss. We demonstrated differential changes of binding site densities in eleven brain regions. Most prominently, we found an up-regulation of GABA(B) and kainate receptor densities in numerous cortical areas of Parkin and DJ-1 knockout mice, as well as increased NMDA but decreased AMPA receptor densities in different brain regions of the Parkin knockout mice. The alterations of three different glutamate receptor types may indicate the potential relevance of the glutamatergic system in the pathogenesis of PD. Furthermore, the cholinergic M1, M2 and nicotinic receptors as well as the adrenergic α2 and the adenosine A(2A) receptors showed differentially increased densities in Parkin and DJ-1 knockout mice. Taken together, knockout of the PD-associated genes Parkin or DJ-1 results in differential changes of neurotransmitter receptor densities, highlighting a possible role of altered non-dopaminergic, and in particular of glutamatergic neurotransmission in PD pathogenesis. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Altered behaviour and cognitive function following combined deletion of Toll-like receptors 2 and 4 in mice.

PubMed

Too, Lay Khoon; McGregor, Iain S; Baxter, Alan G; Hunt, Nicholas H

2016-04-15

Activation of the immune system due to infection or aging is increasingly linked to impaired neuropsychological function. Toll-like receptors 2 and 4 (TLR2, TLR4) are well-characterised for their role in inflammatory events, and their combined activation has been implicated in neurological diseases. We therefore determined whether TLR2 and TLR4 double gene knockout (GKO) mice showed modified behaviour and cognitive function during a 16-day test sequence that employed the automated IntelliCage test system. The IntelliCage features a home cage environment in which groups of mice live and where water reward is gained through performing various tasks centred on drinking stations in each corner of the apparatus. All mice were tested twice, one month apart (the first sequence termed "R1"and the second "R2"). There were fewer corner visits and nosepokes in TLR2/4 GKO compared to wild-type mice during early exploration in R1, suggesting elevated neophobia in GKO mice. Reduced exploration persisted over subsequent test modules during the dark phase. TLR2/4 GKO mice also displayed increased corner visits during drinking sessions compared to non-drinking sessions, but this was not associated with increased drinking. In subsequent, more complex test modules, TLR2/4 GKO mice had unimpaired spatial learning, but showed markedly poorer performance in a visual discrimination reversal task compared to wild-type mice. These results indicated subtle impairments in behaviour and cognitive functions due to double deficiency in TLR2 and TLR4. These finding are highly relevant to understanding the combined actions of TLR2 and TLR4 on neurological status in a range of different disease conditions. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice*

PubMed Central

Sahu, Bhubanananda; Sun, Wenyu; Perusek, Lindsay; Parmar, Vipulkumar; Le, Yun-Zheng; Griswold, Michael D.; Palczewski, Krzysztof; Maeda, Akiko

2015-01-01

Regeneration of the visual chromophore, 11-cis-retinal, is a crucial step in the visual cycle required to sustain vision. This cycle consists of sequential biochemical reactions that occur in photoreceptor cells and the retinal pigmented epithelium (RPE). Oxidation of 11-cis-retinol to 11-cis-retinal is accomplished by a family of enzymes termed 11-cis-retinol dehydrogenases, including RDH5 and RDH11. Double deletion of Rdh5 and Rdh11 does not limit the production of 11-cis-retinal in mice. Here we describe a third retinol dehydrogenase in the RPE, RDH10, which can produce 11-cis-retinal. Mice with a conditional knock-out of Rdh10 in RPE cells (Rdh10 cKO) displayed delayed 11-cis-retinal regeneration and dark adaption after bright light illumination. Retinal function measured by electroretinogram after light exposure was also delayed in Rdh10 cKO mice as compared with controls. Double deletion of Rdh5 and Rdh10 (cDKO) in mice caused elevated 11/13-cis-retinyl ester content also seen in Rdh5−/−Rdh11−/− mice as compared with Rdh5−/− mice. Normal retinal morphology was observed in 6-month-old Rdh10 cKO and cDKO mice, suggesting that loss of Rdh10 in the RPE does not negatively affect the health of the retina. Compensatory expression of other retinol dehydrogenases was observed in both Rdh5−/− and Rdh10 cKO mice. These results indicate that RDH10 acts in cooperation with other RDH isoforms to produce the 11-cis-retinal chromophore needed for vision. PMID:26391396

Targeted deletion of miR-132/-212 impairs memory and alters the hippocampal transcriptome.

PubMed

Hansen, Katelin F; Sakamoto, Kensuke; Aten, Sydney; Snider, Kaitlin H; Loeser, Jacob; Hesse, Andrea M; Page, Chloe E; Pelz, Carl; Arthur, J Simon C; Impey, Soren; Obrietan, Karl

2016-02-01

miR-132 and miR-212 are structurally related microRNAs that have been found to exert powerful modulatory effects within the central nervous system (CNS). Notably, these microRNAs are tandomly processed from the same noncoding transcript, and share a common seed sequence: thus it has been difficult to assess the distinct contribution of each microRNA to gene expression within the CNS. Here, we employed a combination of conditional knockout and transgenic mouse models to examine the contribution of the miR-132/-212 gene locus to learning and memory, and then to assess the distinct effects that each microRNA has on hippocampal gene expression. Using a conditional deletion approach, we show that miR-132/-212 double-knockout mice exhibit significant cognitive deficits in spatial memory, recognition memory, and in tests of novel object recognition. Next, we utilized transgenic miR-132 and miR-212 overexpression mouse lines and the miR-132/-212 double-knockout line to explore the distinct effects of these two miRNAs on the transcriptional profile of the hippocampus. Illumina sequencing revealed that miR-132/-212 deletion increased the expression of 1138 genes; Venn analysis showed that 96 of these genes were also downregulated in mice overexpressing miR-132. Of the 58 genes that were decreased in animals overexpressing miR-212, only four of them were also increased in the knockout line. Functional gene ontology analysis of downregulated genes revealed significant enrichment of genes related to synaptic transmission, neuronal proliferation, and morphogenesis, processes known for their roles in learning, and memory formation. These data, coupled with previous studies, firmly establish a role for the miR-132/-212 gene locus as a key regulator of cognitive capacity. Further, although miR-132 and miR-212 share a seed sequence, these data indicate that these miRNAs do not exhibit strongly overlapping mRNA targeting profiles, thus indicating that these two genes may function in a complex, nonredundant manner to shape the transcriptional profile of the CNS. The dysregulation of miR-132/-212 expression could contribute to signaling mechanisms that are involved in an array of cognitive disorders. © 2016 Hansen et al.; Published by Cold Spring Harbor Laboratory Press.

Targeted deletion of miR-132/-212 impairs memory and alters the hippocampal transcriptome

PubMed Central

Hansen, Katelin F.; Sakamoto, Kensuke; Aten, Sydney; Snider, Kaitlin H.; Loeser, Jacob; Hesse, Andrea M.; Page, Chloe E.; Pelz, Carl; Arthur, J. Simon C.; Impey, Soren

2016-01-01

miR-132 and miR-212 are structurally related microRNAs that have been found to exert powerful modulatory effects within the central nervous system (CNS). Notably, these microRNAs are tandomly processed from the same noncoding transcript, and share a common seed sequence: thus it has been difficult to assess the distinct contribution of each microRNA to gene expression within the CNS. Here, we employed a combination of conditional knockout and transgenic mouse models to examine the contribution of the miR-132/-212 gene locus to learning and memory, and then to assess the distinct effects that each microRNA has on hippocampal gene expression. Using a conditional deletion approach, we show that miR-132/-212 double-knockout mice exhibit significant cognitive deficits in spatial memory, recognition memory, and in tests of novel object recognition. Next, we utilized transgenic miR-132 and miR-212 overexpression mouse lines and the miR-132/-212 double-knockout line to explore the distinct effects of these two miRNAs on the transcriptional profile of the hippocampus. Illumina sequencing revealed that miR-132/-212 deletion increased the expression of 1138 genes; Venn analysis showed that 96 of these genes were also downregulated in mice overexpressing miR-132. Of the 58 genes that were decreased in animals overexpressing miR-212, only four of them were also increased in the knockout line. Functional gene ontology analysis of downregulated genes revealed significant enrichment of genes related to synaptic transmission, neuronal proliferation, and morphogenesis, processes known for their roles in learning, and memory formation. These data, coupled with previous studies, firmly establish a role for the miR-132/-212 gene locus as a key regulator of cognitive capacity. Further, although miR-132 and miR-212 share a seed sequence, these data indicate that these miRNAs do not exhibit strongly overlapping mRNA targeting profiles, thus indicating that these two genes may function in a complex, nonredundant manner to shape the transcriptional profile of the CNS. The dysregulation of miR-132/-212 expression could contribute to signaling mechanisms that are involved in an array of cognitive disorders. PMID:26773099

Knockout of Epstein-Barr Virus BPLF1 Retards B-Cell Transformation and Lymphoma Formation in Humanized Mice

PubMed Central

Li, Guangming; Montgomery, Stephanie A.; Montgomery, Nathan D.; Su, Lishan; Pagano, Joseph S.

2015-01-01

ABSTRACT BPLF1 of Epstein-Barr virus (EBV) is classified as a late lytic cycle protein but is also found in the viral tegument, suggesting its potential involvement at both initial and late stages of viral infection. BPLF1 possesses both deubiquitinating and deneddylating activity located in its N-terminal domain and is involved in processes that affect viral infectivity, viral DNA replication, DNA repair, and immune evasion. A recently constructed EBV BPLF1-knockout (KO) virus was used in conjunction with a humanized mouse model that can be infected with EBV, enabling the first characterization of BPLF1 function in vivo. Results demonstrate that the BPLF1-knockout virus is approximately 90% less infectious than wild-type (WT) virus. Transformation of human B cells, a hallmark of EBV infection, was delayed and reduced with BPLF1-knockout virus. Humanized mice infected with EBV BPLF1-knockout virus showed less weight loss and survived longer than mice infected with equivalent infectious units of WT virus. Additionally, splenic tumors formed in 100% of mice infected with WT EBV but in only 25% of mice infected with BPLF1-KO virus. Morphological features of spleens containing tumors were similar to those in EBV-induced posttransplant lymphoproliferative disease (PTLD) and were almost identical to cases seen in human diffuse large B-cell lymphoma. The presence of EBV genomes was detected in all mice that developed tumors. The results implicate BPLF1 in human B-cell transformation and tumor formation in humanized mice. PMID:26489865

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

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

Chen, Yanyan; The Hamner Institutes for Health Sciences, Research Triangle Park, NC; Xu, Yuanyuan, E-mail: yyxu@cmu.edu.cn

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-doublemore » knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.« less

Neutral endopeptidase (EC 3.4.24.11) terminates colitis by degrading substance P.

PubMed

Sturiale, S; Barbara, G; Qiu, B; Figini, M; Geppetti, P; Gerard, N; Gerard, C; Grady, E F; Bunnett, N W; Collins, S M

1999-09-28

Neurogenic inflammation is regulated by sensory nerves and characterized by extravasation of plasma proteins and infiltration of neutrophils from post-capillary venules and arteriolar vasodilatation. Although it is well established that substance P (SP) interacts with the neurokinin 1 receptor (NK1R) to initiate neurogenic inflammation, the mechanisms that terminate inflammation are unknown. We examined whether neutral endopeptidase (NEP), a cell-surface enzyme that degrades SP in the extracellular fluid, terminates neurogenic inflammation in the colon. In NEP knockout mice, the SP concentration in the colon was approximately 2.5-fold higher than in wild-type mice, suggesting increased bioavailability of SP. The extravasation of Evans blue-labeled plasma proteins in the colon of knockout mice under basal conditions was approximately 4-fold higher than in wild-type mice. This elevated plasma leak was attenuated by recombinant NEP or the NK1R antagonist SR140333, and is thus caused by diminished degradation of SP. To determine whether deletion of NEP predisposes mice to uncontrolled inflammation, we compared dinitrobenzene sulfonic acid-induced colitis in wild-type and knockout mice. The severity of colitis, determined by macroscopic and histologic scoring and by myeloperoxidase activity, was markedly worse in knockout than wild-type mice after 3 and 7 days. The exacerbated inflammation in knockout mice was prevented by recombinant NEP and SR140333. Thus, NEP maintains low levels of SP in the extracellular fluid under basal conditions and terminates its proinflammatory effects. Because we have previously shown that intestinal inflammation results in down-regulation of NEP and diminished degradation of SP, our present results suggest that defects in NEP expression contribute to uncontrolled inflammation.

Neutral endopeptidase (EC 3.4.24.11) terminates colitis by degrading substance P

PubMed Central

Sturiale, S.; Barbara, G.; Qiu, B.; Figini, M.; Geppetti, P.; Gerard, N.; Gerard, C.; Grady, E. F.; Bunnett, N. W.; Collins, S. M.

1999-01-01

Neurogenic inflammation is regulated by sensory nerves and characterized by extravasation of plasma proteins and infiltration of neutrophils from post-capillary venules and arteriolar vasodilatation. Although it is well established that substance P (SP) interacts with the neurokinin 1 receptor (NK1R) to initiate neurogenic inflammation, the mechanisms that terminate inflammation are unknown. We examined whether neutral endopeptidase (NEP), a cell-surface enzyme that degrades SP in the extracellular fluid, terminates neurogenic inflammation in the colon. In NEP knockout mice, the SP concentration in the colon was ≈2.5-fold higher than in wild-type mice, suggesting increased bioavailability of SP. The extravasation of Evans blue-labeled plasma proteins in the colon of knockout mice under basal conditions was ≈4-fold higher than in wild-type mice. This elevated plasma leak was attenuated by recombinant NEP or the NK1R antagonist SR140333, and is thus caused by diminished degradation of SP. To determine whether deletion of NEP predisposes mice to uncontrolled inflammation, we compared dinitrobenzene sulfonic acid-induced colitis in wild-type and knockout mice. The severity of colitis, determined by macroscopic and histologic scoring and by myeloperoxidase activity, was markedly worse in knockout than wild-type mice after 3 and 7 days. The exacerbated inflammation in knockout mice was prevented by recombinant NEP and SR140333. Thus, NEP maintains low levels of SP in the extracellular fluid under basal conditions and terminates its proinflammatory effects. Because we have previously shown that intestinal inflammation results in down-regulation of NEP and diminished degradation of SP, our present results suggest that defects in NEP expression contribute to uncontrolled inflammation. PMID:10500232

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

PubMed

Hou, Congzhe; Ding, Lingcui; Zhang, Jian; Jin, Yecheng; Sun, Chen; Li, Zhenzu; Sun, Xiaoyang; Zhang, Tingting; Zhang, Aizhen; Li, Huashun; Gao, Jiangang

2014-11-01

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs. Copyright © 2014. Published by Elsevier Inc.

CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor*

PubMed Central

Matsushita, Yuki; Sakamoto, Kei; Tamamura, Yoshihiro; Shibata, Yasuaki; Minamizato, Tokutaro; Kihara, Tasuku; Ito, Masako; Katsube, Ken-ichi; Hiraoka, Shuichi; Koseki, Haruhiko; Harada, Kiyoshi; Yamaguchi, Akira

2013-01-01

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy. PMID:23653360

Thrombospondin-1 deficiency causes a shift from fibroproliferative to inflammatory kidney disease and delays onset of renal failure.

PubMed

Zeisberg, Michael; Tampe, Björn; LeBleu, Valerie; Tampe, Desiree; Zeisberg, Elisabeth M; Kalluri, Raghu

2014-10-01

Thrombospondin-1 (TSP1) is a multifunctional matricellular protein known to promote progression of chronic kidney disease. To gain insight into the underlying mechanisms through which TSP1 accelerates chronic kidney disease, we compared disease progression in Col4a3 knockout (KO) mice, which develop spontaneous kidney failure, with that of Col4a3;Tsp1 double-knockout (DKO) mice. Decline of excretory renal function was significantly delayed in the absence of TSP1. Although Col4a3;Tsp1 DKO mice did progress toward end-stage renal failure, their kidneys exhibited distinct histopathological lesions, compared with creatinine level-matched Col4a3 KO mice. Although kidneys of both Col4a3 KO and Col4a3;Tsp1 DKO mice exhibited a widened tubulointerstitium, predominant lesions in Col4a3 KO kidneys were collagen deposition and fibroblast accumulation, whereas in Col4a3;Tsp1 DKO kidney inflammation was predominant, with less collagen deposition. Altered disease progression correlated with impaired activation of transforming growth factor-β1 (TGF-β1) in vivo and in vitro in the absence of TSP1. In summary, our findings suggest that TSP1 contributes to progression of chronic kidney disease by catalyzing activation of latent TGF-β1, resulting in promotion of a fibroproliferative response over an inflammatory response. Furthermore, the findings suggest that fibroproliferative and inflammatory lesions are independent entities, both of which contribute to decline of renal function. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

CRISPR-Mediated Triple Knockout of SLAMF1, SLAMF5 and SLAMF6 Supports Positive Signaling Roles in NKT Cell Development.

PubMed

Huang, Bonnie; Gomez-Rodriguez, Julio; Preite, Silvia; Garrett, Lisa J; Harper, Ursula L; Schwartzberg, Pamela L

2016-01-01

The SLAM family receptors contribute to diverse aspects of lymphocyte biology and signal via the small adaptor molecule SAP. Mutations affecting SAP lead to X-linked lymphoproliferative syndrome Type 1, a severe immunodysregulation characterized by fulminant mononucleosis, dysgammaglobulinemia, and lymphoproliferation/lymphomas. Patients and mice having mutations affecting SAP also lack germinal centers due to a defect in T:B cell interactions and are devoid of invariant NKT (iNKT) cells. However, which and how SLAM family members contribute to these phenotypes remains uncertain. Three SLAM family members: SLAMF1, SLAMF5 and SLAMF6, are highly expressed on T follicular helper cells and germinal center B cells. SLAMF1 and SLAMF6 are also implicated in iNKT development. Although individual receptor knockout mice have limited iNKT and germinal center phenotypes compared to SAP knockout mice, the generation of multi-receptor knockout mice has been challenging, due to the genomic linkage of the genes encoding SLAM family members. Here, we used Cas9/CRISPR-based mutagenesis to generate mutations simultaneously in Slamf1, Slamf5 and Slamf6. Genetic disruption of all three receptors in triple-knockout mice (TKO) did not grossly affect conventional T or B cell development and led to mild defects in germinal center formation post-immunization. However, the TKO worsened defects in iNKT cells development seen in SLAMF6 single gene-targeted mice, supporting data on positive signaling and potential redundancy between these receptors.

Generation of ER{alpha}-floxed and knockout mice using the Cre/LoxP system

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

Antonson, P., E-mail: per.antonson@ki.se; Omoto, Y.; Humire, P.

2012-08-10

Highlights: Black-Right-Pointing-Pointer ER{alpha} floxed and knockout mice were generated. Black-Right-Pointing-Pointer Disruption of the ER{alpha} gene results in sterility in both male and female mice. Black-Right-Pointing-Pointer ER{alpha}{sup -/-} mice have ovaries with hemorrhagic follicles and hypoplastic uterus. Black-Right-Pointing-Pointer Female ER{alpha}{sup -/-} mice develop obesity. -- Abstract: Estrogen receptor alpha (ER{alpha}) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ER{alpha} mouse line that can be used to knock out ER{alpha} in selected tissues by using the Cre/LoxP system. In this study, we established amore » new ER{alpha} knockout mouse line by crossing the floxed ER{alpha} mice with Cre deleter mice. Here we show that genetic disruption of the ER{alpha} gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ER{alpha} is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.« less

The phosphatase JKAP/DUSP22 inhibits T-cell receptor signalling and autoimmunity by inactivating Lck.

PubMed

Li, Ju-Pi; Yang, Chia-Yu; Chuang, Huai-Chia; Lan, Joung-Liang; Chen, Der-Yuan; Chen, Yi-Ming; Wang, Xiaohong; Chen, Alice J; Belmont, John W; Tan, Tse-Hua

2014-04-09

JNK pathway-associated phosphatase (JKAP, also known as DUSP22 or JSP-1) is a JNK activator. The in vivo role of JKAP in immune regulation remains unclear. Here we report that JKAP directly inactivates Lck by dephosphorylating tyrosine-394 residue during T-cell receptor (TCR) signalling. JKAP-knockout T cells display enhanced cell proliferation and cytokine production. JKAP-knockout mice show enhanced T-cell-mediated immune responses and are more susceptible to experimental autoimmune encephalomyelitis (EAE). In addition, the recipient mice that are adoptively transferred with JKAP-knockout T cells show exacerbated EAE symptoms. Aged JKAP-knockout mice spontaneously develop inflammation and autoimmunity. Thus, our results indicate that JKAP is an important phosphatase that inactivates Lck in the TCR signalling turn-off stage, leading to suppression of T-cell-mediated immunity and autoimmunity.

MicroRNA-590 Inhibits Lipoprotein Lipase Expression and Prevents Atherosclerosis in apoE Knockout Mice

PubMed Central

Lv, Yun-Cheng; Wang, Zong-Bao; Yao, Feng; Xie, Wei; Tan, Yu-Lin; Li, Liang; Zhang, Min; Lan, Gang; Gong, Duo; Cheng, Hai-Peng; Zhong, Hui-Juan; Liu, Dan; Huang, Chong; Li, Zhao-Xia; Zheng, Xi-Long; Yin, Wei-Dong; Tang, Chao-Ke

2015-01-01

Recent studies have suggested that miR-590 may play critical roles in cardiovascular disease. This study was designed to determine the effects of miR-590 on lipoprotein lipase (LPL) expression and development of atherosclerosis in apolipoprotein E knockout (apoE−/−) mice and explore the potential mechanisms. En face analysis of the whole aorta revealed that miR-590 significantly decreased aortic atherosclerotic plaque size and lipid content in apoE−/− mice. Double immunofluorescence staining in cross-sections of the proximal aorta showed that miR-590 agomir reduced CD68 and LPL expression in macrophages in atherosclerotic lesions. MiR-590 agomir down-regulated LPL mRNA and protein expression as analyzed by RT-qPCR and western blotting analyses, respectively. Consistently, miR-590 decreased the expression of CD36 and scavenger receptor A1 (SRA1) mRNA and protein. High-performance liquid chromatography (HPLC)analysis confirmed that treatment with miR-590 agomir reduced lipid levels either in plasma orinabdominal cavity macrophages of apoE−/− mice. ELISA analysis showed that miR-590 agomir decreased plasma levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β)and interleukin-6 (IL-6). In contrast, treatment with miR-590 antagomir prevented or reversed these effects. Taken together, these results reveal a novel mechanism of miR-590 effects, and may provide new insights into the development of strategies for attenuating lipid accumulation and pro-inflammatory cytokine secretion. PMID:26397958

Functional Redundancy of MyD88-dependent Signaling Pathways in a Murine Model of Histidyl-tRNA Synthetase-induced Myositis

PubMed Central

Fernandez, Irina; Harlow, Lisa; Zang, Yunjuan; Liu-Bryan, Ru; Ridgway, William M.; Clemens, Paula R.; Ascherman, Dana P.

2013-01-01

We have previously shown that intramuscular administration of bacterially expressed murine histidyl-tRNA synthetase (HRS) triggers florid muscle inflammation (relative to appropriate control proteins) in various congenic strains of mice. Because severe disease develops even in the absence of adaptive immune responses to HRS, we sought to identify innate immune signaling components contributing to our model of HRS-induced myositis. In vitro stimulation assays demonstrated HRS-mediated activation of HEK293 cells transfected with either TLR2 or TLR4, revealing an excitatory capacity exceeding that of other bacterially expressed fusion proteins. Corresponding to this apparent functional redundancy of TLR signaling pathways, HRS immunization of B6.TLR2−/− and B6.TLR4−/− single knockout mice yielded significant lymphocytic infiltration of muscle tissue comparable to that produced in C57BL/6 WT mice. In contrast, concomitant elimination of TLR2 and TLR4 signaling in B6.TLR2−/−.TLR4−/− double knockout mice markedly reduced the severity of HRS-induced muscle inflammation. Complementary subfragment analysis demonstrated that amino acids 60–90 of HRS were absolutely required for in vitro as well as in vivo signaling via these MyD88-dependent TLR pathways—effects mediated, in part, through preferential binding of exogenous ligands capable of activating specific TLRs. Collectively, these experiments indicate that multiple MyD88-dependent signaling cascades contribute to this model of HRS-induced myositis, underscoring the antigenic versatility of HRS and confirming the importance of innate immunity in this system. PMID:23842751

GPR39 (zinc receptor) knockout mice exhibit depression-like behavior and CREB/BDNF down-regulation in the hippocampus.

PubMed

Młyniec, Katarzyna; Budziszewska, Bogusława; Holst, Birgitte; Ostachowicz, Beata; Nowak, Gabriel

2014-10-31

Zinc may act as a neurotransmitter in the central nervous system by activation of the GPR39 metabotropic receptors. In the present study, we investigated whether GPR39 knockout would cause depressive-like and/or anxiety-like behavior, as measured by the forced swim test, tail suspension test, and light/dark test. We also investigated whether lack of GPR39 would change levels of cAMP response element-binding protein (CREB),brain-derived neurotrophic factor (BDNF) and tropomyosin related kinase B (TrkB) protein in the hippocampus and frontal cortex of GPR39 knockout mice subjected to the forced swim test, as measured by Western-blot analysis. In this study, GPR39 knockout mice showed an increased immobility time in both the forced swim test and tail suspension test, indicating depressive-like behavior and displayed anxiety-like phenotype. GPR39 knockout mice had lower CREB and BDNF levels in the hippocampus, but not in the frontal cortex, which indicates region specificity for the impaired CREB/BDNF pathway (which is important in antidepressant response) in the absence of GPR39. There were no changes in TrkB protein in either structure. In the present study, we also investigated activity in the hypothalamus-pituitary-adrenal axis under both zinc- and GPR39-deficient conditions. Zinc-deficient mice had higher serum corticosterone levels and lower glucocorticoid receptor levels in the hippocampus and frontal cortex. There were no changes in the GPR39 knockout mice in comparison with the wild-type control mice, which does not support a role of GPR39 in hypothalamus-pituitary-adrenal axis regulation. The results of this study indicate the involvement of the GPR39 Zn(2+)-sensing receptor in the pathophysiology of depression with component of anxiety. © The Author 2015. Published by Oxford University Press on behalf of CINP.

Effect of Diet High in Coconut Oil on Cardiovascular Disease Risk in ApoE Knockout and Wild Type Mice (Mus musculus)

DTIC Science & Technology

2016-04-07

Objective: We evaluated the risk of cardiovascular disease in both control and proatherosclerotic mice consuming diets high in coconut oil. Methods...The mice were weighed and randomly assigned to receive a custom diet with either coconut oil or milk fat. Both diets were formulated to have the...significant differences were seen between knockout and wildtype mice in aorta score regardless of diet, and in liver score with coconut oil diet

Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out.

PubMed

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

2012-05-01

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

Altered synaptic phospholipid signaling in PRG-1 deficient mice induces exploratory behavior and motor hyperactivity resembling psychiatric disorders.

PubMed

Schneider, Patrick; Petzold, Sandra; Sommer, Angela; Nitsch, Robert; Schwegler, Herbert; Vogt, Johannes; Roskoden, Thomas

2018-01-15

Plasticity related gene 1 (PRG-1) is a neuron specific membrane protein located at the postsynaptic density of glutamatergic synapses. PRG-1 modulates signaling pathways of phosphorylated lipid substrates such as lysophosphatidic acid (LPA). Deletion of PRG-1 increases presynaptic glutamate release probability leading to neuronal over-excitation. However, due to its cortical expression, PRG-1 deficiency leading to increased glutamatergic transmission is supposed to also affect motor pathways. We therefore analyzed the effects of PRG-1 function on exploratory and motor behavior using homozygous PRG-1 knockout (PRG-1 -/- ) mice and PRG-1/LPA 2 -receptor double knockout (PRG-1 -/- /LPA 2 -/- ) mice in two open field settings of different size and assessing motor behavior in the Rota Rod test. PRG-1 -/- mice displayed significantly longer path lengths and higher running speed in both open field conditions. In addition, PRG-1 -/- mice spent significantly longer time in the larger open field and displayed rearing and self-grooming behavior. Furthermore PRG-1 -/- mice displayed stereotypical behavior resembling phenotypes of psychiatric disorders in the smaller sized open field arena. Altogether, this behavior is similar to the stereotypical behavior observed in animal models for psychiatric disease of autistic spectrum disorders which reflects a disrupted balance between glutamatergic and GABAergic synapses. These differences indicate an altered excitation/inhibition balance in neuronal circuits in PRG-1 -/- mice as recently shown in the somatosensory cortex [38]. In contrast, PRG-1 -/- /LPA 2 -/- did not show significant changes in behavior in the open field suggesting that these specific alterations were abolished when the LPA 2 -receptor was lacking. Our findings indicate that PRG-1 deficiency led to over-excitability caused by an altered LPA/LPA 2 -R signaling inducing a behavioral phenotype typically observed in animal models for psychiatric disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of the CCR-5/MIP-1 α Axis in the Pathogenesis of Rocio Virus Encephalitis in a Mouse Model

PubMed Central

Chávez, Juliana H.; França, Rafael F. O.; Oliveira, Carlo J. F.; de Aquino, Maria T. P.; Farias, Kleber J. S.; Machado, Paula R. L.; de Oliveira, Thelma F. M.; Yokosawa, Jonny; Soares, Edson G.; da Silva, João S.; da Fonseca, Benedito A. L.; Figueiredo, Luiz T. M.

2013-01-01

Rocio virus (ROCV) caused an outbreak of human encephalitis during the 1970s in Brazil and its immunopathogenesis remains poorly understood. CC-chemokine receptor 5 (CCR5) is a chemokine receptor that binds to macrophage inflammatory protein (MIP-1 α). Both molecules are associated with inflammatory cells migration during infections. In this study, we demonstrated the importance of the CCR5 and MIP-1 α, in the outcome of viral encephalitis of ROCV-infected mice. CCR5 and MIP-1 α knockout mice survived longer than wild-type (WT) ROCV-infected animals. In addition, knockout mice had reduced inflammation in the brain. Assessment of brain viral load showed mice virus detection five days post-infection in wild-type and CCR5−/− mice, while MIP-1 α−/− mice had lower viral loads seven days post-infection. Knockout mice required a higher lethal dose than wild-type mice as well. The CCR5/MIP-1 α axis may contribute to migration of infected cells to the brain and consequently affect the pathogenesis during ROCV infection. PMID:24080631

Effect of mCOUP-TF1 deficiency on the glossopharyngeal and vagal sensory ganglia.

PubMed

Ichikawa, H; Lin, S-C; Tsai, S Y; Tsai, M-J; Sugimoto, T

2004-07-16

Immunohistochemistry for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase and calbindin D-28k was performed on the glossopharyngeal and vagal ganglia in mCOUP-TFI knockout mice to know the effect of its deficiency on different types of primary sensory neurons. In wild type and heterozygous mice, the glossopharyngeal and vagal ganglia contained abundant CGRP-, tyrosine hydroxylase- and calbindin D-28k-immunoreactive (IR) neurons. In the ganglia of mCOUP-TFI knockout mice, a 38% decrease of CGRP-IR neurons was detected. However, the number of tyrosine hydroxylase- or calbindin D-28k-neurons was not altered by the mCOUP-TFI deficiency. In the tongue of knockout mice, the number of CGRP-IR nerve fibers decreased compared to wild-type and heterozygous mice. The development of CGRP-IR petrosal neurons, which supply innervation of the tongue, may depend on mCOUP-TFI.

Blueberries reduce lipid peroxidation and boost antioxidant enzymes in apoe knockout mice

USDA-ARS?s Scientific Manuscript database

ApoE knockout (ApoE-/-) mice fed AIN-93G diet (CD) formulated to contain 1 % freeze-dried whole wild blueberries (CD1 percent BB) were found to have significantly less atherosclerotic lesions in aorta. Biomarkers of lipid peroxidation, including F2-isoprostanes, hydroxyoctadecadienoic acids (HODEs) ...

Differential action of methamphetamine on tyrosine hydroxylase and dopamine transport in the nigrostriatal pathway of μ-opioid receptor knockout mice.

PubMed

Park, Sang Won; He, Zhi; Shen, Xine; Roman, Richard J; Ma, Tangeng

2012-06-01

Extensive anatomical and functional interactions exist between central dopaminergic and opioidergic systems and both systems are proposed to be targets for amphetamine-like drugs. We have previously reported that μ-opioid receptor (μ-OR) knockout mice are resistant to the loss of dopamine in the striatum and the development of behavioral sensitization induced by repeated methamphetamine (METH) treatment. The present study assessed whether METH-treated μ-OR knockout mice exhibit a differential response of the expression of dopamine transporter and tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis and maintaining dopamine levels. Mice daily received intraperitoneal injection of METH (0, 0.6, 2.5, or 10 mg/kg) for 7 days and sacrificed on day 11 (4 days after the last injection). The expression of TH protein in the striatum and the levels of TH mRNA and number of TH positive neurons in the substantia nigra were reduced in wild-type mice treated with METH (2.5 and 10 mg/kg), but not in the μ-OR knockout mice. In contrast, METH exposure at the highest dose (10 mg/kg) reduced dopamine transporter levels in both strains of mice. These results suggest that the μ-OR contributes to METH-induced loss of dopamine and behavioral sensitization by decreasing the expression of TH.

Loss of TLR2 Worsens Spontaneous Colitis in MDR1A Deficiency through Commensally Induced Pyroptosis

PubMed Central

Ey, Birgit; Eyking, Annette; Klepak, Magdalena; Salzman, Nita H.; Göthert, Joachim R.; Rünzi, Michael; Schmid, Kurt W.; Gerken, Guido; Podolsky, Daniel K.

2013-01-01

Variants of the multidrug resistance gene (MDR1/ABCB1) have been associated with increased susceptibility to severe ulcerative colitis (UC). In this study, we investigated the role of TLR/IL-1R signaling pathways including the common adaptor MyD88 in the pathogenesis of chronic colonic inflammation in MDR1A deficiency. Double- or triple-null mice lacking TLR2, MD-2, MyD88, and MDR1A were generated in the FVB/N background. Deletion of TLR2 in MDR1A deficiency resulted in fulminant pancolitis with early expansion of CD11b+ myeloid cells and rapid shift toward TH1-dominant immune responses in the lamina propria. Colitis exacerbation in TLR2/MDR1A double-knockout mice required the unaltered commensal microbiota and the LPS coreceptor MD-2. Blockade of IL-1β activity by treatment with IL-1R antagonist (IL-1Ra; Anakinra) inhibited colitis acceleration in TLR2/MDR1A double deficiency; intestinal CD11b+Ly6C+-derived IL-1β production and inflammation entirely depended on MyD88. TLR2/MDR1A double-knockout CD11b+ myeloid cells expressed MD-2/TLR4 and hyperresponded to nonpathogenic Escherichia coli or LPS with reactive oxygen species production and caspase-1 activation, leading to excessive cell death and release of proinflammatory IL-1β, consistent with pyroptosis. Inhibition of reactive oxygen species–mediated lysosome degradation suppressed LPS hyperresponsiveness. Finally, active UC in patients carrying the TLR2-R753Q and MDR1-C3435T polymorphisms was associated with increased nuclear expression of caspase-1 protein and cell death in areas of acute inflammation, compared with active UC patients without these variants. In conclusion, we show that the combined defect of two UC susceptibility genes, MDR1A and TLR2, sets the stage for spontaneous and uncontrolled colitis progression through MD-2 and IL-1R signaling via MyD88, and we identify commensally induced pyroptosis as a potential innate immune effector in severe UC pathogenesis. PMID:23636052

Loss of TLR2 worsens spontaneous colitis in MDR1A deficiency through commensally induced pyroptosis.

PubMed

Ey, Birgit; Eyking, Annette; Klepak, Magdalena; Salzman, Nita H; Göthert, Joachim R; Rünzi, Michael; Schmid, Kurt W; Gerken, Guido; Podolsky, Daniel K; Cario, Elke

2013-06-01

Variants of the multidrug resistance gene (MDR1/ABCB1) have been associated with increased susceptibility to severe ulcerative colitis (UC). In this study, we investigated the role of TLR/IL-1R signaling pathways including the common adaptor MyD88 in the pathogenesis of chronic colonic inflammation in MDR1A deficiency. Double- or triple-null mice lacking TLR2, MD-2, MyD88, and MDR1A were generated in the FVB/N background. Deletion of TLR2 in MDR1A deficiency resulted in fulminant pancolitis with early expansion of CD11b(+) myeloid cells and rapid shift toward TH1-dominant immune responses in the lamina propria. Colitis exacerbation in TLR2/MDR1A double-knockout mice required the unaltered commensal microbiota and the LPS coreceptor MD-2. Blockade of IL-1β activity by treatment with IL-1R antagonist (IL-1Ra; Anakinra) inhibited colitis acceleration in TLR2/MDR1A double deficiency; intestinal CD11b(+)Ly6C(+)-derived IL-1β production and inflammation entirely depended on MyD88. TLR2/MDR1A double-knockout CD11b(+) myeloid cells expressed MD-2/TLR4 and hyperresponded to nonpathogenic Escherichia coli or LPS with reactive oxygen species production and caspase-1 activation, leading to excessive cell death and release of proinflammatory IL-1β, consistent with pyroptosis. Inhibition of reactive oxygen species-mediated lysosome degradation suppressed LPS hyperresponsiveness. Finally, active UC in patients carrying the TLR2-R753Q and MDR1-C3435T polymorphisms was associated with increased nuclear expression of caspase-1 protein and cell death in areas of acute inflammation, compared with active UC patients without these variants. In conclusion, we show that the combined defect of two UC susceptibility genes, MDR1A and TLR2, sets the stage for spontaneous and uncontrolled colitis progression through MD-2 and IL-1R signaling via MyD88, and we identify commensally induced pyroptosis as a potential innate immune effector in severe UC pathogenesis.

Physiological roles of A1 and A2A adenosine receptors in regulating heart rate, body temperature, and locomotion as revealed using knockout mice and caffeine.

PubMed

Yang, Jiang-Ning; Chen, Jiang-Fan; Fredholm, Bertil B

2009-04-01

Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O(2)C) were studied in awake mice lacking one or both of the adenosine A(1) or A(2A) receptors (A(1)R or A(2A)R, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A(1)R knockout (A(1)RKO) mice but lower in A(2A)RKO mice and intermediate in A(1)-A(2A)R double KO mice. A single dose of an unselective beta-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A(1)Rs had little effect on Temp, whereas deletion of A(2A)Rs increased it in females and decreased it in males. A(1)-A(2A)RKO mice had lower Temp than WT mice. LA was unaltered in A(1)RKO mice and lower in A(2A)RKO and A(1)-A(2A)RKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A(2A)R. Caffeine ingestion also increased LA in an A(2A)R-dependent manner in male mice. Caffeine ingestion significantly increased O(2)C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A(1)R or A(2A)R blockade. Selective A(2B) antagonism had little or no effect. Thus A(1)R and A(2A)R influence HR, Temp, LA, and O(2)C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A(2A)R plays an important role in the modulation of O(2)C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A(1)R and A(2A)R.

Vaccination with recombinant adenoviruses expressing Ebola virus glycoprotein elicits protection in the interferon alpha/beta receptor knock-out mouse.

PubMed

O'Brien, Lyn M; Stokes, Margaret G; Lonsdale, Stephen G; Maslowski, David R; Smither, Sophie J; Lever, Mark S; Laws, Thomas R; Perkins, Stuart D

2014-03-01

The resistance of adult immunocompetent mice to infection with ebolaviruses has led to the development of alternative small animal models that utilise immunodeficient mice, for example the interferon α/β receptor knock-out mouse (IFNR(-/-)). IFNR(-/-) mice have been shown to be susceptible to infection with ebolaviruses by multiple routes but it is not known if this murine model is suitable for testing therapeutics that rely on the generation of an immune response for efficacy. We have tested recombinant adenovirus vectors for their ability to protect IFNR(-/-) mice from challenge with Ebola virus and have analysed the humoral response generated after immunisation. The recombinant vaccines elicited good levels of protection in the knock-out mouse and the antibody response in IFNR(-/-) mice was similar to that observed in vaccinated wild-type mice. These results indicate that the IFNR(-/-) mouse is a relevant small animal model for studying ebolavirus-specific therapeutics. Copyright © 2014. Published by Elsevier Inc.

Co-expression of HLA-B7 and HLA-B27 alleles is associated with B7-restricted immunodominant responses following influenza infection.

PubMed

Akram, Ali; Inman, Robert D

2013-12-01

It is recognized that host response following viral infection is characterized by immunodominance, but deciphering the different factors contributing to immunodominance has proved a challenge due to concurrent expression of multiple MHC class I alleles. To address this, we generated H2-K(-/-)/D(-/-) double-knockout transgenic mice expressing either one or two human MHC-I alleles. We hypothesized that co-expression of different allele combinations figures critically in immunodominance and examined this in influenza-infected, double Tg MHC-I mice. In A2/B7 or A2/B27 mice, using ELISpot assays with the A2-restricted matrix I.58-66, the B7-restricted NP418-426 or the B27-restricted NP383-391 influenza A (flu) epitopes, we observed the expected recognition of both peptides for both alleles. In contrast, in flu-infected B7/B27 mice, a significantly reduced level of B27/NP383-restricted CTL response was detected while there was no change in the B7/NP418-restricted CTL response. Flu-specific tetramer studies revealed a partial deletion of Vβ8.1(+) NP383/B27-restricted CD8(+) T cells, and a diminished Vβ12(+) CD8(+) T-cell expansion in B7/B27 Tg mice. Using HLA Tg chimeric mice, we confirmed these findings. These findings shed light on the immune consequences of co-dominant expression of MHC-I alleles for host immune response to pathogens. © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Green tea polyphenol treatment attenuates atherosclerosis in high-fat diet-fed apolipoprotein E-knockout mice via alleviating dyslipidemia and up-regulating autophagy

PubMed Central

Jiang, Jinjin; Yu, Pengxin; Zhang, Guofu; Zhang, Guanghui; Liu, Xiaoting

2017-01-01

Background: Green tea polyphenol (GTP) is a polyphenol source from green tea that has drawn wide attention owing to epidemiological evidence of its beneficial effects in the prevention of cardiovascular disease; the underlying molecular mechanisms of these effects are not well understood. This study aimed to investigate the effects of GTP treatment on autophagy regulation in the vessel wall and lipid metabolism of HFD-fed male ApoE-knockout mice. Methods: Adult male ApoE-knockout mice (n = 30) fed with a high-fat diet (HFD) were treated with either vehicle or GTP (3.2 or 6.4 g/L) administered via drinking water for 15 weeks, and C57BL/6J mice fed with standard chow diet (STD) were used as the control group. Metabolic parameters, expression of key mRNAs and proteins of hepatic lipid metabolism and autophagy in the vessel wall of mice were determined after the 15-week treatment. Results: A HFD induced atherosclerosis formation and lipid metabolism disorders as well as reduced autophagy expression in the vessel wall of ApoE-knockout mice, but GTP treatment alleviated the lipid metabolism disorders, decreased the oxLDL levels in serum, and increased the mRNA and protein expressions of hepatic PPARα and autophagy markers (LC3, Beclin1 and p62) in the vessel wall of ApoE-knockout mice. Conclusions: Our findings suggest that GTP supplementation showed marked suppression of atherogenesis through improved lipid metabolism as well as through a direct impact on oxLDL and autophagy flux in the vessel wall. PMID:28777810

Impairments in the Initiation of Maternal Behavior in Oxytocin Receptor Knockout Mice

PubMed Central

Rich, Megan E.; deCárdenas, Emily J.; Lee, Heon-Jin; Caldwell, Heather K.

2014-01-01

Oxytocin (Oxt) acting through its single receptor subtype, the Oxtr, is important for the coordination of physiology and behavior associated with parturition and maternal care. Knockout mouse models have been helpful in exploring the contributions of Oxt to maternal behavior, including total body Oxt knockout (Oxt −/−) mice, forebrain conditional Oxtr knockout (Oxtr FB/FB) mice, and total body Oxtr knockout (Oxtr −/−) mice. Since Oxtr −/− mice are unable to lactate, maternal behavior has only been examined in virgin females, or in dams within a few hours of parturition, and there have been no studies that have examined their anxiety-like and depression-like behavior following parturition. To improve our understanding of how the absence of Oxt signaling affects maternal behavior, mood and anxiety, we designed a study using Oxtr −/− mice that separated nursing behavior from other aspects of maternal care, such as licking and grooming by thelectomizing (i.e. removing the nipples) of Oxtr +/+ mice and sham-thelectomizing Oxtr −/− mice, and pairing both genotypes with a wet nurse. We then measured pup abandonment, maternal behavior, and postpartum anxiety-like and depression-like behaviors. We hypothesized that genetic disruption of the Oxtr would impact maternal care, mood and anxiety. Specifically, we predicted that Oxtr −/− dams would have impaired maternal care and increased anxiety-like and depression-like behaviors in the postpartum period. We found that Oxtr −/− dams had significantly higher levels of pup abandonment compared to controls, which is consistent with previous work in Oxtr FB/FB mice. Interestingly, Oxtr −/− dams that initiated maternal care did not differ from wildtype controls in measures of maternal behavior. We also did not find any evidence of altered anxiety-like or depressive-like behavior in the postpartum period of Oxtr −/− dams. Thus, our data suggest that Oxt lowers the threshold for the initiation of maternal behavior. PMID:24892749

Reversal of disease-related pathologies in the fragile X mouse model by selective activation of GABAB receptors with arbaclofen.

PubMed

Henderson, Christina; Wijetunge, Lasani; Kinoshita, Mika Nakamoto; Shumway, Matthew; Hammond, Rebecca S; Postma, Friso R; Brynczka, Christopher; Rush, Roger; Thomas, Alexia; Paylor, Richard; Warren, Stephen T; Vanderklish, Peter W; Kind, Peter C; Carpenter, Randall L; Bear, Mark F; Healy, Aileen M

2012-09-19

Fragile X syndrome (FXS), the most common inherited cause of intellectual disability and autism, results from the transcriptional silencing of FMR1 and loss of the mRNA translational repressor protein fragile X mental retardation protein (FMRP). Patients with FXS exhibit changes in neuronal dendritic spine morphology, a pathology associated with altered synaptic function. Studies in the mouse model of fragile X have shown that loss of FMRP causes excessive synaptic protein synthesis, which results in synaptic dysfunction and altered spine morphology. We tested whether the pharmacologic activation of the γ-aminobutyric acid type B (GABA(B)) receptor could correct or reverse these phenotypes in Fmr1-knockout mice. Basal protein synthesis, which is elevated in the hippocampus of Fmr1-knockout mice, was corrected by the in vitro application of the selective GABA(B) receptor agonist STX209 (arbaclofen, R-baclofen). STX209 also reduced to wild-type values the elevated AMPA receptor internalization in Fmr1-knockout cultured neurons, a known functional consequence of increased protein synthesis. Acute administration of STX209 in vivo, at doses that modify behavior, decreased mRNA translation in the cortex of Fmr1-knockout mice. Finally, the chronic administration of STX209 in juvenile mice corrected the increased spine density in Fmr1-knockout mice without affecting spine density in wild-type mice. Thus, activation of the GABA(B) receptor with STX209 corrected synaptic abnormalities considered central to fragile X pathophysiology, a finding that suggests that STX209 may be a potentially effective therapy to treat the core symptoms of FXS.

SLAP deficiency enhances number and function of regulatory T cells preventing chronic autoimmune arthritis in SKG mice.

PubMed

Peterson, Lisa K; Shaw, Laura A; Joetham, Anthony; Sakaguchi, Shimon; Gelfand, Erwin W; Dragone, Leonard L

2011-02-15

To test if manipulating TCR complex-mediated signaling (TCR signaling) could treat autoimmune disease, we generated the double SKG Src-like adapter protein (SLAP) knockout (DSSKO) mouse model. The SKG mutation in ZAP70 and SLAP have opposing functions on the regulation of TCR signaling. The combination of these two mutations alters TCR signaling in the context of a defined genetic background, uniform environmental conditions, and a well-characterized signaling disruption. In contrast to SKG mice, DSSKO mice do not develop zymosan-induced chronic autoimmune arthritis. This arthritis prevention is not due to significant alterations in thymocyte development or repertoire selection but instead enhanced numbers of regulatory T cells (Tregs) and decreased numbers of Th17 cells skewing the ratio of Tregs to autoreactive effector T cells. Treg depletion and/or functional blockade led to the development of arthritis in DSSKO mice. In vitro suppression of effector T cell proliferation was also enhanced, demonstrating that DSSKO mice have increased numbers of Tregs with increased function. Understanding how TCR signals influence development, expansion, and function of Tregs in DSSKO mice could advance our ability to manipulate Treg biology to treat ultimately autoimmune disease.

SLAP Deficiency Enhances Number and Function of Regulatory T Cells Preventing Chronic Autoimmune Arthritis in SKG Mice

PubMed Central

Peterson, Lisa K.; Shaw, Laura A.; Joetham, Anthony; Sakaguchi, Shimon; Gelfand, Erwin W.; Dragone, Leonard L.

2011-01-01

To test if manipulating TCR complex-mediated signaling (TCR signaling) could treat autoimmune disease, we generated the double SKG Src-like adapter protein (SLAP) knockout (DSSKO) mouse model. The SKG mutation in ZAP70 and SLAP have opposing functions on the regulation of TCR signaling. The combination of these two mutations alters TCR signaling in the context of a defined genetic background, uniform environmental conditions, and a well-characterized signaling disruption. In contrast to SKG mice, DSSKO mice do not develop zymosan-induced chronic autoimmune arthritis. This arthritis prevention is not due to significant alterations in thymocyte development or repertoire selection but instead enhanced numbers of regulatory T cells (Tregs) and decreased numbers of Th17 cells skewing the ratio of Tregs to autoreactive effector T cells. Treg depletion and/or functional blockade led to the development of arthritis in DSSKO mice. In vitro suppression of effector T cell proliferation was also enhanced, demonstrating that DSSKO mice have increased numbers of Tregs with increased function. Understanding how TCR signals influence development, expansion, and function of Tregs in DSSKO mice could advance our ability to manipulate Treg biology to treat ultimately autoimmune disease. PMID:21248251

Glucagon Receptor Knockout Prevents Insulin-Deficient Type 1 Diabetes in Mice

PubMed Central

Lee, Young; Wang, May-Yun; Du, Xiu Quan; Charron, Maureen J.; Unger, Roger H.

2011-01-01

OBJECTIVE To determine the role of glucagon action in the metabolic phenotype of untreated insulin deficiency. RESEARCH DESIGN AND METHODS We compared pertinent clinical and metabolic parameters in glucagon receptor-null (Gcgr−/−) mice and wild-type (Gcgr+/+) controls after equivalent destruction of β-cells. We used a double dose of streptozotocin to maximize β-cell destruction. RESULTS Gcgr+/+ mice became hyperglycemic (>500 mg/dL), hyperketonemic, polyuric, and cachectic and had to be killed after 6 weeks. Despite comparable β-cell destruction in Gcgr−/− mice, none of the foregoing clinical or laboratory manifestations of diabetes appeared. There was marked α-cell hyperplasia and hyperglucagonemia (∼1,200 pg/mL), but hepatic phosphorylated cAMP response element binding protein and phosphoenolpyruvate carboxykinase mRNA were profoundly reduced compared with Gcgr+/+ mice with diabetes—evidence that glucagon action had been effectively blocked. Fasting glucose levels and oral and intraperitoneal glucose tolerance tests were normal. Both fasting and nonfasting free fatty acid levels and nonfasting β-hydroxy butyrate levels were lower. CONCLUSIONS We conclude that blocking glucagon action prevents the deadly metabolic and clinical derangements of type 1 diabetic mice. PMID:21270251

Mice lacking MKP-1 and MKP-5 Reveal Hierarchical Regulation of Regenerative Myogenesis.

PubMed

Shi, Hao; Gatzke, Florian; Molle, Julia M; Lee, Han Bin; Helm, Emma T; Oldham, Jessie J; Zhang, Lei; Gerrard, David E; Bennett, Anton M

2015-11-12

The relative contribution of the MAP kinase phosphatases (MKPs) in the integration of MAP kinase-dependent signaling during regenerative myogenesis has yet to be fully investigated. MKP-1 and MKP-5 maintain skeletal muscle homeostasis by providing positive and negative effects on regenerative myogenesis, respectively. In order to define the hierarchical contributions of MKP-1 and MKP-5 in the regulation of regenerative myogenesis we genetically ablated both MKPs in mice. MKP-1/MKP 5-deficient double-knockout (MKP1/5- DKO) mice were viable, and upon skeletal muscle injury, were severely impaired in their capacity to regenerate skeletal muscle. Satellite cells were fewer in number in MKP1/5-DKO mice and displayed a reduced proliferative capacity as compared with those derived from wild-type mice. MKP1/5-DKO mice exhibited increased inflammation and the macrophage M1 to M2 transition during the resolution of inflammation was impaired following injury. These results demonstrate that the actions of MKP-1 to positively regulate myogenesis predominate over those of MKP-5, which negatively regulates myogenesis. Hence, MKP-1 and MKP-5 function to maintain skeletal muscle homeostasis through non-overlapping and opposing signaling pathways.

Mice lacking MKP-1 and MKP-5 Reveal Hierarchical Regulation of Regenerative Myogenesis

PubMed Central

Shi, Hao; Gatzke, Florian; Molle, Julia M.; Lee, Han Bin; Helm, Emma T.; Oldham, Jessie J.; Zhang, Lei; Gerrard, David E.; Bennett, Anton M.

2015-01-01

The relative contribution of the MAP kinase phosphatases (MKPs) in the integration of MAP kinase-dependent signaling during regenerative myogenesis has yet to be fully investigated. MKP-1 and MKP-5 maintain skeletal muscle homeostasis by providing positive and negative effects on regenerative myogenesis, respectively. In order to define the hierarchical contributions of MKP-1 and MKP-5 in the regulation of regenerative myogenesis we genetically ablated both MKPs in mice. MKP-1/MKP 5-deficient double-knockout (MKP1/5- DKO) mice were viable, and upon skeletal muscle injury, were severely impaired in their capacity to regenerate skeletal muscle. Satellite cells were fewer in number in MKP1/5-DKO mice and displayed a reduced proliferative capacity as compared with those derived from wild-type mice. MKP1/5-DKO mice exhibited increased inflammation and the macrophage M1 to M2 transition during the resolution of inflammation was impaired following injury. These results demonstrate that the actions of MKP-1 to positively regulate myogenesis predominate over those of MKP-5, which negatively regulates myogenesis. Hence, MKP-1 and MKP-5 function to maintain skeletal muscle homeostasis through non-overlapping and opposing signaling pathways. PMID:27064463

Urea transporter knockout mice and their renal phenotypes.

PubMed

Fenton, Robert A; Yang, Baoxue

2014-01-01

Urea transporter gene knockout mice have been created for the study of the urine-concentrating mechanism. The major findings in studies of the renal phenotype of these mice are as follows: (1) Urea accumulation in the inner medullary interstitium is dependent on intrarenal urea recycling mediated by urea transporters; (2) urea transporters are essential for preventing urea-induced osmotic diuresis and thus for water conservation; (3) NaCl concentration in the inner medullary interstitium is not significantly affected by the absence of IMCD, descending limb of Henle and descending vasa recta urea transporters. Studies in urea transporter knockout mouse models have highlighted the essential role of urea for producing maximally concentrated urine.

Angiopoietin-1 deficiency increases tumor metastasis in mice.

PubMed

Michael, Iacovos P; Orebrand, Martina; Lima, Marta; Pereira, Beatriz; Volpert, Olga; Quaggin, Susan E; Jeansson, Marie

2017-08-11

Angipoietin-1 activation of the tyrosine kinase receptor Tek expressed mainly on endothelial cells leads to survival and stabilization of endothelial cells. Studies have shown that Angiopoietin-1 counteracts permeability induced by a number of stimuli. Here, we test the hypothesis that loss of Angiopoietin-1/Tek signaling in the vasculature would increase metastasis. Angiopoietin-1 was deleted in mice just before birth using floxed Angiopoietin-1 and Tek mice crossed to doxycycline-inducible bitransgenic ROSA-rtTA/tetO-Cre mice. By crossing Angiopoietin-1 knockout mice to the MMTV-PyMT autochthonous mouse breast cancer model, we investigated primary tumor growth and metastasis to the lung. Furthermore, we utilized B16F10 melanoma cells subcutaneous and experimental lung metastasis models in Angiopoietin-1 and Tek knockout mice. We found that primary tumor growth in MMTV-PyMT mice was unaffected, while metastasis to the lung was significantly increased in Angiopoietin-1 knockout MMTV-PyMT mice. In addition, angiopoietin-1 deficient mice exhibited a significant increase in lung metastasis of B16F10 melanoma cells, compared to wild type mice 3 weeks after injection. Additional experiments showed that this was likely an early event due to increased attachment or extravasation of tumor cells, since seeding of tumor cells was significantly increased 4 and 24 h post tail vein injection. Finally, using inducible Tek knockout mice, we showed a significant increase in tumor cell seeding to the lung, suggesting that Angiopoietin-1/Tek signaling is important for vascular integrity to limit metastasis. This study show that loss of the Angiopoietin-1/Tek vascular growth factor system leads to increased metastasis without affecting primary tumor growth.

Effects of long- and short-term darbepoetin-α treatment on oxidative stress, inflammation and endothelial injury in ApoE knockout mice.

PubMed

Özdemir, Evrim Dursun; Hanikoglu, Aysegul; Cort, Aysegul; Ozben, Beste; Suleymanlar, Gultekin; Ozben, Tomris

2017-07-01

Atherosclerosis and atherosclerosis-related complications are the main cause of death in the world. Vascular injury in response to inflammation and enhanced oxidant stress promotes endothelial dysfunction and leads to atherosclerotic lesions. Low-dose treatment with darbepoetin-α may be a potential therapeutic tool for endothelial injury and atherosclerosis. In order to study the effect of darbepoetin-α on endothelial injury and atherosclerosis, we used ApoE-/- mice as the atherosclerotic mice model. We monitored atherosclerosis and plaque formation histochemically in ApoE knockout mice at early and late stages of atherosclerosis. Darbepoetin-α was injected intraperitoneally at a dose of 0.1 μg/kg to ApoE-/- mice. The results of 2 ApoE-/- mice groups injected with darbepoetin-α (early and late stages of atherosclerosis) were compared to the results of the corresponding saline injected ApoE-/- mice groups and the control (C57BL/6) mice. Lipid profile (total cholesterol, triglyceride), inflammation (CRP, IL-6, histamine), endothelial injury (ICAM-1, selectin) and oxidative stress markers (lipid peroxidation, protein oxidation) were significantly increased in 4 atherosclerotic groups compared to the control group. Short-term darbepoetin-α had no marked effects on indicators of inflammation and endothelial injury in the ApoE knockout mice groups compared to the ApoE knockout mice not treated with darbepoetin-α, however, darbepoetin-α significantly decreased 8-isoprostane and protein carbonyl content. Long term darbepoetin-α treatment reduced oxidative stress in ApoE-/- mice. This study contributes to understanding and elucidating the biochemical changes occurring during early and late stages of atherosclerosis development regarding lipid profile, inflammation, endothelial injury and oxidative stress markers.

Olfactory discrimination deficits in mice lacking the dopamine transporter or the D2 dopamine receptor.

PubMed

Tillerson, Jennifer L; Caudle, W Michael; Parent, Jack M; Gong, C; Schallert, Timothy; Miller, Gary W

2006-09-15

Previous pharmacological studies have implicated dopamine as a modulator of olfactory bulb processing. Several disorders characterized by altered dopamine homeostasis in olfaction-related brain regions display olfactory deficits. To further characterize the role of dopamine in olfactory processing, we subjected dopamine transporter knockout mice (DAT -/-) and dopamine receptor 2 knockout mice (D2 -/-) to a battery of olfactory tests. In addition to behavioral characterization, several neurochemical markers of olfactory bulb integrity and function were examined. DAT -/- mice displayed an olfactory discrimination deficit, but did not differ detectably from DAT wildtype (DAT +/+) mice in odor habituation, olfactory sensitivity, or odor recognition memory. Neurochemically, DAT -/- mice have decreased D2 receptor staining in the periglomerular layer of the olfactory bulb and increased tyrosine hydroxylase immunoreactivity compared to DAT +/+ controls. D2 -/- mice exhibited the same olfactory deficit as the DAT -/- mice, further supporting the role of dopamine at the D2 synapse in olfactory discrimination processing. The findings presented in this paper reinforce the functional significance of dopamine and more specifically the D2 receptor in olfactory discrimination and may help explain the behavioral phenotype in the DAT and D2 knockout mice.

Neutral endopeptidase knockout induces hyperalgesia in a model of visceral pain, an effect related to bradykinin and nitric oxide.

PubMed

Fischer, Hanspeter S; Zernig, Gerald; Hauser, Kurt F; Gerard, Craig; Hersh, Louis B; Saria, Alois

2002-01-01

Neutral endopeptidase (EC3.4.24.11, NEP, enkephalinase) is a zinc-metalloendopeptidase, cleaving a variety of substrates like enkephalins, substance P, and bradykinin. In the brain, NEP is a key enzyme in the degradation of enkephalins. Pharmacological inhibition of NEP-activity causes analgesia resulting from enhanced extracellular enkephalin concentrations. Recently, transgenic mice lacking the enzyme NEP have been developed (Lu, 1995). The present study was designed to investigate the nociceptive behavior of these NEP-knockout mice. Interestingly, NEP-deficient mice did not respond with decreased pain perception, but exhibited hyperalgesia in the hot-plate jump, warm-water tail-withdrawal, and mostnotablyin theacetic-acid writhing test. Inhibition of aminopeptidase N by bestatin reduced writhing in both strains, whereas NEP-inhibition by thiorphan reduced writhing selectively in wild-type mice. Naloxone increased writhing in wild-type but not in knockouts, whereas the bradykinin B2-receptor antagonist HOE140 reduced writhing selectively in NEP-knockouts. Similarly, the nitric oxide synthase inhibitor L-NAME reduced writhing in NEP-knockouts. These results indicate that genetic elimination of NEP, in contrast to pharmacological inhibition, leads to bradykinin-induced hyperalgesia instead of enkephalin-mediated analgesia. Nitric oxide (NO) is suggested to be involved in this process.

Intimal cushions and endothelial nuclear elongation around mouse aortic branches and their spatial correspondence with patterns of lipid deposition

PubMed Central

Bond, Andrew R.; Ni, Chih-Wen; Jo, Hanjoong

2010-01-01

Spatial variation in hemodynamic stresses acting on the arterial wall may explain the nonuniform distribution of atherosclerosis. In thoracic aortas of LDL receptor/apolipoprotein E double knockout mice, lesions develop preferentially around the entire circumference of intercostal branch ostia, regardless of age, with the highest prevalence occurring upstream. Additional chevron-shaped lesions occur further upstream of the ostia. This pattern differs from the age-related ones occurring in people and rabbits. In the present study, patterns of near-wall blood flow around intercostal ostia in wild-type mice were estimated from the morphology of endothelial nuclei, which were shown in vitro to elongate in response to elevated shear stress and to align with the flow, and wall structure was assessed from confocal and scanning electron microscopy. A triangular intimal cushion surrounded the upstream part of most ostia. Nuclear length-to-width ratios were lowest over this cushion and highest at the sides of branches, regardless of age. Nuclear orientations were consistent with flow diverging around the branch. The pattern of nuclear morphology differed from the age-related ones observed in rabbits. The intimal cushion and the distribution of shear stress inferred from these observations can partly account for the pattern of lesions observed in knockout mice. Nuclear elongation in nonbranch regions was approximately constant across animals of different size, demonstrating the existence of a mechanism by which endothelial cells compensate for the dependence of mean aortic wall shear stress on body mass. PMID:19933414

Activation of PPARγ Ameliorates Spatial Cognitive Deficits through Restoring Expression of AMPA Receptors in Seipin Knock-Out Mice.

PubMed

Zhou, Libin; Chen, Tingting; Li, Guoxi; Wu, Chaoming; Wang, Conghui; Li, Lin; Sha, Sha; Chen, Lei; Liu, George; Chen, Ling

2016-01-27

A characteristic phenotype of congenital generalized lipodystrophy 2 (CGL2) that is caused by loss-of-function of seipin gene is mental retardation. Here, we show that seipin deficiency in hippocampal CA1 pyramidal cells caused the reduction of peroxisome proliferator-activated receptor gamma (PPARγ). Twelve-week-old systemic seipin knock-out mice and neuronal seipin knock-out (seipin-nKO) mice, but not adipose seipin knock-out mice, exhibited spatial cognitive deficits as assessed by the Morris water maze and Y-maze, which were ameliorated by the treatment with the PPARγ agonist rosiglitazone (rosi). In addition, seipin-nKO mice showed the synaptic dysfunction and the impairment of NMDA receptor-dependent LTP in hippocampal CA1 regions. The density of AMPA-induced current (IAMPA) in CA1 pyramidal cells and GluR1/GluR2 expression were significantly reduced in seipin-nKO mice, whereas the NMDA-induced current (INMDA) and NR1/NR2 expression were not altered. Rosi treatment in seipin-nKO mice could correct the decrease in expression and activity of AMPA receptor (AMPAR) and was accompanied by recovered synaptic function and LTP induction. Furthermore, hippocampal ERK2 and CREB phosphorylation in seipin-nKO mice were reduced and this could be rescued by rosi treatment. Rosi treatment in seipin-nKO mice elevated BDNF concentration. The MEK inhibitor U0126 blocked rosi-restored AMPAR expression and LTP induction in seipin-nKO mice, but the Trk family inhibitor K252a did not. These findings indicate that the neuronal seipin deficiency selectively suppresses AMPAR expression through reducing ERK-CREB activities, leading to the impairment of LTP and spatial memory, which can be rescued by PPARγ activation. Congenital generalized lipodystrophy 2 (CGL2), caused by loss-of-function mutation of seipin gene, is characterized by mental retardation. By the generation of systemic or neuronal seipin knock-out mice, the present study provides in vivo evidence that neuronal seipin deficiency causes deficits in spatial memory and hippocampal LTP induction. Neuronal seipin deficiency selectively suppresses AMPA receptor expression, ERK-CREB phosphorylation with the decline of PPARγ. The PPARγ agonist rosiglitazone can ameliorate spatial cognitive deficits and rescue the LTP induction in seipin knock-out mice by restoring AMPA receptor expression and ERK-CREB activities. Copyright © 2016 the authors 0270-6474/16/361242-12$15.00/0.

Lipid-lowering effects of anti-angiopoietin-like 4 antibody recapitulate the lipid phenotype found in angiopoietin-like 4 knockout mice

PubMed Central

Desai, Urvi; Lee, E-Chiang; Chung, Kyu; Gao, Cuihua; Gay, Jason; Key, Billie; Hansen, Gwenn; Machajewski, Dennis; Platt, Kenneth A.; Sands, Arthur T.; Schneider, Matthias; Van Sligtenhorst, Isaac; Suwanichkul, Adisak; Vogel, Peter; Wilganowski, Nat; Wingert, June; Zambrowicz, Brian P.; Landes, Greg; Powell, David R.

2007-01-01

We used gene knockout mice to explore the role of Angiopoietin-like-4 (Angptl4) in lipid metabolism as well as to generate anti-Angptl4 mAbs with pharmacological activity. Angptl4 −/− mice had lower triglyceride (TG) levels resulting both from increased very low-density lipoprotein (VLDL) clearance and decreased VLDL production and had modestly lower cholesterol levels. Also, both Angptl4 −/− suckling mice and adult mice fed a high-fat diet showed reduced viability associated with lipogranulomatous lesions of the intestines and their draining lymphatics and mesenteric lymph nodes. Treating C57BL/6J, ApoE −/−, LDLr −/−, and db/db mice with the anti-Angptl4 mAb 14D12 recapitulated the lipid and histopathologic phenotypes noted in Angptl4 −/− mice. This demonstrates that the knockout phenotype reflects not only the physiologic function of the Angptl4 gene but also predicts the pharmacologic consequences of Angptl4 protein inhibition with a neutralizing antibody in relevant models of human disease. PMID:17609370

The role of system Xc- in methamphetamine-induced dopaminergic neurotoxicity in mice.

PubMed

Dang, Duy-Khanh; Shin, Eun-Joo; Tran, Hai-Quyen; Kim, Dae-Joong; Jeong, Ji Hoon; Jang, Choon-Gon; Nah, Seung-Yeol; Sato, Hideyo; Nabeshima, Toshitaka; Yoneda, Yukio; Kim, Hyoung-Chun

2017-09-01

The cystine/glutamate antiporter (system Xc - , Sxc) transports cystine into cell in exchange for glutamate. Since xCT is a specific subunit of Sxc, we employed xCT knockout mice and investigated whether this antiporter affected methamphetamine (MA)-induced dopaminergic neurotoxicity. MA treatment significantly increased striatal oxidative burdens in wild type mice. xCT inhibitor [i.e., S-4-carboxy-phenylglycine (CPG), sulfasalazine] or an xCT knockout significantly protected against these oxidative burdens. MA-induced increases in Iba-1 expression and Iba-1-labeled microglial immunoreactivity (Iba-1-IR) were significantly attenuated by CPG or sulfasalazine administration or xCT knockout. CPG or sulfasalazine significantly attenuated MA-induced TUNEL-positive cell populations in the striatum of Taconic ICR mice. The decrease in excitatory amino acid transporter-2 (or glutamate transporter-1) expression and increase in glutamate release were attenuated by CPG, sulfasalazine or xCT knockout. In addition, CPG, sulfasalazine or xCT knockout significantly protected against dopaminergic loss (i.e., decreases in tyrosine hydroxylase expression and immunoreactivity, and an increase in dopamine turnover rate) induced by MA. However, CPG, sulfasalazine or xCT knockout did not significantly affect the impaired glutathione system [i.e., decrease in reduced glutathione (GSH) and increase in oxidized glutathione (GSSG)] induced by MA. Our results suggest that Sxc mediates MA-induced neurotoxicity via facilitating oxidative stress, microgliosis, proapoptosis, and glutamate-related toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in mice.

PubMed

Schlüter, O M; Fornai, F; Alessandrí, M G; Takamori, S; Geppert, M; Jahn, R; Südhof, T C

2003-01-01

In humans, mutations in the alpha-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson's disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. alpha-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves alpha-synuclein, we generated alpha-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting alpha-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the alpha-synuclein gene. As described previously, deletion of alpha-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, alpha-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the alpha-synuclein gene exhibited no protection. Furthermore, alpha-synuclein knockout mice but not the mice with the alpha-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that alpha-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.

Transcription factor YY1 can control AID-mediated mutagenesis in mice.

PubMed

Zaprazna, Kristina; Basu, Arindam; Tom, Nikola; Jha, Vibha; Hodawadekar, Suchita; Radova, Lenka; Malcikova, Jitka; Tichy, Boris; Pospisilova, Sarka; Atchison, Michael L

2018-02-01

Activation-induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off-target non-Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock-out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID-mediated mutations. Using mice that overexpress AID (IgκAID yy1 f/f ) or that express normal AID levels (yy1 f/f ), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the Sμ switch region, and reduced AID-mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1-AID physical interaction which blocks AID ubiquitination. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

SPERM MOTILITY IN HSF1 KNOCKOUT MICE AFTER HEAT SHOCK IS ASSOCIATED WITH FERTILITY DEFICITS

EPA Science Inventory

SPERM MOTILITY IN HSF1 KNOCKOUT MICE AFTER HEAT SHOCK IS ASSOCIATED WITH FERTILITY DEFICITS. L.F. Strader*, S.D. Perreault, J.C. Luft*, and D.J. Dix*. US EPA/ORD, Reproductive Toxicology Div., Research Triangle Park, NC
Heat shock proteins (HSPs) protect cells from environm...

RETINOIC ACID INDUCTION OF CLEFT PALATE IN EGF AND TGF-ALPHA KNOCKOUT MICE: STAGE SPECIFIC INFLUENCES OF GROWTH FACTOR EXPRESSION

EPA Science Inventory

ABBOTT, B. D., LEFFLER, K.E. AND BUCKALEW, A.R, Reproductive Toxicology Division, NHEERL, ORD, US EPA, Research Triangle Park, North Carolina. Retinoic acid induction of cleft palate (CP) in EGF and TGF knockout mice: Stage specific influences of growth factor expression.
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Role of fructose and fructokinase in acute dehydration-induced vasopressin gene expression and secretion in mice

PubMed Central

Roncal-Jimenez, Carlos A.; Lanaspa-Garcia, Miguel A.; Oppelt, Sarah A.; Kuwabara, Masanari; Jensen, Thomas; Milagres, Tamara; Andres-Hernando, Ana; Ishimoto, Takuji; Garcia, Gabriela E.; Johnson, Ginger; MacLean, Paul S.; Sanchez-Lozada, Laura-Gabriela; Tolan, Dean R.; Johnson, Richard J.

2016-01-01

Fructose stimulates vasopressin in humans and can be generated endogenously by activation of the polyol pathway with hyperosmolarity. We hypothesized that fructose metabolism in the hypothalamus might partly control vasopressin responses after acute dehydration. Wild-type and fructokinase-knockout mice were deprived of water for 24 h. The supraoptic nucleus was evaluated for vasopressin and markers of the aldose reductase-fructokinase pathway. The posterior pituitary vasopressin and serum copeptin levels were examined. Hypothalamic explants were evaluated for vasopressin secretion in response to exogenous fructose. Water restriction increased serum and urine osmolality and serum copeptin in both groups of mice, although the increase in copeptin in wild-type mice was larger than that in fructokinase-knockout mice. Water-restricted, wild-type mice showed an increase in vasopressin and aldose reductase mRNA, sorbitol, fructose and uric acid in the supraoptic nucleus. In contrast, fructokinase-knockout mice showed no change in vasopressin or aldose reductase mRNA, and no changes in sorbitol or uric acid, although fructose levels increased. With water restriction, vasopressin in the pituitary of wild-type mice was significantly less than that of fructokinase-knockout mice, indicating that fructokinase-driven vasopressin secretion overrode synthesis. Fructose increased vasopressin release in hypothalamic explants that was not observed in fructokinase-knockout mice. In situ hybridization documented fructokinase mRNA in the supraoptic nucleus, paraventricular nucleus and suprachiasmatic nucleus. Acute dehydration activates the aldose reductase-fructokinase pathway in the hypothalamus and partly drives the vasopressin response. Exogenous fructose increases vasopressin release in hypothalamic explants dependent on fructokinase. Nevertheless, circulating vasopressin is maintained and urinary concentrating is not impaired. NEW & NOTEWORTHY This study increases our understanding of the mechanisms leading to vasopressin release under conditions of water restriction (acute dehydration). Specifically, these studies suggest that the aldose reductase-fructokinase pathways may be involved in vasopressin synthesis in the hypothalamus and secretion by the pituitary in response to acute dehydration. Nevertheless, mice undergoing water restriction remain capable of maintaining sufficient vasopressin (copeptin) levels to allow normal urinary concentration. Further studies of the aldose reductase-fructokinase system in vasopressin regulation appear indicated. PMID:27852737

Role of fructose and fructokinase in acute dehydration-induced vasopressin gene expression and secretion in mice.

PubMed

Song 宋志林, Zhilin; Roncal-Jimenez, Carlos A; Lanaspa-Garcia, Miguel A; Oppelt, Sarah A; Kuwabara, Masanari; Jensen, Thomas; Milagres, Tamara; Andres-Hernando, Ana; Ishimoto, Takuji; Garcia, Gabriela E; Johnson, Ginger; MacLean, Paul S; Sanchez-Lozada, Laura-Gabriela; Tolan, Dean R; Johnson, Richard J

2017-02-01

Fructose stimulates vasopressin in humans and can be generated endogenously by activation of the polyol pathway with hyperosmolarity. We hypothesized that fructose metabolism in the hypothalamus might partly control vasopressin responses after acute dehydration. Wild-type and fructokinase-knockout mice were deprived of water for 24 h. The supraoptic nucleus was evaluated for vasopressin and markers of the aldose reductase-fructokinase pathway. The posterior pituitary vasopressin and serum copeptin levels were examined. Hypothalamic explants were evaluated for vasopressin secretion in response to exogenous fructose. Water restriction increased serum and urine osmolality and serum copeptin in both groups of mice, although the increase in copeptin in wild-type mice was larger than that in fructokinase-knockout mice. Water-restricted, wild-type mice showed an increase in vasopressin and aldose reductase mRNA, sorbitol, fructose and uric acid in the supraoptic nucleus. In contrast, fructokinase-knockout mice showed no change in vasopressin or aldose reductase mRNA, and no changes in sorbitol or uric acid, although fructose levels increased. With water restriction, vasopressin in the pituitary of wild-type mice was significantly less than that of fructokinase-knockout mice, indicating that fructokinase-driven vasopressin secretion overrode synthesis. Fructose increased vasopressin release in hypothalamic explants that was not observed in fructokinase-knockout mice. In situ hybridization documented fructokinase mRNA in the supraoptic nucleus, paraventricular nucleus and suprachiasmatic nucleus. Acute dehydration activates the aldose reductase-fructokinase pathway in the hypothalamus and partly drives the vasopressin response. Exogenous fructose increases vasopressin release in hypothalamic explants dependent on fructokinase. Nevertheless, circulating vasopressin is maintained and urinary concentrating is not impaired. This study increases our understanding of the mechanisms leading to vasopressin release under conditions of water restriction (acute dehydration). Specifically, these studies suggest that the aldose reductase-fructokinase pathways may be involved in vasopressin synthesis in the hypothalamus and secretion by the pituitary in response to acute dehydration. Nevertheless, mice undergoing water restriction remain capable of maintaining sufficient vasopressin (copeptin) levels to allow normal urinary concentration. Further studies of the aldose reductase-fructokinase system in vasopressin regulation appear indicated. Copyright © 2017 the American Physiological Society.

Genetic evidence that thyroid hormone is indispensable for prepubertal insulin-like growth factor-I expression and bone acquisition in mice.

PubMed

Xing, Weirong; Govoni, Kristen E; Donahue, Leah Rae; Kesavan, Chandrasekhar; Wergedal, Jon; Long, Carlin; Bassett, J H Duncan; Gogakos, Apostolos; Wojcicka, Anna; Williams, Graham R; Mohan, Subburaman

2012-05-01

Understanding how bone growth is regulated by hormonal and mechanical factors during early growth periods is important for optimizing the attainment of peak bone mass to prevent or postpone the occurrence of fragility fractures later in life. Using genetic mouse models that are deficient in thyroid hormone (TH) (Tshr(-/-) and Duox2(-/-)), growth hormone (GH) (Ghrhr(lit/lit)), or both (Tshr(-/-); Ghrhr(lit/lit)), we demonstrate that there is an important period prior to puberty when the effects of GH are surprisingly small and TH plays a critical role in the regulation of skeletal growth. Daily administration of T3/T4 during days 5 to 14, the time when serum levels of T3 increase rapidly in mice, rescued the skeletal deficit in TH-deficient mice but not in mice lacking both TH and GH. However, treatment of double-mutant mice with both GH and T3/T4 rescued the bone density deficit. Increased body fat in the TH-deficient as well as TH/GH double-mutant mice was rescued by T3/T4 treatment during days 5 to 14. In vitro studies in osteoblasts revealed that T3 in the presence of TH receptor (TR) α1 bound to a TH response element in intron 1 of the IGF-I gene to stimulate transcription. In vivo studies using TRα and TRβ knockout mice revealed evidence for differential regulation of insulin-like growth factor (IGF)-I expression by the two receptors. Furthermore, blockade of IGF-I action partially inhibited the biological effects of TH, thus suggesting that both IGF-I-dependent and IGF-I-independent mechanisms contribute to TH effects on prepubertal bone acquisition. Copyright © 2012 American Society for Bone and Mineral Research.

Abrogated Freud-1/Cc2d1a Repression of 5-HT1A Autoreceptors Induces Fluoxetine-Resistant Anxiety/Depression-Like Behavior.

PubMed

Vahid-Ansari, Faranak; Daigle, Mireille; Manzini, M Chiara; Tanaka, Kenji F; Hen, René; Geddes, Sean D; Béïque, Jean-Claude; James, Jonathan; Merali, Zul; Albert, Paul R

2017-12-06

Freud-1/Cc2d1a represses the gene transcription of serotonin-1A (5-HT1A) autoreceptors, which negatively regulate 5-HT tone. To test the role of Freud-1 in vivo , we generated mice with adulthood conditional knock-out of Freud-1 in 5-HT neurons ( cF1ko ). In cF1ko mice, 5-HT1A autoreceptor protein, binding and hypothermia response were increased, with reduced 5-HT content and neuronal activity in the dorsal raphe. The cF1ko mice displayed increased anxiety- and depression-like behavior that was resistant to chronic antidepressant (fluoxetine) treatment. Using conditional Freud-1/5-HT1A double knock-out ( cF1/1A dko ) to disrupt both Freud-1 and 5-HT1A genes in 5-HT neurons, no increase in anxiety- or depression-like behavior was seen upon knock-out of Freud-1 on the 5-HT1A autoreceptor-negative background; rather, a reduction in depression-like behavior emerged. These studies implicate transcriptional dysregulation of 5-HT1A autoreceptors by the repressor Freud-1 in anxiety and depression and provide a clinically relevant genetic model of antidepressant resistance. Targeting specific transcription factors, such as Freud-1, to restore transcriptional balance may augment response to antidepressant treatment. SIGNIFICANCE STATEMENT Altered regulation of the 5-HT1A autoreceptor has been implicated in human anxiety, major depression, suicide, and resistance to antidepressants. This study uniquely identifies a single transcription factor, Freud-1, as crucial for 5-HT1A autoreceptor expression in vivo Disruption of Freud-1 in serotonin neurons in mice links upregulation of 5-HT1A autoreceptors to anxiety/depression-like behavior and provides a new model of antidepressant resistance. Treatment strategies to reestablish transcriptional regulation of 5-HT1A autoreceptors could provide a more robust and sustained antidepressant response. Copyright © 2017 the authors 0270-6474/17/3711967-12$15.00/0.

Elevated progranulin contributes to synaptic and learning deficit due to loss of fragile X mental retardation protein.

PubMed

Zhang, Kun; Li, Yu-Jiao; Guo, Yanyan; Zheng, Kai-Yin; Yang, Qi; Yang, Le; Wang, Xin-Shang; Song, Qian; Chen, Tao; Zhuo, Min; Zhao, Ming-Gao

2017-12-01

Fragile X syndrome is an inheritable form of intellectual disability caused by loss of fragile X mental retardation protein (FMRP, encoded by the FMR1 gene). Absence of FMRP caused overexpression of progranulin (PGRN, encoded by GRN), a putative tumour necrosis factor receptor ligand. In the present study, we found that progranulin mRNA and protein were upregulated in the medial prefrontal cortex of Fmr1 knock-out mice. In Fmr1 knock-out mice, elevated progranulin caused insufficient dendritic spine pruning and late-phase long-term potentiation in the medial prefrontal cortex of Fmr1 knock-out mice. Partial progranulin knock-down restored spine morphology and reversed behavioural deficits, including impaired fear memory, hyperactivity, and motor inflexibility in Fmr1 knock-out mice. Progranulin increased levels of phosphorylated glutamate ionotropic receptor GluA1 and nuclear factor kappa B in cultured wild-type neurons. Tumour necrosis factor receptor 2 antibody perfusion blocked the effects of progranulin on GluA1 phosphorylation; this result indicates that tumour necrosis factor receptor 2 is required for progranulin-mediated GluA1 phosphorylation and late-phase long-term potentiation expression. However, high basal level of progranulin in Fmr1 knock-out mice prevented further facilitation of synaptic plasticity by exogenous progranulin. Partial downregulation of progranulin or tumour necrosis factor receptor 2/nuclear factor kappa B signalling restored synaptic plasticity and memory deficits in Fmr1 knock-out mice. These findings suggest that elevated PGRN is linked to cognitive deficits of fragile X syndrome, and the progranulin/tumour necrosis factor receptor 2 signalling pathway may be a putative therapeutic target for improving cognitive deficits in fragile X syndrome. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A deafness mechanism of digenic Cx26 (GJB2) and Cx30 (GJB6) mutations: Reduction of endocochlear potential by impairment of heterogeneous gap junctional function in the cochlear lateral wall.

PubMed

Mei, Ling; Chen, Jin; Zong, Liang; Zhu, Yan; Liang, Chun; Jones, Raleigh O; Zhao, Hong-Bo

2017-12-01

Digenic Connexin26 (Cx26, GJB2) and Cx30 (GJB6) heterozygous mutations are the second most frequent cause of recessive deafness in humans. However, the underlying deafness mechanism remains unclear. In this study, we created different double Cx26 and Cx30 heterozygous (Cx26 +/- /Cx30 +/- ) mouse models to investigate the underlying pathological changes and deafness mechanism. We found that double Cx26 +/- /Cx30 +/- heterozygous mice had hearing loss. Endocochlear potential (EP), which is a driving force for hair cells producing auditory receptor current, was reduced. However, unlike Cx26 homozygous knockout (Cx26 -/- ) mice, the cochlea in Cx26 +/- /Cx30 +/- mice displayed normal development and had no apparent hair cell degeneration. Gap junctions (GJs) in the cochlea form two independent networks: the epithelial cell GJ network in the organ of Corti and the connective tissue GJ network in the cochlear lateral wall. We further found that double heterozygous deletion of Cx26 and Cx30 in the epithelial cells did not reduce EP and had normal hearing, suggesting that Cx26 +/- /Cx30 +/- may mainly impair gap junctional functions in the cochlear lateral wall and lead to EP reduction and hearing loss. Most of Cx26 and Cx30 in the cochlear lateral wall co-expressed in the same gap junctional plaques. Moreover, sole Cx26 +/- or Cx30 +/- heterozygous mice had no hearing loss. These data further suggest that digenic Cx26 and Cx30 mutations may impair heterozygous coupling of Cx26 and Cx30 in the cochlear lateral wall to reduce EP, thereby leading to hearing loss. Copyright © 2017 Elsevier Inc. All rights reserved.

Deletion of Suppressor of Cytokine Signaling 3 from Forebrain Neurons Delays Infertility and Onset of Hypothalamic Leptin Resistance in Response to a High Caloric Diet

PubMed Central

McEwen, Hayden J. L.; Inglis, Megan A.; Quennell, Janette H.; Grattan, David R.

2016-01-01

The cellular processes that cause high caloric diet (HCD)-induced infertility are poorly understood but may involve upregulation of suppressor of cytokine signaling (SOCS-3) proteins that are associated with hypothalamic leptin resistance. Deletion of SOCS-3 from brain cells is known to protect mice from diet-induced obesity, but the effects on HCD-induced infertility are unknown. We used neuron-specific SOCS3 knock-out mice to elucidate this and the effects on regional hypothalamic leptin resistance. As expected, male and female neuron-specific SOCS3 knock-out mice were protected from HCD-induced obesity. While female wild-type mice became infertile after 4 months of HCD feeding, infertility onset in knock-out females was delayed by 4 weeks. Similarly, knock-out mice had delayed leptin resistance development in the medial preoptic area and anteroventral periventricular nucleus, regions important for generation of the surge of GnRH and LH that induces ovulation. We therefore tested whether the suppressive effects of HCD on the estradiol-induced GnRH/LH surge were overcome by neuron-specific SOCS3 knock-out. Although only 20% of control HCD-mice experienced a preovulatory-like LH surge, LH surges could be induced in almost all neuron-specific SOCS3 knock-out mice on this diet. In contrast to females, HCD-fed male mice did not exhibit any fertility decline compared with low caloric diet-fed males despite their resistance to the satiety effects of leptin. These data show that deletion of SOCS3 delays the onset of leptin resistance and infertility in HCD-fed female mice, but given continued HCD feeding this state does eventually occur, presumably in response to other mechanisms inhibiting leptin signal transduction. SIGNIFICANCE STATEMENT Obesity is commonly associated with infertility in humans and other animals. Treatments for human infertility show a decreased success rate with increasing body mass index. A hallmark of obesity is an increase in circulating leptin levels; despite this, the brain responds as if there were low levels of leptin, leading to increased appetite and suppressed fertility. Here we show that leptin resistant infertility is caused in part by the leptin signaling molecule SOCS3. Deletion of SOCS3 from brain neurons delays the onset of diet-induced infertility. PMID:27383590

Deletion of Suppressor of Cytokine Signaling 3 from Forebrain Neurons Delays Infertility and Onset of Hypothalamic Leptin Resistance in Response to a High Caloric Diet.

PubMed

McEwen, Hayden J L; Inglis, Megan A; Quennell, Janette H; Grattan, David R; Anderson, Greg M

2016-07-06

The cellular processes that cause high caloric diet (HCD)-induced infertility are poorly understood but may involve upregulation of suppressor of cytokine signaling (SOCS-3) proteins that are associated with hypothalamic leptin resistance. Deletion of SOCS-3 from brain cells is known to protect mice from diet-induced obesity, but the effects on HCD-induced infertility are unknown. We used neuron-specific SOCS3 knock-out mice to elucidate this and the effects on regional hypothalamic leptin resistance. As expected, male and female neuron-specific SOCS3 knock-out mice were protected from HCD-induced obesity. While female wild-type mice became infertile after 4 months of HCD feeding, infertility onset in knock-out females was delayed by 4 weeks. Similarly, knock-out mice had delayed leptin resistance development in the medial preoptic area and anteroventral periventricular nucleus, regions important for generation of the surge of GnRH and LH that induces ovulation. We therefore tested whether the suppressive effects of HCD on the estradiol-induced GnRH/LH surge were overcome by neuron-specific SOCS3 knock-out. Although only 20% of control HCD-mice experienced a preovulatory-like LH surge, LH surges could be induced in almost all neuron-specific SOCS3 knock-out mice on this diet. In contrast to females, HCD-fed male mice did not exhibit any fertility decline compared with low caloric diet-fed males despite their resistance to the satiety effects of leptin. These data show that deletion of SOCS3 delays the onset of leptin resistance and infertility in HCD-fed female mice, but given continued HCD feeding this state does eventually occur, presumably in response to other mechanisms inhibiting leptin signal transduction. Obesity is commonly associated with infertility in humans and other animals. Treatments for human infertility show a decreased success rate with increasing body mass index. A hallmark of obesity is an increase in circulating leptin levels; despite this, the brain responds as if there were low levels of leptin, leading to increased appetite and suppressed fertility. Here we show that leptin resistant infertility is caused in part by the leptin signaling molecule SOCS3. Deletion of SOCS3 from brain neurons delays the onset of diet-induced infertility. Copyright © 2016 the authors 0270-6474/16/367142-12$15.00/0.

Attenuated Stress Response to Acute Restraint and Forced Swimming Stress in Arginine Vasopressin 1b Receptor Subtype (Avpr1b) Receptor Knockout Mice and Wild-Type Mice Treated with a Novel Avpr1b Receptor Antagonist

PubMed Central

Roper, J A; Craighead, M; O’Carroll, A-M; Lolait, S J

2010-01-01

Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin-releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic-pituitary-adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild-type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress-induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild-type mice that had been pre-treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint-stressed knockout male mice, and in Avpr1b-antagonist-treated male wild-type mice. By contrast, plasma CORT levels were not reduced in acutely restraint-stressed female knockout animals, or in female wild-type animals pre-treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic-pituitary-adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors. PMID:20846299

Attenuated stress response to acute restraint and forced swimming stress in arginine vasopressin 1b receptor subtype (Avpr1b) receptor knockout mice and wild-type mice treated with a novel Avpr1b receptor antagonist.

PubMed

Roper, J A; Craighead, M; O'Carroll, A-M; Lolait, S J

2010-11-01

Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin-releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic-pituitary-adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild-type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress-induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild-type mice that had been pre-treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint-stressed knockout male mice, and in Avpr1b-antagonist-treated male wild-type mice. By contrast, plasma CORT levels were not reduced in acutely restraint-stressed female knockout animals, or in female wild-type animals pre-treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic-pituitary-adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors. © 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.

Analysis of knockout mice suggests a role for VGF in the control of fat storage and energy expenditure.

PubMed

Watson, Elizabeth; Fargali, Samira; Okamoto, Haruka; Sadahiro, Masato; Gordon, Ronald E; Chakraborty, Tandra; Sleeman, Mark W; Salton, Stephen R

2009-10-28

Previous studies of mixed background mice have demonstrated that targeted deletion of Vgf produces a lean, hypermetabolic mouse that is resistant to diet-, lesion-, and genetically-induced obesity. To investigate potential mechanism(s) and site(s) of action of VGF, a neuronal and endocrine secreted protein and neuropeptide precursor, we further analyzed the metabolic phenotypes of two independent VGF knockout lines on C57Bl6 backgrounds. Unlike hyperactive VGF knockout mice on a mixed C57Bl6-129/SvJ background, homozygous mutant mice on a C57Bl6 background were hypermetabolic with similar locomotor activity levels to Vgf+/Vgf+ mice, during day and night cycles, indicating that mechanism(s) other than hyperactivity were responsible for their increased energy expenditure. In Vgf-/Vgf- knockout mice, morphological analysis of brown and white adipose tissues (BAT and WAT) indicated decreased fat storage in both tissues, and decreased adipocyte perimeter and area in WAT. Changes in gene expression measured by real-time RT-PCR were consistent with increased fatty acid oxidation and uptake in BAT, and increased lipolysis, decreased lipogenesis, and brown adipocyte differentiation in WAT, suggesting that increased sympathetic nervous system activity in Vgf-/Vgf- mice may be associated with or responsible for alterations in energy expenditure and fat storage. In addition, uncoupling protein 1 (UCP1) and UCP2 protein levels, mitochondrial number, and mitochondrial cristae density were upregulated in Vgf-/Vgf- BAT. Using immunohistochemical and histochemical techniques, we detected VGF in nerve fibers innervating BAT and Vgf promoter-driven reporter expression in cervical and thoracic spinal ganglia that project to and innervate the chest wall and tissues including BAT. Moreover, VGF peptide levels were quantified by radioimmunoassay in BAT, and were found to be down-regulated by a high fat diet. Lastly, despite being hypermetabolic, VGF knockout mice were cold intolerant. We propose that VGF and/or VGF-derived peptides modulate sympathetic outflow pathways to regulate fat storage and energy expenditure.

ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice

PubMed Central

Brzozowska, Natalia; Li, Kong M.; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S.

2016-01-01

Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b−∕−), Bcrp knockout (Abcg2−∕−), combined P-gp/Bcrp knockout (Abcb1a/b−∕−Abcg2−∕−) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders. PMID:27257556

ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice.

PubMed

Brzozowska, Natalia; Li, Kong M; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S; Arnold, Jonathon C

2016-01-01

Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b (-∕-)), Bcrp knockout (Abcg2 (-∕-)), combined P-gp/Bcrp knockout (Abcb1a/b (-∕-) Abcg2 (-∕-)) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.

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

Zika Virus Infection in Mice Causes Panuveitis with Shedding of Virus in Tears.

PubMed

Miner, Jonathan J; Sene, Abdoulaye; Richner, Justin M; Smith, Amber M; Santeford, Andrea; Ban, Norimitsu; Weger-Lucarelli, James; Manzella, Francesca; Rückert, Claudia; Govero, Jennifer; Noguchi, Kevin K; Ebel, Gregory D; Diamond, Michael S; Apte, Rajendra S

2016-09-20

Zika virus (ZIKV) is an emerging flavivirus that causes congenital abnormalities and Guillain-Barré syndrome. ZIKV infection also results in severe eye disease characterized by optic neuritis, chorioretinal atrophy, and blindness in newborns and conjunctivitis and uveitis in adults. We evaluated ZIKV infection of the eye by using recently developed mouse models of pathogenesis. ZIKV-inoculated mice developed conjunctivitis, panuveitis, and infection of the cornea, iris, optic nerve, and ganglion and bipolar cells in the retina. This phenotype was independent of the entry receptors Axl or Mertk, given that Axl(-/-), Mertk(-/-), and Axl(-/-)Mertk(-/-) double knockout mice sustained levels of infection similar to those of control animals. We also detected abundant viral RNA in tears, suggesting that virus might be secreted from lacrimal glands or shed from the cornea. This model provides a foundation for studying ZIKV-induced ocular disease, defining mechanisms of viral persistence, and developing therapeutic approaches for viral infections of the eye. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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

A saturation hypothesis to explain both enhanced and impaired learning with enhanced plasticity

PubMed Central

Nguyen-Vu, TD Barbara; Zhao, Grace Q; Lahiri, Subhaneil; Kimpo, Rhea R; Lee, Hanmi; Ganguli, Surya; Shatz, Carla J; Raymond, Jennifer L

2017-01-01

Across many studies, animals with enhanced synaptic plasticity exhibit either enhanced or impaired learning, raising a conceptual puzzle: how enhanced plasticity can yield opposite learning outcomes? Here, we show that the recent history of experience can determine whether mice with enhanced plasticity exhibit enhanced or impaired learning in response to the same training. Mice with enhanced cerebellar LTD, due to double knockout (DKO) of MHCI H2-Kb/H2-Db (KbDb−/−), exhibited oculomotor learning deficits. However, the same mice exhibited enhanced learning after appropriate pre-training. Theoretical analysis revealed that synapses with history-dependent learning rules could recapitulate the data, and suggested that saturation may be a key factor limiting the ability of enhanced plasticity to enhance learning. Optogenetic stimulation designed to saturate LTD produced the same impairment in WT as observed in DKO mice. Overall, our results suggest that the recent history of activity and the threshold for synaptic plasticity conspire to effect divergent learning outcomes. DOI: http://dx.doi.org/10.7554/eLife.20147.001 PMID:28234229

Deficiency of iNOS-derived NO accelerates lipid accumulation-independent liver fibrosis in non-alcoholic steatohepatitis mouse model.

PubMed

Nozaki, Yuichi; Fujita, Koji; Wada, Koichiro; Yoneda, Masato; Kessoku, Takaomi; Shinohara, Yoshiyasu; Imajo, Kento; Ogawa, Yuji; Nakamuta, Makoto; Saito, Satoru; Masaki, Naohiko; Nagashima, Yoji; Terauchi, Yasuo; Nakajima, Atsushi

2015-04-01

Although many of the factors and molecules closely associated with non-alcoholic steatohepatitis (NASH) have been reported, the role of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) on the progression of NASH remains unclear. We therefore investigated the role of iNOS-derived NO in NASH pathogenesis with a long-term follow-up study using systemic iNOS-knockout mice under high-fat diet (HFD) conditions. iNOS-knockout and wild-type mice were fed a basal or HFD for 10 or 48 weeks. Lipid accumulation, fibrosis, and inflammation were evaluated, and various factors and molecules closely associated with NASH were analyzed. Marked fibrosis and inflammation (indicators of NASH) were observed in the livers of iNOS-knockout mice compared to wild-type mice after 48 weeks of a HFD; however, lipid accumulation in iNOS-knockout mice livers was less than in the wild-type. Increased expressions of various cytokines that are transcriptionally controlled by NF-kB in iNOS-deficient mice livers were observed during HFD conditions. iNOS-derived NO may play a protective role against the progression to NASH during an HFD by preventing fibrosis and inflammation, which are mediated by NF-kB activation in Kupffer cells. A lack of iNOS-derived NO accelerates progression to NASH without excessive lipid accumulation.

Effects of dopamine D1-like and D2-like antagonists on cocaine discrimination in muscarinic receptor knockout mice.

PubMed

Thomsen, Morgane; Caine, Simon Barak

2016-04-05

Muscarinic and dopamine brain systems interact intimately, and muscarinic receptor ligands, like dopamine ligands, can modulate the reinforcing and discriminative stimulus (S(D)) effects of cocaine. To enlighten the dopamine/muscarinic interactions as they pertain to the S(D) effects of cocaine, we evaluated whether muscarinic M1, M2 or M4 receptors are necessary for dopamine D1 and/or D2 antagonist mediated modulation of the S(D) effects of cocaine. Knockout mice lacking M1, M2, or M4 receptors, as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline in a food-reinforced drug discrimination procedure. Effects of pretreatments with the dopamine D1 antagonist SCH 23390 and the dopamine D2 antagonist eticlopride were evaluated. In intact mice, both SCH 23390 and eticlopride attenuated the cocaine discriminative stimulus effect, as expected. SCH 23390 similarly attenuated the cocaine discriminative stimulus effect in M1 knockout mice, but not in mice lacking M2 or M4 receptors. The effects of eticlopride were comparable in each knockout strain. These findings demonstrate differences in the way that D1 and D2 antagonists modulate the S(D) effects of cocaine, D1 modulation being at least partially dependent upon activity at the inhibitory M2/M4 muscarinic subtypes, while D2 modulation appeared independent of these systems. Copyright © 2016 Elsevier B.V. All rights reserved.

TRC8-dependent degradation of hepatitis C virus immature core protein regulates viral propagation and pathogenesis

PubMed Central

Aizawa, Sayaka; Okamoto, Toru; Sugiyama, Yukari; Kouwaki, Takahisa; Ito, Ayano; Suzuki, Tatsuya; Ono, Chikako; Fukuhara, Takasuke; Yamamoto, Masahiro; Okochi, Masayasu; Hiraga, Nobuhiko; Imamura, Michio; Chayama, Kazuaki; Suzuki, Ryosuke; Shoji, Ikuo; Moriishi, Kohji; Moriya, Kyoji; Koike, Kazuhiko; Matsuura, Yoshiharu

2016-01-01

Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin–proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCV core protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells. PMID:27142248

Linking cellular zinc status to body weight and fat mass: mapping quantitative trait loci in Znt7 knockout mice

USDA-ARS?s Scientific Manuscript database

Zinc transporter 7 (Znt7, Slc30a7) knockout (KO) mice display abnormalities in body weight gain and body adiposity. Regulation of body weight and fatness is complex, involving multiple genetic and environmental factors. To understand how zinc homeostasis influences body weight gain and fat deposit a...

Hypercholesterolemia is associated with a T helper (Th) 1/Th2 switch of the autoimmune response in atherosclerotic apo E-knockout mice.

PubMed Central

Zhou, X; Paulsson, G; Stemme, S; Hansson, G K

1998-01-01

Atherosclerosis is an inflammatory-fibrotic response to accumulation of cholesterol in the artery wall. In hypercholesterolemia, low density lipoproteins (LDL) accumulate and are oxidized to proinflammatory compounds in the arterial intima, leading to activation of endothelial cells, macrophages, and T lymphocytes. We have studied immune cell activation and the autoimmune response to oxidized LDL in atherosclerotic apo E-knockout mice. Autoantibodies to oxidized LDL exhibited subclass specificities indicative of T cell help, and the increase in antibody titers in peripheral blood was associated with increased numbers of cytokine-expressing T cells in the spleen. In addition to T cell-dependent antibodies, IgM antibodies to oxidized LDL were also increased in apo E-knockout mice. This suggests that both T cell-dependent and T cell-independent epitopes may be present on oxidized LDL. In moderate hypercholesterolemia, IgG antibodies were largely of the IgG2a isotype, suggesting that T cell help was provided by proinflammatory T helper (Th) 1 cells, which are prominent components of atherosclerotic lesions. In severe hypercholesterolemia induced by cholesterol feeding of apo E-knockout mice, a switch to Th2-dependent help was evident. It was associated with a loss of IFN-gamma-producing Th1 cells in the spleen, whereas IL-4-producing Th2 cells were more resistant to hypercholesterolemia. IFN-gamma but not IL-4 mRNA was detected in atherosclerotic lesions of moderately hypercholesterolemic apo E-knockout mice, but IL-4 mRNA appeared in the lesions when mice were made severely hypercholesterolemic by cholesterol feeding. These data show that IFN-gamma-producing Th1 cells infiltrate atherosclerotic lesions and provide T cell help for autoimmune responses to oxidized LDL in apo E-knockout mice. However, severe hypercholesterolemia is associated with a switch from Th1 to Th2, which results not only in the formation of IgG1 autoantibodies to oxidized LDL, but also in the appearance of Th2-type cytokines in the atherosclerotic lesions. Since the two subsets of T cells counteract each other, this switch may have important consequences for the inflammatory/immune process in atherosclerosis. PMID:9541503

Reduced expression of the Ca(2+) transporter protein PMCA2 slows Ca(2+) dynamics in mouse cerebellar Purkinje neurones and alters the precision of motor coordination.

PubMed

Empson, Ruth M; Turner, Paul R; Nagaraja, Raghavendra Y; Beesley, Philip W; Knöpfel, Thomas

2010-03-15

Cerebellar Purkinje neurones (PNs) express high levels of the plasma membrane calcium ATPase, PMCA2, a transporter protein critical for the clearance of calcium from excitable cells. Genetic deletion of one PMCA2 encoding gene in heterozygous PMCA2 knock-out (PMCA2(+/-) mice enabled us to determine how PMCA2 influences PN calcium regulation without the complication of the severe morphological changes associated with complete PMCA2 knock-out (PMCA2(-/-) in these cells. The PMCA2(+/-) cerebellum expressed half the normal levels of PMCA2 and this nearly doubled the time taken for PN dendritic calcium transients to recover (mean fast and slow recovery times increased from 70 ms to 110 ms and from 600 ms to 1100 ms). The slower calcium recovery had distinct consequences for PMCA2(+/-) PN physiology. The PNs exhibited weaker climbing fibre responses, prolonged outward Ca(2+)-dependent K(+) current (mean fast and slow recovery times increased from 136 ms to 192 ms and from 595 ms to 1423 ms) and a slower mean frequency of action potential firing (7.4 Hz compared with 15.8 Hz). Our findings were consistent with prolonged calcium accumulation in the cytosol of PMCA2(+/-) Purkinje neurones. Although PMCA2(+/-) mice exhibited outwardly normal behaviour and little change in their gait pattern, when challenged to run on a narrow beam they exhibited clear deficits in hindlimb coordination. Training improved the motor performance of both PMCA2(+/-) and wild-type mice, although PMCA2(+/-) mice were always impaired. We conclude that reduced calcium clearance perturbs calcium dynamics in PN dendrites and that this is sufficient to disrupt the accuracy of cerebellar processing and motor coordination.

Lipocalin 2 Plays an Important Role in Regulating Inflammation in Retinal Degeneration.

PubMed

Parmar, Tanu; Parmar, Vipul M; Perusek, Lindsay; Georges, Anouk; Takahashi, Masayo; Crabb, John W; Maeda, Akiko

2018-05-01

It has become increasingly important to understand how retinal inflammation is regulated because inflammation plays a role in retinal degenerative diseases. Lipocalin 2 (LCN2), an acute stress response protein with multiple innate immune functions, is increased in ATP-binding cassette subfamily A member 4 ( Abca4 ) -/- retinol dehydrogenase 8 ( Rdh8 ) -/- double-knockout mice, an animal model for Stargardt disease and age-related macular degeneration (AMD). To examine roles of LCN2 in retinal inflammation and degeneration, Lcn2 -/- Abca4 -/- Rdh8 -/- triple-knockout mice were generated. Exacerbated inflammation following light exposure was observed in Lcn2 -/- Abca4 -/- Rdh8 -/- mice as compared with Abca4 -/- Rdh8 -/- mice, with upregulation of proinflammatory genes and microglial activation. RNA array analyses revealed an increase in immune response molecules such as Ccl8 , Ccl2 , and Cxcl10 To further probe a possible regulatory role for LCN2 in retinal inflammation, we examined the in vitro effects of LCN2 on NF-κB signaling in human retinal pigmented epithelial (RPE) cells differentiated from induced pluripotent stem cells derived from healthy donors. We found that LCN2 induced expression of antioxidant enzymes heme oxygenase 1 and superoxide dismutase 2 in these RPE cells and could inhibit the cytotoxic effects of H 2 O 2 and LPS. ELISA revealed increased LCN2 levels in plasma of patients with Stargardt disease, retinitis pigmentosa, and age-related macular degeneration as compared with healthy controls. Finally, overexpression of LCN2 in RPE cells displayed protection from cell death. Overall these results suggest that LCN2 is involved in prosurvival responses during cell stress and plays an important role in regulating inflammation during retinal degeneration. Copyright © 2018 by The American Association of Immunologists, Inc.

Nox2 Knockout Delays Infarct Progression and Increases Vascular Recovery through Angiogenesis in Mice following Ischaemic Stroke with Reperfusion

PubMed Central

McCann, Sarah K.; Dusting, Gregory J.; Roulston, Carli L.

2014-01-01

Evidence suggests the NADPH oxidases contribute to ischaemic stroke injury and Nox2 is the most widely studied subtype in the context of stroke. There is still conjecture however regarding the benefits of inhibiting Nox2 to improve stroke outcome. The current study aimed to examine the temporal effects of genetic Nox2 deletion on neuronal loss after ischaemic stroke using knockout (KO) mice with 6, 24 and 72 hour recovery. Transient cerebral ischaemia was induced via intraluminal filament occlusion and resulted in reduced infarct volumes in Nox2 KO mice at 24 h post-stroke compared to wild-type controls. No protection was evident at either 6 h or 72 h post-stroke, with both genotypes exhibiting similar volumes of damage. Reactive oxygen species were detected using dihydroethidium and were co-localised with neurons and microglia in both genotypes using immunofluorescent double-labelling. The effect of Nox2 deletion on vascular damage and recovery was also examined 24 h and 72 h post-stroke using an antibody against laminin. Blood vessel density was decreased in the ischaemic core of both genotypes 24 h post-stroke and returned to pre-stroke levels only in Nox2 KO mice by 72 h. Overall, these results are the first to show that genetic Nox2 deletion merely delays the progression of neuronal loss after stroke but does not prevent it. Additionally, we show for the first time that Nox2 deletion increases re-vascularisation of the damaged brain by 72 h, which may be important in promoting endogenous brain repair mechanisms that rely on re-vascularisation. PMID:25375101

Prenatal nicotine exposure increases hyperventilation in α4-knock-out mice during mild asphyxia.

PubMed

Avraam, Joanne; Cohen, Gary; Drago, John; Frappell, Peter B

2015-03-01

Prenatal nicotine exposure alters breathing and ventilatory responses to stress through stimulation of nicotine acetylcholine receptors (nAChRs). We tested the hypothesis that α4-containing nAChRs are involved in mediating the effects of prenatal nicotine exposure on ventilatory and metabolic responses to intermittent mild asphyxia (MA). Using open-flow plethysmography, we measured ventilation (V̇(E)) and rate of O2 consumption ( V̇(O2)) of wild-type (WT) and α4-knock-out (KO) mice, at postnatal (P) days 1-2 and 7-8, with and without prenatal nicotine exposure (6 mg kg(-1) day(-1) beginning on embryonic day 14). Mice were exposed to seven 2 min cycles of mild asphyxia (10% O2 and 5% CO2), each interspersed with 2 min of air. Compared to WT, α4 KO mice had increased air V̇(E) and V̇(O2) at P7-8, but not P1-2. Irrespective of age, genotype had no effect on the hyperventilatory response (increase in V̇(E)/V̇(O2)) to MA. At P1-2, nicotine suppressed air V̇(E) and V̇(O2) in both genotypes but did not affect the hyperventilatory response to MA. At P7-8 nicotine suppressed air V̇(E) and V̇(O2) of only α4 KO's but also significantly enhanced V̇(E) during MA (nearly double that of WT; p<0.001). This study has revealed complex effects of α4 nAChR deficiency and prenatal nicotine exposure on ventilatory and metabolic interactions and responses to stress. Copyright © 2015 Elsevier B.V. All rights reserved.

Toll-like receptors 2 and 4 contribute to sepsis-induced depletion of spleen dendritic cells.

PubMed

Pène, Frédéric; Courtine, Emilie; Ouaaz, Fatah; Zuber, Benjamin; Sauneuf, Bertrand; Sirgo, Gonzalo; Rousseau, Christophe; Toubiana, Julie; Balloy, Viviane; Chignard, Michel; Mira, Jean-Paul; Chiche, Jean-Daniel

2009-12-01

Depletion of dendritic cells (DC) in secondary lymphoid organs is a hallmark of sepsis-induced immune dysfunction. In this setting, we investigated if Toll-like receptor (TLR)-dependent signaling might modulate the maturation process and the survival of DC. Using a model of sublethal polymicrobial sepsis induced by cecal ligation and puncture, we investigated the quantitative and functional features of spleen DC in wild-type, TLR2(-/-), TLR4(-/-), and TLR2(-/-) TLR4(-/-) mice. By 24 h, a decrease in the relative percentage of CD11c(high) spleen DC occurred in wild-type mice but was prevented in TLR2(-/-), TLR4(-/-), and TLR2(-/-) TLR4(-/-) mice. In wild-type mice, sepsis dramatically affected both CD11c(+) CD8alpha(+) and CD11c(+) CD8alpha(-) subsets. In all three types of knockout mice studied, the CD11c(+) CD8alpha(+) subset followed a depletion pattern similar to that for wild-type mice. In contrast, the loss of CD11c(+) CD8alpha(-) cells was attenuated in TLR2(-/-) and TLR4(-/-) mice and completely prevented in TLR2(-/-) TLR4(-/-) mice. Accordingly, apoptosis of spleen DC was increased in septic wild-type mice and inhibited in knockout mice. In addition we characterized the functional features of spleen DC obtained from septic mice. As shown by increased expression of major histocompatibility complex class II and CD86, polymicrobial sepsis induced maturation of DC, with subsequent increased capacity to prime T lymphocytes, similarly in wild-type and knockout mice. In response to CpG DNA stimulation, production of interleukin-12 was equally impaired in DC obtained from wild-type and knockout septic mice. In conclusion, although dispensable for the DC maturation process, TLR2 and TLR4 are involved in the mechanisms leading to depletion of spleen DC following polymicrobial sepsis.

High-salt in addition to high-fat diet may enhance inflammation and fibrosis in liver steatosis induced by oxidative stress and dyslipidemia in mice.

PubMed

Uetake, Yuzaburo; Ikeda, Hitoshi; Irie, Rie; Tejima, Kazuaki; Matsui, Hiromitsu; Ogura, Sayoko; Wang, Hong; Mu, ShengYu; Hirohama, Daigoro; Ando, Katsuyuki; Sawamura, Tatsuya; Yatomi, Yutaka; Fujita, Toshiro; Shimosawa, Tatsuo

2015-02-13

It is widely known that salt is an accelerating factor for the progression of metabolic syndrome and causes cardiovascular diseases, most likely due to its pro-oxidant properties. We hypothesized that excessive salt intake also facilitates the development of nonalcoholic steatohepatitis (NASH), which is frequently associated with metabolic syndrome. We examined the exacerbating effect of high-salt diet on high-fat diet-induced liver injury in a susceptible model to oxidative stress, apoE knockout and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) transgenic mice. High-salt diet led to NASH in high-fat diet-fed LOX-1 transgenic/apoE knockout mice without affecting high-fat diet-induced dyslipidemia or hepatic triglyceride accumulation. Additionally, a high-salt and high-fat diet stimulated oxidative stress production and inflammatory reaction to a greater extent than did a high-fat diet in the liver of LOX-1 transgenic/apoE knockout mice. We demonstrated that high-salt diet exacerbated NASH in high-fat diet-fed LOX-1 transgenic /apoE knockout mice and that this effect was associated with the stimulation of oxidative and inflammatory processes; this is the first study to suggest the important role of excessive salt intake in the development of NASH.

Loss of Dok-1 and Dok-2 in mice causes severe experimental colitis accompanied by reduced expression of IL-17A and IL-22

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

Waseda, Masazumi; Arimura, Sumimasa; Shimura, Eri

Appropriate immune responses and mucosal barrier functions are required for the maintenance of intestinal homeostasis. Defects in this defense system may lead to inflammatory disorders such as inflammatory bowel disease. Downstream of tyrosine kinases 1 (Dok-1) and its closest homolog, Dok-2, are preferentially expressed in immune cells, and play essential roles in the negative regulation of multiple signaling pathways in both innate and adaptive immunity. However, the function of these proteins in intestinal homeostasis remained unclear. Here we show that Dok-1/-2 double knockout (DKO) mice were highly susceptible to dextran sodium sulfate (DSS)-induced colitis compared with Dok-1 or Dok-2 singlemore » KO and wild type (WT) mice. Furthermore, DSS-treated Dok-1/-2 DKO mice exhibited increased colonic tissue damage accompanied by reduced proliferation of the epithelial cells relative to WT controls, suggesting that Dok-1/-2 DKO mice have defects in the repair of intestinal epithelial lesions. In addition, the levels of the Th17 cytokines IL-17A and IL-22, which have protective roles in DSS-induced colitis, were reduced in DSS-treated Dok-1/-2 DKO mice compared with WT mice. Taken together, our results demonstrate that Dok-1 and Dok-2 negatively regulate intestinal inflammation, apparently through the induction of IL-17A and IL-22 expression. - Highlights: • Dok-1 and Dok-2 play a cooperative role in protection against DSS-induced colitis. • Dok-1/-2 double KO (DKO) mice show extensive ulceration of the colon after DSS treatment. • Proliferation of colonic epithelium is inhibited in DSS-treated Dok-1/-2 DKO mice. • Expression of IL-17A and IL-22 is reduced in the colon of DSS-treated Dok-1/-2 DKO mice.« less

Regeneration of the Exocrine Pancreas Is Delayed in Telomere-Dysfunctional Mice

PubMed Central

von Figura, Guido; Wagner, Martin; Nalapareddy, Kodandaramireddy; Hartmann, Daniel; Kleger, Alexander; Guachalla, Luis Miguel; Rolyan, Harshvardhan; Adler, Guido; Rudolph, Karl Lenhard

2011-01-01

Introduction Telomere shortening is a cell-intrinsic mechanism that limits cell proliferation by induction of DNA damage responses resulting either in apoptosis or cellular senescence. Shortening of telomeres has been shown to occur during human aging and in chronic diseases that accelerate cell turnover, such as chronic hepatitis. Telomere shortening can limit organ homeostasis and regeneration in response to injury. Whether the same holds true for pancreas regeneration in response to injury is not known. Methods In the present study, pancreatic regeneration after acute cerulein-induced pancreatitis was studied in late generation telomerase knockout mice with short telomeres compared to telomerase wild-type mice with long telomeres. Results Late generation telomerase knockout mice exhibited impaired exocrine pancreatic regeneration after acute pancreatitis as seen by persistence of metaplastic acinar cells and markedly reduced proliferation. The expression levels of p53 and p21 were not significantly increased in regenerating pancreas of late generation telomerase knockout mice compared to wild-type mice. Conclusion Our results indicate that pancreatic regeneration is limited in the context of telomere dysfunction without evidence for p53 checkpoint activation. PMID:21364961

Deficiency of prolyl oligopeptidase in mice disturbs synaptic plasticity and reduces anxiety-like behaviour, body weight, and brain volume.

PubMed

Höfling, Corinna; Kulesskaya, Natalia; Jaako, Külli; Peltonen, Iida; Männistö, Pekka T; Nurmi, Antti; Vartiainen, Nina; Morawski, Markus; Zharkovsky, Alexander; Võikar, Vootele; Roßner, Steffen; García-Horsman, J Arturo

2016-06-01

Prolyl oligopeptidase (PREP) has been implicated in neurodegeneration and neuroinflammation and has been considered a drug target to enhance memory in dementia. However, the true physiological role of PREP is not yet understood. In this paper, we report the phenotyping of a mouse line where the PREP gene has been knocked out. This work indicates that the lack of PREP in mice causes reduced anxiety but also hyperactivity. The cortical volumes of PREP knockout mice were smaller than those of wild type littermates. Additionally, we found increased expression of diazepam binding inhibitor protein in the cortex and of the somatostatin receptor-2 in the hippocampus of PREP knockout mice. Furthermore, immunohistochemistry and tail suspension test revealed lack of response of PREP knockout mice to lipopolysaccharide insult. Further analysis revealed significantly increased levels of polysialylated-neural cell adhesion molecule in PREP deficient mice. These findings might be explained as possible alteration in brain plasticity caused by PREP deficiency, which in turn affect behaviour and brain development. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

Core features of frontotemporal dementia recapitulated in progranulin knockout mice

PubMed Central

Ghoshal, N.; Dearborn, J.T.; Wozniak, D.F.; Cairns, N.J.

2011-01-01

Frontotemporal dementia (FTD) is typified by behavioral and cognitive changes manifested as altered social comportment and impaired memory performance. To investigate the neurodegenerative consequences of progranulin gene (GRN) mutations, which cause an inherited form of FTD, we used previously generated progranulin knockout mice (Grn-/-). Specifically, we characterized two cohorts of early and later middle-age wild type and knockout mice using a battery of tests to assess neurological integrity and behavioral phenotypes analogous to FTD. The Grn-/- mice exhibited reduced social engagement and learning and memory deficits. Immunohistochemical approaches were used to demonstrate the presence of lesions characteristic of frontotemporal lobar degeneration (FTLD) with GRN mutation including ubiquitination, microgliosis, and reactive astrocytosis, the pathological substrate of FTD. Importantly, Grn-/- mice also have decreased overall survival compared to Grn+/+ mice. These data suggest that the Grn-/- mouse reproduces some core features of FTD with respect to behavior, pathology, and survival. This murine model may serve as a valuable in vivo model of FTLD with GRN mutation through which molecular mechanisms underlying the disease can be further dissected. PMID:21933710

The Brain Proteome of the Ubiquitin Ligase Peli1 Knock-Out Mouse during Experimental Autoimmune Encephalomyelitis.

PubMed

Lereim, Ragnhild Reehorst; Oveland, Eystein; Xiao, Yichuan; Torkildsen, Øivind; Wergeland, Stig; Myhr, Kjell-Morten; Sun, Shao-Cong; Berven, Frode S

2016-09-01

The ubiquitin ligase Peli1 has previously been suggested as a potential treatment target in multiple sclerosis. In the multiple sclerosis disease model, experimental autoimmune encephalomyelitis, Peli1 knock-out led to less activated microglia and less inflammation in the central nervous system. Despite being important in microglia, Peli1 expression has also been detected in glial and neuronal cells. In the present study the overall brain proteomes of Peli1 knock-out mice and wild-type mice were compared prior to experimental autoimmune encephalomyelitis induction, at onset of the disease and at disease peak. Brain samples from the frontal hemisphere, peripheral from the extensive inflammatory foci, were analyzed using TMT-labeling of sample pools, and the discovered proteins were verified in individual mice using label-free proteomics. The greatest proteomic differences between Peli1 knock-out and wild-type mice were observed at the disease peak. In Peli1 knock-out a higher degree of antigen presentation, increased activity of adaptive and innate immune cells and alterations to proteins involved in iron metabolism were observed during experimental autoimmune encephalomyelitis. These results unravel global effects to the brain proteome when abrogating Peli1 expression, underlining the importance of Peli1 as a regulator of the immune response also peripheral to inflammatory foci during experimental autoimmune encephalomyelitis. The proteomics data is available in PRIDE with accession PXD003710.

p21{sup WAF1/Cip1/Sdi1} knockout mice respond to doxorubicin with reduced cardiotoxicity

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

Terrand, Jerome; Xu, Beibei; Morrissy, Steve

2011-11-15

Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21{sup WAF1/Cip1/Sdi1} (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significantmore » changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFN{gamma} and TNF{alpha} in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice. -- Highlights: Black-Right-Pointing-Pointer Doxorubicin induces p21 elevation in the myocardium. Black-Right-Pointing-Pointer Doxorubicin causes dilated cardiomyopathy in wild type mice. Black-Right-Pointing-Pointer p21 Knockout mice are resistant against doxorubicin induced cardiomyopathy. Black-Right-Pointing-Pointer Lack of inflammatory response correlates with the resistance in p21 knockout mice.« less

One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs.

PubMed

Zuo, Erwei; Cai, Yi-Jun; Li, Kui; Wei, Yu; Wang, Bang-An; Sun, Yidi; Liu, Zhen; Liu, Jiwei; Hu, Xinde; Wei, Wei; Huo, Xiaona; Shi, Linyu; Tang, Cheng; Liang, Dan; Wang, Yan; Nie, Yan-Hong; Zhang, Chen-Chen; Yao, Xuan; Wang, Xing; Zhou, Changyang; Ying, Wenqin; Wang, Qifang; Chen, Ren-Chao; Shen, Qi; Xu, Guo-Liang; Li, Jinsong; Sun, Qiang; Xiong, Zhi-Qi; Yang, Hui

2017-07-01

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.

The role of KCNQ1/KCNE1 K(+) channels in intestine and pancreas: lessons from the KCNE1 knockout mouse.

PubMed

Warth, R; Garcia Alzamora, M; Kim, J K; Zdebik, A; Nitschke, R; Bleich, M; Gerlach, U; Barhanin, J; Kim, S J

2002-03-01

KCNE1 (IsK, minK) co-assembles with KCNQ1 (KvLQT1) to form voltage-dependent K(+) channels. Both KCNQ1 and KCNE1 are expressed in epithelial cells of gut and exocrine pancreas. We examined the role of KCNQ1/KCNE1 in Cl(-) secretion in small and large intestine and exocrine pancreas using the KCNE1 knockout mouse. Immunofluorescence revealed a similar basolateral localization of KCNQ1 in jejunum and colon of KCNE1 wild-type and knockout mice. Electrogenic Cl(-) secretion in the colon was not affected by gene disruption of KCNE1; in jejunum forskolin-induced short-circuit current was some 40% smaller but without being significantly different. Inhibition of KCNQ1 channels by 293B (IC(50) 1 micromol l(-1)) and by IKS224 (IC(50) 14 nmol l(-1)) strongly diminished intestinal Cl(-) secretion. In exocrine pancreas of wild-type mice, KCNQ1 was predominantly located at the basolateral membrane. In KCNE1 knockout mice, however, the basolateral staining was less pronounced and the distribution of secretory granules was irregular. A slowly activating and 293B-sensitive K(+) current was activated via cholinergic stimulation in pancreatic acinar cells of wild-type mice. In KCNE1 knockout mice this K(+) current was strongly reduced. In conclusion intestinal Cl(-) secretion is independent from KCNE1 but requires KCNQ1. In mouse pancreatic acini KCNQ1 probably co-assembled with KCNE1 leads to a voltage-dependent K(+) current that might be of importance for electrolyte and enzyme secretion.

Effects of vitamin D receptor knockout on cornea epithelium gap junctions.

PubMed

Lu, Xiaowen; Watsky, Mitchell A

2014-05-06

Gap junctions are present in all corneal cell types and have been shown to have a critical role in cell phenotype determination. Vitamin D has been shown to influence cell differentiation, and recent work demonstrates the presence of vitamin D in the ocular anterior segment. This study measured and compared gap junction diffusion coefficients among different cornea epithelium phenotypes and in keratocytes using a noninvasive technique, fluorescence recovery after photobleaching (FRAP), and examined the influence of vitamin D receptor (VDR) knockout on epithelial gap junction communication in intact corneas. Previous gap junction studies in cornea epithelium and keratocytes were performed using cultured cells or ex vivo invasive techniques. These invasive techniques were unable to measure diffusion coefficients and likely were disruptive to normal cell physiology. Corneas from VDR knockout and control mice were stained with 5(6)-carboxyfluorescein diacetate (CFDA). Gap junction diffusion coefficients of the corneal epithelium phenotypes and of keratocytes, residing in intact corneas, were detected using FRAP. Diffusion coefficients equaled 18.7, 9.8, 5.6, and 4.2 μm(2)/s for superficial squamous cells, middle wing cells, basal cells, and keratocytes, respectively. Corneal thickness, superficial cell size, and the superficial squamous cell diffusion coefficient of 10-week-old VDR knockout mice were significantly lower than those of control mice (P < 0.01). The superficial cell diffusion coefficient of heterozygous mice was significantly lower than control mice (P < 0.05). Our results demonstrate differences in gap junction dye spread among the epithelial cell phenotypes, mirroring the epithelial developmental axis. The VDR knockout influences previously unreported cell-to-cell communication in superficial epithelium.

Differential gene expression in Ndph-knockout mice in retinal development.

PubMed

Schäfer, Nikolaus F; Luhmann, Ulrich F O; Feil, Silke; Berger, Wolfgang

2009-02-01

Mutations in the NDP gene impair angiogenesis in the eyes of patients diagnosed with a type of blindness belonging to the group of exudative vitreoretinopathies. This study was conducted to investigate the differential gene expression caused by the absence of Norrin (the NDP protein) in the developing mouse retina and to elucidate early pathogenic events. A comparative gene expression analysis was performed on postnatal day (p)7 retinas from a knockout mouse model for Norrie disease using gene microarrays. Subsequently, results were verified by quantitative real-time PCR analyses. Immunohistochemistry was performed for the vascular permeability marker plasmalemma vesicle associated protein (Plvap). Our study identified expression differences in Ndph(y/-) versus wild-type mice retinas at p7. Gene transcription of the neutral amino acid transporter Slc38a5, apolipoprotein D (ApoD), and angiotensin II receptor-like 1 (Agtrl1) was decreased in the knockout mouse, whereas transcript levels of adrenomedullin (Adm) and of the plasmalemma vesicle associated protein (Plvap) were increased in comparison to the wild-type. In addition, ectopic expression of Plvap was found in the developing retinal vasculature of Norrin-knockout mice on the protein level. These data provide molecular evidence for a role of Norrin in the development of the retinal vasculature. Expression of two genes, Plvap and Slc38a5, is considerably altered in retinal development of Norrin-knockout mice and may reflect or contribute to the pathogenesis of the disease. In particular, ectopic expression of Plvap is consistent with hallmark disease symptoms in mice and humans.

Vascular changes in the cerebellum of Norrin /Ndph knockout mice correlate with high expression of Norrin and Frizzled-4.

PubMed

Luhmann, Ulrich F O; Neidhardt, John; Kloeckener-Gruissem, Barbara; Schäfer, Nikolaus F; Glaus, Esther; Feil, Silke; Berger, Wolfgang

2008-05-01

X-linked Norrie disease, familial exudative vitreoretinopathy (FEVR), Coat's disease and retinopathy of prematurity are severe human eye diseases and can all be caused by mutations in the Norrie disease pseudoglioma gene. They all show vascular defects and characteristic features of retinal hypoxia. Only Norrie disease displays additional neurological symptoms, which are sensorineural hearing loss and mental retardation. In the present study, we analysed transcript levels of the ligand Norrin (Ndph) and its two receptors Frizzled-4 (Fzd4) and LDL-related protein receptor 5 (Lrp5) in six different brain regions (cerebellum, cortex, hippocampus, olfactory bulb, pituitary and brain stem) of 6- to 8-month-old wild-type and Ndph knockout mice by quantitative real-time PCR. No effect of the Ndph knockout allele on Fzd4 or Lrp5 receptor expression was found. Furthermore, no alterations of the transcript levels of three hypoxia-regulated angiogenic factors (Vegfa, Itgrb3 and Tie1) were observed in the absence of Norrin. Interestingly, we identified significant differences in Ndph, Fzd4 and Lrp5 transcript levels in brain regions of wild-type mice and observed highest expression of Norrin and frizzled-4 in cerebellum. Transcript analyses were correlated with morphological data obtained from cerebellum and immunohistochemical studies of blood vessels in different brain regions. Vessel density was reduced in the cerebellum of Ndph knockout mice but the number of Purkinje and granular cells was not altered. This provides the first description of a brain phenotype in Ndph knockout mice, which will help to elucidate the role of Norrin in the brain.

Double knockout of carbonic anhydrase II (CAII) and Na(+)-Cl(-) cotransporter (NCC) causes salt wasting and volume depletion.

PubMed

Xu, Jie; Barone, Sharon; Brooks, Mary-Beth; Soleimani, Manoocher

2013-01-01

The thiazide-sensitive Na(+)-Cl(-) cotransporter NCC and the Cl(-)/HCO3(-)exchanger pendrin are expressed on apical membranes of distal cortical nephron segments and mediate salt absorption, with pendrin working in tandem with the epithelial Na(+) channel (ENaC) and the Na(+)-dependent chloride/bicarbonate exchanger (NDCBE), whereas NCC is working by itself. A recent study showed that NCC and pendrin compensate for loss of each other under basal conditions, therefore masking the role that each plays in salt reabsorption. Carbonic anhydrase II (CAII, CA2 or CAR2) plays an important role in acid-base transport and salt reabsorption in the proximal convoluted tubule and acid-base transport in the collecting duct. Animals with CAII deletion show remodeling of intercalated cells along with the downregulation of pendrin. NCC KO mice on the other hand show significant upregulation of pendrin and ENaC. Neither model shows any significant salt wasting under baseline conditions. We hypothesized that the up-regulation of pendrin is essential for the prevention of salt wasting in NCC KO mice. To test this hypothesis, we generated NCC/CAII double KO (dKO) mice by crossing mice with single deletion of NCC and CAII. The NCC/CAII dKO mice displayed significant downregulation of pendrin, along with polyuria and salt wasting. As a result, the dKO mice developed volume depletion, which was associated with the inability to concentrate urine. We conclude that the upregulation of pendrin is essential for the prevention of salt and water wasting in NCC deficient animals and its downregulation or inactivation will result in salt wasting, impaired water conservation and volume depletion in the setting of NCC inactivation or inhibition. © 2014 S. Karger AG, Basel.

Bone Is a Major Target of PTH/PTHrP Receptor Signaling in Regulation of Fetal Blood Calcium Homeostasis

PubMed Central

Hirai, Takao; Kobayashi, Tatsuya; Nishimori, Shigeki; Karaplis, Andrew C.; Goltzman, David

2015-01-01

The blood calcium concentration during fetal life is tightly regulated within a narrow range by highly interactive homeostatic mechanisms that include transport of calcium across the placenta and fluxes in and out of bone; the mechanisms of this regulation are poorly understood. Our findings that endochondral bone-specific PTH/PTHrP receptor (PPR) knockout (KO) mice showed significant reduction of fetal blood calcium concentration compared with that of control littermates at embryonic day 18.5 led us to focus on bone as a possibly major determinant of fetal calcium homeostasis. We found that the fetal calcium concentration of Runx2 KO mice was significantly higher than that of control littermates, suggesting that calcium flux into bone had a considerable influence on the circulating calcium concentration. Moreover, Runx2:PTH double mutant fetuses showed calcium levels similar to those of Runx2 KO mice, suggesting that part of the fetal hypocalcemia in PTH KO mice was caused by the increment of the mineralized bone mass allowed by the formation of osteoblasts. Finally, Rank:PTH double mutant mice had a blood calcium concentration even lower than that of the either Rank KO or PTH KO mice alone at embryonic day 18.5. These observations in our genetic models suggest that PTH/PTHrP receptor signaling in bones has a significant role of the regulation of fetal blood calcium concentration and that both placental transport and osteoclast activation contribute to PTH's hypercalcemic action. They also show that PTH-independent deposition of calcium in bone is the major controller of fetal blood calcium level. PMID:26052897

The importance of immunohistochemical analyses in evaluating the phenotype of Kv channel knockout mice.

PubMed

Menegola, Milena; Clark, Eliana; Trimmer, James S

2012-06-01

To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

Accumulated α-synuclein affects the progression of GM2 gangliosidoses.

PubMed

Suzuki, Kyoko; Yamaguchi, Akira; Yamanaka, Shoji; Kanzaki, Seiichi; Kawashima, Masato; Togo, Takashi; Katsuse, Omi; Koumitsu, Noriko; Aoki, Naoya; Iseki, Eizo; Kosaka, Kenji; Yamaguchi, Kayoko; Hashimoto, Makoto; Aoki, Ichiro; Hirayasu, Yoshio

2016-10-01

The accumulation of α-synuclein (ASyn) has been observed in several lysosomal storage diseases (LSDs) but it remains unclear if ASyn accumulation contributes to LSD pathology. ASyn also accumulates in the neurons of Sandhoff disease (SD) patients and SD model mice (Hexb-/- ASyn+/+ mice). SD is a lysosomal storage disorder caused by the absence of a functional β-subunit on the β-hexosaminidase A and B enzymes, which leads to the accumulation of ganglioside in the central nervous system. Here, we explored the role of accumulated ASyn in the progression of Hexb-/- mice by creating a Hexb-/- ASyn-/- double-knockout mice. Our results show that Hexb-/- ASyn-/- mice demonstrated active microglia levels and less dopaminergic neuron loss, without altering the neuronal storage of ganglioside. The autophagy and ubiquitin proteasome pathways are defective in the neurons of Hexb-/- ASyn+/+ mice. In ultrastructural physiological studies, the mitochondria structures look degenerated and dysfunctional. As a result, expression of manganese superoxide dismutase 2 are reduced, and reactive oxygen species-mediated oxidative damage in the neurons of Hexb-/- ASyn+/+ mice. Interestingly, these dysfunctions improved in Hexb-/- ASyn-/- mice. But any clinical improvement were hardly observed in Hexb-/- ASyn-/- mice. Taken together, these findings suggest that ASyn accumulation plays an important role in the pathogenesis of neuropathy in SD and other LSDs, and is therefore a target for novel therapies. Copyright © 2016 Elsevier Inc. All rights reserved.

Interleukin-6 promotes systemic lupus erythematosus progression with Treg suppression approach in a murine systemic lupus erythematosus model.

PubMed

Mao, Xiaoli; Wu, Yunyun; Diao, Huitian; Hao, Jianlei; Tian, Gaofei; Jia, Zhenghu; Li, Zheng; Xiong, Sidong; Wu, Zhenzhou; Wang, Puyue; Zhao, Liqing; Yin, Zhinan

2014-11-01

Our aim is to reveal the role of interleukin 6 (IL-6) in the pathogenesis of systemic lupus erythematosus (SLE) in a murine model of SLE. Normal female C57BL/6 mice were immunized with syngeneic-activated lymphocyte-derived DNA (ALD-DNA) to induce SLE. Non-immunized mice were used as control. SLE-associated markers, including anti-double-stranded DNA (anti-dsDNA) Abs, urine protein, and kidney histopathology, were assayed to ensure the induction of the disease. Compared with control mice, ALD-DNA immunized mice exhibited high levels of anti-dsDNA Abs, IL-6 expression in vivo and in vitro. We also found that IL-6 knockout (IL-6KO) mice were resistant to ALD-DNA-induced SLE. The activation of CD4(+) T cells in immunized IL-6KO mice was lower than in immunized wild-type (Wt) mice. Intracellular cytokine staining showed that Foxp3 expression in immunized IL-6KO mice was higher than in immunized Wt mice, which might be associated with the disease severity. We further discovered that ALD-DNA-stimulated dendritic cells supernatants could result in higher IL-6 and TNF-α expression and could suppress Foxp3 expression. In addition, blocking IL-6 could up-regulate Foxp3 expression. Therefore, our findings show that IL-6 promotes the progression of SLE via suppressing Treg differentiation.

Genetic reduction of mitochondrial complex I function does not lead to loss of dopamine neurons in vivo.

PubMed

Kim, Hyung-Wook; Choi, Won-Seok; Sorscher, Noah; Park, Hyung Joon; Tronche, François; Palmiter, Richard D; Xia, Zhengui

2015-09-01

Inhibition of mitochondrial complex I activity is hypothesized to be one of the major mechanisms responsible for dopaminergic neuron death in Parkinson's disease. However, loss of complex I activity by systemic deletion of the Ndufs4 gene, one of the subunits comprising complex I, does not cause dopaminergic neuron death in culture. Here, we generated mice with conditional Ndufs4 knockout in dopaminergic neurons (Ndufs4 conditional knockout mice [cKO]) to examine the effect of complex I inhibition on dopaminergic neuron function and survival during aging and on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in vivo. Ndufs4 cKO mice did not show enhanced dopaminergic neuron loss in the substantia nigra pars compacta or dopamine-dependent motor deficits over the 24-month life span. These mice were just as susceptible to MPTP as control mice. However, compared with control mice, Ndufs4 cKO mice exhibited an age-dependent reduction of dopamine in the striatum and increased α-synuclein phosphorylation in dopaminergic neurons of the substantia nigra pars compacta. We also used an inducible Ndufs4 knockout mouse strain (Ndufs4 inducible knockout) in which Ndufs4 is conditionally deleted in all cells in adult to examine the effect of adult onset, complex I inhibition on MPTP sensitivity of dopaminergic neurons. The Ndufs4 inducible knockout mice exhibited similar sensitivity to MPTP as control littermates. These data suggest that mitochondrial complex I inhibition in dopaminergic neurons does contribute to dopamine loss and the development of α-synuclein pathology. However, it is not sufficient to cause cell-autonomous dopaminergic neuron death during the normal life span of mice. Furthermore, mitochondrial complex I inhibition does not underlie MPTP toxicity in vivo in either cell autonomous or nonautonomous manner. These results provide strong evidence that inhibition of mitochondrial complex I activity is not sufficient to cause dopaminergic neuron death during aging nor does it contribute to dopamine neuron toxicity in the MPTP model of Parkinson's disease. These findings suggest the existence of alternative mechanisms of dopaminergic neuron death independent of mitochondrial complex I inhibition. Copyright © 2015 Elsevier Inc. All rights reserved.

EFFECTS OF HEAT AND BROMOCHLOROACETIC ACID ON MALE REPRODUCTION IN HEAT SHOCK FACTOR-1 GENE KNOCKOUT MICE

EPA Science Inventory

Effects of heat and bromochloroacetic acid on male reproduction in heat shock factor-1 gene knockout mice.
Luft JC1, IJ Benjamin2, JB Garges1 and DJ Dix1. 1Reproductive Toxicology Division, USEPA, RTP, NC, 27711 and 2Dept of Internal Medicine, Univ.of Texas Southwestern Med C...

Survival and Injury Outcome After TBI: Influence of Pre- and Post-Exposure to Caffeine

DTIC Science & Technology

2012-10-01

A1Rs. This notion is supported by findings that TBI in A1R knockout mice led to lethal status epilepticus (SE) (Kochanek et al., 2006). Likewise, A1R...G.E., Dixon, C.E., Schnermann, J., Jackson, E.K., 2006. Adenosine A1 receptor knockout mice develop lethal status epilepti- cus after experimental

Disrupting Hepatocyte Cyp51 from Cholesterol Synthesis Leads to Progressive Liver Injury in the Developing Mouse and Decreases RORC Signalling

NASA Astrophysics Data System (ADS)

Urlep, Žiga; Lorbek, Gregor; Perše, Martina; Jeruc, Jera; Juvan, Peter; Matz-Soja, Madlen; Gebhardt, Rolf; Björkhem, Ingemar; Hall, Jason A.; Bonneau, Richard; Littman, Dan R.; Rozman, Damjana

2017-01-01

Development of mice with hepatocyte knockout of lanosterol 14α-demethylase (HCyp51-/-) from cholesterol synthesis is characterized by the progressive onset of liver injury with ductular reaction and fibrosis. These changes begin during puberty and are generally more aggravated in the knockout females. However, a subgroup of (pre)pubertal knockout mice (runts) exhibits a pronounced male prevalent liver dysfunction characterized by downregulated amino acid metabolism and elevated Casp12. RORC transcriptional activity is diminished in livers of all runt mice, in correlation with the depletion of potential RORC ligands subsequent to CYP51 disruption. Further evidence for this comes from the global analysis that identified a crucial overlap between hepatic Cyp51-/- and Rorc-/- expression profiles. Additionally, the reduction in RORA and RORC transcriptional activity was greater in adult HCyp51-/- females than males, which correlates well with their downregulated amino and fatty acid metabolism. Overall, we identify a global and sex-dependent transcriptional de-regulation due to the block in cholesterol synthesis during development of the Cyp51 knockout mice and provide in vivo evidence that sterol intermediates downstream of lanosterol may regulate the hepatic RORC activity.

Lithium ameliorates altered glycogen synthase kinase-3 and behavior in a mouse model of fragile X syndrome.

PubMed

Yuskaitis, Christopher J; Mines, Marjelo A; King, Margaret K; Sweatt, J David; Miller, Courtney A; Jope, Richard S

2010-02-15

Fragile X syndrome (FXS), the most common form of inherited mental retardation and a genetic cause of autism, results from mutated fragile X mental retardation-1 (Fmr1). This study examined the effects on glycogen synthase kinase-3 (GSK3) of treatment with a metabotropic glutamate receptor (mGluR) antagonist, MPEP, and the GSK3 inhibitor, lithium, in C57Bl/6 Fmr1 knockout mice. Increased mGluR signaling may contribute to the pathology of FXS, and the mGluR5 antagonist MPEP increased inhibitory serine-phosphorylation of brain GSK3 selectively in Fmr1 knockout mice but not in wild-type mice. Inhibitory serine-phosphorylation of GSK3 was lower in Fmr1 knockout, than wild-type, mouse brain regions and was increased by acute or chronic lithium treatment, which also increased hippocampal brain-derived neurotrophic factor levels. Fmr1 knockout mice displayed alterations in open-field activity, elevated plus-maze, and passive avoidance, and these differences were ameliorated by chronic lithium treatment. These findings support the hypothesis that impaired inhibition of GSK3 contributes to the pathogenesis of FXS and support GSK3 as a potential therapeutic target.

B7-2 Expressed on EL4 Lymphoma Suppresses Antitumor Immunity by an Interleukin 4–dependent Mechanism

PubMed Central

Stremmel, C.; Greenfield, E.A.; Howard, E.; Freeman, G.J.; Kuchroo, V.K.

1999-01-01

For T cells to become functionally activated they require at least two signals. The B7 costimulatory molecules B7-1 and B7-2 provide the “second signal” pivotal for T cell activation. In this report, we studied the relative roles of B7-1 and B7-2 molecules in the induction of antitumor immunity to the T cell thymoma, EL4. We generated EL4 tumor cells that expressed B7-1, B7-2, and B7-1+B7-2 by transfecting murine cDNAs. Our results demonstrate that EL4–B7-1 cells are completely rejected in syngeneic mice. Unlike EL4–B7-1 cells, we find that EL4–B7-2 cells are not rejected but progressively grow in the mice. A B7-1– and B7-2–EL4 double transfectant was generated by introducing B7-2 cDNA into the EL4–B7-1 tumor line that regressed in vivo. The EL4–B7-1+B7-2 double transfectant was not rejected when implanted into syngeneic mice but progressively grew to produce tumors. The double transfectant EL4 cells could costimulate T cell proliferation that could be blocked by anti–B7-1 antibodies, anti–B7-2 antibodies, or hCTLA4 immunoglobulin, showing that the B7-1 and B7-2 molecules expressed on the EL4 cells were functional. In vivo, treatment of mice implanted with double-transfected EL4 cells with anti–B7-2 monoclonal antibody resulted in tumor rejection. Furthermore, the EL4–B7-2 and EL4–B7-1+B7-2 cells, but not the wild-type EL4 cells, were rejected in interleukin 4 (IL-4) knockout mice. Our data suggests that B7-2 expressed on some T cell tumors inhibits development of antitumor immunity, and IL-4 appears to play a critical role in abrogation of the antitumor immune response. PMID:10075975

B7-2 expressed on EL4 lymphoma suppresses antitumor immunity by an interleukin 4-dependent mechanism.

PubMed

Stremmel, C; Greenfield, E A; Howard, E; Freeman, G J; Kuchroo, V K

1999-03-15

For T cells to become functionally activated they require at least two signals. The B7 costimulatory molecules B7-1 and B7-2 provide the "second signal" pivotal for T cell activation. In this report, we studied the relative roles of B7-1 and B7-2 molecules in the induction of antitumor immunity to the T cell thymoma, EL4. We generated EL4 tumor cells that expressed B7-1, B7-2, and B7-1+B7-2 by transfecting murine cDNAs. Our results demonstrate that EL4-B7-1 cells are completely rejected in syngeneic mice. Unlike EL4-B7-1 cells, we find that EL4-B7-2 cells are not rejected but progressively grow in the mice. A B7-1- and B7-2-EL4 double transfectant was generated by introducing B7-2 cDNA into the EL4-B7-1 tumor line that regressed in vivo. The EL4-B7-1+B7-2 double transfectant was not rejected when implanted into syngeneic mice but progressively grew to produce tumors. The double transfectant EL4 cells could costimulate T cell proliferation that could be blocked by anti-B7-1 antibodies, anti-B7-2 antibodies, or hCTLA4 immunoglobulin, showing that the B7-1 and B7-2 molecules expressed on the EL4 cells were functional. In vivo, treatment of mice implanted with double-transfected EL4 cells with anti-B7-2 monoclonal antibody resulted in tumor rejection. Furthermore, the EL4-B7-2 and EL4-B7-1+B7-2 cells, but not the wild-type EL4 cells, were rejected in interleukin 4 (IL-4) knockout mice. Our data suggests that B7-2 expressed on some T cell tumors inhibits development of antitumor immunity, and IL-4 appears to play a critical role in abrogation of the antitumor immune response.

Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology

PubMed Central

Hüttemann, Maik; Lee, Icksoo; Gao, Xiufeng; Pecina, Petr; Pecinova, Alena; Liu, Jenney; Aras, Siddhesh; Sommer, Natascha; Sanderson, Thomas H.; Tost, Monica; Neff, Frauke; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Naton, Beatrix; Rathkolb, Birgit; Rozman, Jan; Favor, Jack; Hans, Wolfgang; Prehn, Cornelia; Puk, Oliver; Schrewe, Anja; Sun, Minxuan; Höfler, Heinz; Adamski, Jerzy; Bekeredjian, Raffi; Graw, Jochen; Adler, Thure; Busch, Dirk H.; Klingenspor, Martin; Klopstock, Thomas; Ollert, Markus; Wolf, Eckhard; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabě de Angelis, Martin; Weissmann, Norbert; Doan, Jeffrey W.; Bassett, David J. P.; Grossman, Lawrence I.

2012-01-01

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (−50 and −29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced Penh and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (−12%), reduced total oxygen consumption rate (−8%), improved glucose tolerance, and reduced grip force (−14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.—Hüttemann, M., Lee, I., Gao, X., Pecina, P., Pecinova, A., Liu, J., Aras, S., Sommer, N., Sanderson, T. H., Tost, M., Neff, F., Aguilar-Pimentel, J. A., Becker, L., Naton, B., Rathkolb, B., Rozman, J., Favor, J., Hans, W., Prehn, C., Puk, O., Schrewe, A., Sun, M., Höfler, H., Adamski, J., Bekeredjian, R., Graw, J., Adler, T., Busch, D. H., Klingenspor, M., Klopstock, T., Ollert, M., Wolf, E., Fuchs, H., Gailus-Durner, V., Hrabě de Angelis, M., Weissmann, N., Doan, J. W., Bassett, D. J. P., Grossman, L. I. Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology. PMID:22730437

High affinity kainate receptor subunits are necessary for ionotropic but not metabotropic signaling

PubMed Central

Fernandes, Herman B.; Catches, Justin S.; Petralia, Ronald S.; Copits, Bryan A.; Xu, Jian; Russell, Theron A.; Swanson, Geoffrey T.; Contractor, Anis

2009-01-01

Summary Kainate receptors are atypical members of the glutamate receptor family which are able to signal through both ionotropic and metabotropic pathways. Of the five individual kainate receptor subunits the high-affinity subunits, GluK4 (KA1) and GluK5 (KA2), are unique in that they do not form functional homomeric receptors in recombinant expression systems, but combine with the primary subunits GluK1-3 (GluR5-7) to form heteromeric assemblies. Here we generated a GluK4 mutant mouse by disrupting the Grik4 gene locus. We found that loss of the GluK4 subunit leads to a significant reduction in synaptic kainate receptor currents. Moreover, ablation of both high-affinity subunits in GluK4/GluK5 double knockout mice leads to a complete loss of pre- and postsynaptic ionotropic function of synaptic kainate receptors. The principal subunits remain at the synaptic plasma membrane, but are distributed away from postsynaptic densities and presynaptic active zones. There is also an alteration in the properties of the remaining kainate receptors, as kainic acid application fails to elicit responses in GluK4/GluK5 knockout neurons. Despite the lack of detectable ionotropic synaptic receptors, the kainate receptor-mediated inhibition of the slow afterhyperpolarization current (IsAHP), which is dependent on metabotropic pathways, was intact in GluK4/GluK5 knockout mice. These results uncover a previously unknown critical role for the high-affinity kainate receptor subunits as obligatory components of ionotropic kainate receptor function, and further, demonstrate that kainate receptor participation in metabotropic signaling pathways does not require their classic role as ion channels. PMID:19778510

Type 1 Deiodinase Regulates ApoA-I Gene Expression and ApoA-I Synthesis Independent of Thyroid Hormone Signaling.

PubMed

Liu, Jing; Hernandez-Ono, Antonio; Graham, Mark J; Galton, Valerie Anne; Ginsberg, Henry N

2016-07-01

Plasma levels of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (ApoA-I) are reduced in individuals with defective insulin signaling. Initial studies using liver-specific insulin receptor (InsR) knockout mice identified reduced expression of type 1 deiodinase (Dio1) as a potentially novel link between defective hepatic insulin signaling and reduced expression of the ApoA-I gene. Our objective was to examine the regulation of ApoA-I expression by Dio1. Acute inactivation of InsR by adenoviral delivery of Cre recombinase to InsR floxed mice reduced HDL-C and expression of both ApoA-I and Dio1. Overexpression of Dio1 in InsR knockout mice restored HDL-C and ApoA-I levels and increased the expression of ApoA-I. Dio1 knockout mice had low expression of ApoA-I and reduced serum levels of HDL-C and ApoA-I. Treatment of C57BL/6J mice with antisense to Dio1 reduced ApoA-I mRNA, HDL-C, and serum ApoA-I. Hepatic 3,5,3'-triiodothyronine content was normal or elevated in InsR knockout mice or Dio1 knockout mice. Knockdown of either InsR or Dio1 by siRNA in HepG2 cells decreased the expression of ApoA-I and ApoA-I synthesis and secretion. siRNA knockdown of InsR or Dio1 decreased activity of a region of the ApoA-I promoter lacking thyroid hormone response elements (region B). Electrophoretic mobility shift assay demonstrated that reduced Dio1 expression decreased the binding of nuclear proteins to region B. Reductions in Dio1 expression reduce the expression of ApoA-I in a 3,5,3'-triiodothyronine-/thyroid hormone response element-independent manner. © 2016 American Heart Association, Inc.

[Effects of aquaporin-4 gene knockout on behavior changes and cerebral morphology during aging in mice].

PubMed

Su, Shengan; Lu, Yunbi; Zhang, Weiping

2013-05-01

To investigate the effects of aquaporin-4 (AQP4) gene knockout on the behavior changes and cerebral morphology during aging in mice,and to compare that of young and aged mice between AQP4 knockout mice (AQP4(-/-)) and wild type mice (AQP4(+/+)). Fifty-eight CD-1 mice were divided into four groups: young (2-3 months old) AQP4(-/-), aged (17-19 months old) AQP4(-/-), young AQP4(+/+) and aged AQP4(+/+). The activity levels and exploring behavior of mice were tested in open field. The neurons were stained with toluidine blue and NeuN, the astrocytes and microglia were stained with GFAP and Iba-1, respectively. The morphological changes of neuron, astrocyte and microglia were then analyzed. Compared with young mice, the total walking distance in open field of aged AQP4(+/+) mice and aged AQP4(-/-) mice decreased 41.2% and 44.1%, respectively (P<0.05); while there was no difference in the ratio of distance and retention time in the central area of open field. The density of neuron in cortex of aged AQP4(+/+) mice and aged AQP4(-/-) mice decreased 19.6% and 15.8%, respectively (P<0.05), while there was no difference in the thickness of neuron cell body in hippocampus CA1 region. The density of astrocyte in hippocampus CA3 region of aged AQP4(+/+) mice and aged AQP4(-/-) mice increased 57.7% and 64.3%, respectively (P<0.001), while there was no difference in the area of astrocyte. The area of microglia in hippocampus CA3 region of aged AQP4(+/+) mice and aged AQP4(-/-) mice increased 46.9% and 52.0%, respectively (P<0.01), while there was no difference in the density of microglia. Compared with AQP4(+/+) mice, the young and aged AQP4(-/-) mice showed smaller area of astrocyte in hippocampus CA3 region, reduced 18.0% in young mice and 23.6% in aged mice. There was no difference between AQP4(+/+) mice and AQP4(-/-) mice for other observed indexes. AQP4 may be involved in change of astrocyte and astrocyte-related behaviors during aging. AQP4 gene knockout may have limited effects on the change of neuron, microglia and most neuronal behaviors in aging process.

TAM receptors affect adult brain neurogenesis by negative regulation of microglial cell activation.

PubMed

Ji, Rui; Tian, Shifu; Lu, Helen J; Lu, Qingjun; Zheng, Yan; Wang, Xiaomin; Ding, Jixiang; Li, Qiutang; Lu, Qingxian

2013-12-15

TAM tyrosine kinases play multiple functional roles, including regulation of the target genes important in homeostatic regulation of cytokine receptors or TLR-mediated signal transduction pathways. In this study, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impairs hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAPK and NF-κB activation and elevated production of proinflammatory cytokines that are detrimental to neuron stem cell proliferation and neuronal differentiation. Injection of LPS causes even more severe inhibition of BrdU incorporation in the Tyro3(-/-)Axl(-/-)Mertk(-/-) triple-knockout (TKO) brains, consistent with the LPS-elicited enhanced expression of proinflammatory mediators, for example, IL-1β, IL-6, TNF-α, and inducible NO synthase, and this effect is antagonized by coinjection of the anti-inflammatory drug indomethacin in wild-type but not TKO brains. Conditioned medium from TKO microglia cultures inhibits neuron stem cell proliferation and neuronal differentiation. IL-6 knockout in Axl(-/-)Mertk(-/-) double-knockout mice overcomes the inflammatory inhibition of neurogenesis, suggesting that IL-6 is a major downstream neurotoxic mediator under homeostatic regulation by TAM receptors in microglia. Additionally, autonomous trophic function of the TAM receptors on the proliferating neuronal progenitors may also promote progenitor differentiation into immature neurons.

Deletion of the alpha-arrestin protein Txnip in mice promotes adiposity and adipogenesis while preserving insulin sensitivity.

PubMed

Chutkow, William A; Birkenfeld, Andreas L; Brown, Jonathan D; Lee, Hui-Young; Frederick, David W; Yoshioka, Jun; Patwari, Parth; Kursawe, Romy; Cushman, Samuel W; Plutzky, Jorge; Shulman, Gerald I; Samuel, Varman T; Lee, Richard T

2010-06-01

Thioredoxin interacting protein (Txnip), a regulator of cellular oxidative stress, is induced by hyperglycemia and inhibits glucose uptake into fat and muscle, suggesting a role for Txnip in type 2 diabetes pathogenesis. Here, we tested the hypothesis that Txnip-null (knockout) mice are protected from insulin resistance induced by a high-fat diet. Txnip gene-deleted (knockout) mice and age-matched wild-type littermate control mice were maintained on a standard chow diet or subjected to 4 weeks of high-fat feeding. Mice were assessed for body composition, fat development, energy balance, and insulin responsiveness. Adipogenesis was measured from ex vivo fat preparations, and in mouse embryonic fibroblasts (MEFs) and 3T3-L1 preadipocytes after forced manipulation of Txnip expression. Txnip knockout mice gained significantly more adipose mass than controls due to a primary increase in both calorie consumption and adipogenesis. Despite increased fat mass, Txnip knockout mice were markedly more insulin sensitive than controls, and augmented glucose transport was identified in both adipose and skeletal muscle. RNA interference gene-silenced preadipocytes and Txnip(-/-) MEFs were markedly adipogenic, whereas Txnip overexpression impaired adipocyte differentiation. As increased adipogenesis and insulin sensitivity suggested aspects of augmented peroxisome proliferator-activated receptor-gamma (PPARgamma) response, we investigated Txnip's regulation of PPARgamma function; manipulation of Txnip expression directly regulated PPARgamma expression and activity. Txnip deletion promotes adiposity in the face of high-fat caloric excess; however, loss of this alpha-arrestin protein simultaneously enhances insulin responsiveness in fat and skeletal muscle, revealing Txnip as a novel mediator of insulin resistance and a regulator of adipogenesis.

Innate immunity of surfactant proteins A and D in urinary tract infection with uropathogenic Escherichia coli

PubMed Central

Hu, Fengqi; Ding, Guohua; Zhang, Zhiyong; Gatto, Louis A.; Hawgood, Samuel; Poulain, Francis R.; Cooney, Robert N.; Wang, Guirong

2015-01-01

To investigate the effects of surfactant proteins A and D (SP-A, SP-D) in urinary tract infection (UTI), SP-A and SP-D double knockout (SP-A/D KO) and wild type (WT) C57BL/6 female mice were infected with uropathogenic Escherichia coli by intravesical inoculation. Compared with WT mice SP-A/D KO mice showed increased susceptibility to UTI as evidenced by higher bacterial CFU, more infiltrating neutrophils and severe pathological changes. Keratinocyte-derived chemokine increased in the kidney of WT mice but not in SP-A/D KO mice 24 h post-infection. Compared to control, level of IL-17 was elevated in the kidney of infected WT and SP-A/D KO mice and the level of IL-17 was higher in the infected SP-A/D KO mice than infected WT mice 24 and 48 h post-infection. Basal level of p38 MAPK phosphorylation in SP-A/D KO mice was higher compared to WT mice. Phosphorylated-p38 level was elevated in the kidney of WT mice post-infection but not in SP-A/D KO mice. Furthermore, in vitro growth of uropathogenic E. coli was inhibited by SP-A and SP-D. We conclude that SP-A and SP-D function as mediators of innate immunity by inhibiting bacterial growth and modulating renal inflammation in part by regulating p38 MAPK-related pathway in murine UTI. PMID:26511057

3,4-methylenedioxymethamphetamine self-administration is abolished in serotonin transporter knockout mice.

PubMed

Trigo, José Manuel; Renoir, Thibault; Lanfumey, Laurence; Hamon, Michel; Lesch, Klaus-Peter; Robledo, Patricia; Maldonado, Rafael

2007-09-15

The neurobiological mechanism underlying the reinforcing effects of 3,4-methylenedioxymethamphetamine (MDMA) remains unclear. The aim of the present study was to determine the contribution of the serotonin transporter (SERT) in MDMA self-administration behavior by using knockout (KO) mice deficient in SERT. Knockout mice and wild-type (WT) littermates were trained to acquire intravenous self-administration of MDMA (0, .03, .06, .125, and .25 mg/kg/infusion) on a fixed ratio 1 (FR1) schedule of reinforcement. Additional groups of mice were trained to obtain food and water to rule out operant responding impairments. Microdialysis studies were performed to evaluate dopamine (DA) and serotonin (5-HT) extracellular levels in the nucleus accumbens (NAC) and prefrontal cortex (PFC), respectively, after acute MDMA (10 mg/kg). None of the MDMA doses tested maintained intravenous self-administration in KO animals, whereas WT mice acquired responding for MDMA. Acquisition of operant responding for food and water was delayed in KO mice, but no differences between genotypes were observed on the last day of training. MDMA increased DA extracellular levels to a similar extent in the NAC of WT and KO mice. Conversely, extracellular concentrations of 5-HT in the PFC were increased following MDMA only in WT mice. These findings provide evidence for the specific involvement of SERT in MDMA reinforcing properties.

β1/2 or M2/3 Receptors Are Required for Different Gastrointestinal Motility Responses Induced by Acupuncture at Heterotopic or Homotopic Acupoints

PubMed Central

Yu, Xiaochun; Cui, Changxiang; Yang, Zhaokun; Shi, Hong; Jing, Xianghong; Zhu, Bing

2016-01-01

Acupuncture at homotopic acupoints or heterotopic acupoints is known to either inhibit or facilitate gastrointestinal motility, depending on the acupoint location. However, little effort has been made to investigate the roles of specific receptors (such as adrenergic and muscarinic acetylcholine receptors) in mediating the effects of acupuncture at heterotopic and homotopic acupoints. Different adrenergic receptor subtypes or cholinergic receptor subtypes are predominantly expressed in various sections of the gut, resulting in variations between the effects of acupuncture at heterotopic or homotopic acupoints on gastrointestinal motility. Here, we investigated the role of β1/β2 receptors and M2/M3 receptors in gastrointestinal motility regulated by acupuncture at ST37, a heterotopic acupoint, and ST25, a homotopic acupoint, by simultaneously recording intraluminal pressures in the distal colon and stomach or jejunum and examining fecal phenol red excretion in β1/2 receptor-knockout mice and M2/3 receptor-knockout mice. We found that knockout of the M2/3 receptor significantly inhibited ST37 acupuncture-induced enhancement of gastric motility, jejunal motility, and colonic motility. Additionally, knocking out of the β1/2 receptor significantly diminished the ST25 acupuncture-induced inhibition of gastric motility and jejunal motility without significantly altering the enhancement of colonic motility induced by acupuncture at ST25. Acupuncture at ST37 significantly accelerated gastrointestinal transition in β1/2 receptor-knockout mice and their wild-type littermates. However, this acceleration of gastrointestinal transition was markedly diminished in M2/3 receptor-knockout mice relative to their wild-type littermates. Acupuncture at ST25 significantly increased gastrointestinal transition in β1/2 receptor-knockout mice and significantly decreased gastrointestinal transition in M2/3 receptor-knockout mice without altering gastrointestinal transition in wild-type littermates of either. Our study revealed that M2/3 receptors are required for the gastrointestinal motility associated with whole gastrointestinal transition enhanced by acupuncture at heterotopic acupoints, whereas β1/2 receptors are required for the same gastrointestinal motility processes inhibited by acupuncture at homotopic acupoints. Therefore, our findings reveal important biological mechanisms underlying acupuncture treatment of disorders involving gastrointestinal motility dysfunction. PMID:27978539

Acute food deprivation reverses morphine-induced locomotion deficits in M5 muscarinic receptor knockout mice.

PubMed

Steidl, Stephan; Lee, Esther; Wasserman, David; Yeomans, John S

2013-09-01

Lesions of the pedunculopontine tegmental nucleus (PPT), one of two sources of cholinergic input to the ventral tegmental area (VTA), block conditioned place preference (CPP) for morphine in drug-naïve rats. M5 muscarinic cholinergic receptors, expressed by midbrain dopamine neurons, are critical for the ability of morphine to increase nucleus accumbens dopamine levels and locomotion, and for morphine CPP. This suggests that M5-mediated PPT cholinergic inputs to VTA dopamine neurons critically contribute to morphine-induced dopamine activation, reward and locomotion. In the current study we tested whether food deprivation, which reduces PPT contribution to morphine CPP in rats, could also reduce M5 contributions to morphine-induced locomotion in mice. Acute 18-h food deprivation reversed the phenotypic differences usually seen between non-deprived wild-type and M5 knockout mice. That is, food deprivation increased morphine-induced locomotion in M5 knockout mice but reduced morphine-induced locomotion in wild-type mice. Food deprivation increased saline-induced locomotion equally in wild-type and M5 knockout mice. Based on these findings, we suggest that food deprivation reduces the contribution of M5-mediated PPT cholinergic inputs to the VTA in morphine-induced locomotion and increases the contribution of a PPT-independent pathway. The contributions of cholinergic, dopaminergic and GABAergic neurons to the effects of acute food deprivation are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Peripheral surgical wounding may induce cognitive impairment through interlukin-6-dependent mechanisms in aged mice.

PubMed

Dong, Yuanlin; Xu, Zhipeng; Huang, Lining; Zhang, Yiying; Xie, Zhongcong

2016-01-01

Post-operative cognitive dysfunction (POCD) is associated with morbidity, mortality and increased cost of medical care. However, the neuropathogenesis and targeted interventions of POCD remain largely to be determined. We have found that the peripheral surgical wounding induces an age-dependent Aβ accumulation, neuroinflammation and cognitive impairment in aged mice. Pro-inflammatory cytokine interlukin-6 (IL-6) has been reported to be associated with cognitive impairment in rodents and humans. However, the role of IL-6 in the neuropathogenesis of POCD is unknown. We therefore employed pharmacological (IL-6 antibody) and genetic (knockout of IL-6) approach to investigate whether IL-6 contributed to the peripheral surgical wounding-induced cognitive impairment in aged mice. Abdominal surgery under local anesthesia (peripheral surgical wounding) was established in 18-month-old wild-type and IL-6 knockout mice ( n = 6 to 10 in each group). Brain level of IL-6 and cognitive function in the mice were determined by western blot, ELISA at the end of procedure, and Fear Conditioning System at 7 days after the procedure. The peripheral surgical wounding increased the level of IL-6 in the hippocampus of aged wild-type, but not IL-6 knockout mice. IL-6 antibody ameliorated the peripheral surgical wounding-induced cognitive impairment in the aged wild-type mice. Finally, the peripheral surgical wounding did not induce cognitive impairment in the aged IL-6 knockout mice. These data suggested that IL-6 would be a required pro-inflammatory cytokine for the peripheral surgical wounding-induced cognitive impairment. Given this, further studies are warranted to investigate the role of IL-6 in the neuropathogenesis and targeted interventions of POCD.

Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis.

PubMed

Lin, Shin-Jen; Ho, Hsin-Chiu; Lee, Yi-Fen; Liu, Ning-Chun; Liu, Su; Li, Gonghui; Shyr, Chih-Rong; Chang, Chawnshang

2012-06-07

Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

Peripheral surgical wounding may induce cognitive impairment through interlukin-6-dependent mechanisms in aged mice

PubMed Central

Dong, Yuanlin; Xu, Zhipeng; Huang, Lining; Zhang, Yiying; Xie, Zhongcong

2016-01-01

Post-operative cognitive dysfunction (POCD) is associated with morbidity, mortality and increased cost of medical care. However, the neuropathogenesis and targeted interventions of POCD remain largely to be determined. We have found that the peripheral surgical wounding induces an age-dependent Aβ accumulation, neuroinflammation and cognitive impairment in aged mice. Pro-inflammatory cytokine interlukin-6 (IL-6) has been reported to be associated with cognitive impairment in rodents and humans. However, the role of IL-6 in the neuropathogenesis of POCD is unknown. We therefore employed pharmacological (IL-6 antibody) and genetic (knockout of IL-6) approach to investigate whether IL-6 contributed to the peripheral surgical wounding-induced cognitive impairment in aged mice. Abdominal surgery under local anesthesia (peripheral surgical wounding) was established in 18-month-old wild-type and IL-6 knockout mice (n = 6 to 10 in each group). Brain level of IL-6 and cognitive function in the mice were determined by western blot, ELISA at the end of procedure, and Fear Conditioning System at 7 days after the procedure. The peripheral surgical wounding increased the level of IL-6 in the hippocampus of aged wild-type, but not IL-6 knockout mice. IL-6 antibody ameliorated the peripheral surgical wounding-induced cognitive impairment in the aged wild-type mice. Finally, the peripheral surgical wounding did not induce cognitive impairment in the aged IL-6 knockout mice. These data suggested that IL-6 would be a required pro-inflammatory cytokine for the peripheral surgical wounding-induced cognitive impairment. Given this, further studies are warranted to investigate the role of IL-6 in the neuropathogenesis and targeted interventions of POCD. PMID:28217289

Morphologic and Histologic Comparison of Hypertrophic Scar in Nude Mice, T-Cell Receptor, and Recombination Activating Gene Knockout Mice.

PubMed

Momtazi, Moein; Ding, Jie; Kwan, Peter; Anderson, Colin C; Honardoust, Dariush; Goekjian, Serge; Tredget, Edward E

2015-12-01

Proliferative scars in nude mice have demonstrated morphologic and histologic similarities to human hypertrophic scar. Gene knockout technology provides the opportunity to study the effect of deleting immune cells in various disease processes. The authors' objective was to test whether grafting human skin onto T-cell receptor (TCR) αβ-/-γδ-/-, recombination activating gene (RAG)-1-/-, and RAG-2γ-/-c-/- mice results in proliferative scars consistent with human hypertrophic scar and to characterize the morphologic, histologic, and cellular changes that occur after removing immune cells. Nude TCRαβ-/-γδ-/-, RAG-1-/-, and RAG-2-/-γc-/- mice (n = 20 per strain) were grafted with human skin and euthanized at 30, 60, 120, and 180 days. Controls (n = 5 per strain) were autografted with mouse skin. Scars and normal skin were harvested at each time point. Sections were stained with hematoxylin and eosin, Masson's trichrome, and immunohistochemistry for anti-human leukocyte antigen-ABC, α-smooth muscle actin, decorin, and biglycan. TCRαβ-/-γδ-/-, RAG-1-/-, and RAG-2-/-γc-/- mice grafted with human skin developed firm, elevated scars with histologic and immunohistochemical similarities to human hypertrophic scar. Autografted controls showed no evidence of pathologic scarring. Knockout animals demonstrated a capacity for scar remodeling not observed in nude mice where reductions in α-smooth muscle actin staining pattern and scar thickness occurred over time. Human skin transplanted onto TCRαβ-/-γδ-/-, RAG-1-/-, and RAG-2-/-γc-/- mice results in proliferative scars with morphologic and histologic features of human hypertrophic scar. Remodeling of proliferative scars generated in knockout animals is analogous to changes in human hypertrophic scar. These animal models may better represent the natural history of human hypertrophic scar.

MAPK phosphotase 5 deficiency contributes to protection against blood-stage Plasmodium yoelii 17XL infection in mice.

PubMed

Cheng, Qianqian; Zhang, Qingfeng; Xu, Xindong; Yin, Lan; Sun, Lin; Lin, Xin; Dong, Chen; Pan, Weiqing

2014-04-15

Cell-mediated immunity plays a crucial role in the development of host resistance to asexual blood-stage malaria infection. However, little is known of the regulatory factors involved in this process. In this study, we investigated the impact of MAPK phosphotase 5 (MKP5) on protective immunity against a lethal Plasmodium yoelii 17XL blood-stage infection using MKP5 knockout C57BL/6 mice. Compared with wild-type control mice, MKP5 knockout mice developed significantly lower parasite burdens with prolonged survival times. We found that this phenomenon correlated with a rapid and strong IFN-γ-dependent cellular immune response during the acute phase of infection. Inactivation of IFN-γ by the administration of a neutralizing Ab significantly reduced the protective effects in MKP5 knockout mice. By analyzing IFN-γ production in innate and adaptive lymphocyte subsets, we observed that MKP5 deficiency specifically enhanced the IFN-γ response mediated by CD4+ T cells, which was attributable to the increased stimulatory capacity of splenic CD11c+ dendritic cells. Furthermore, following vaccination with whole blood-stage soluble plasmodial Ag, MKP5 knockout mice acquired strongly enhanced Ag-specific immune responses and a higher level of protection against subsequent P. yoelii 17XL challenge. Finally, we found the enhanced response mediated by MKP5 deficiency resulted in a lethal consequence in mice when infected with nonlethal P. yoelii 17XNL. Thus, our data indicate that MKP5 is a potential regulator of immune resistance against Plasmodium infection in mice, and that an understanding of the role of MKP5 in manipulating anti-malaria immunity may provide valuable information on the development of better control strategies for human malaria.

Nonessential role of beta3 and beta5 integrin subunits for efficient clearance of cellular debris after light-induced photoreceptor degeneration.

PubMed

Joly, Sandrine; Samardzija, Marijana; Wenzel, Andreas; Thiersch, Markus; Grimm, Christian

2009-03-01

During light-induced photoreceptor degeneration, large amounts of cellular debris are formed that must be cleared from the subretinal space. The integrins alphavbeta5 and alphavbeta3 are involved in the normal physiological process of phagocytosis in the retina. This study was conducted to investigate the question of whether the lack of beta5 and/or beta3 integrin subunits might influence the course of retinal degeneration and/or clearance of photoreceptor debris induced by acute exposure to light. Wild-type, beta5(-/-) and beta3(-/-) single-knockout, and beta3(-/-)/beta5(-/-) Ccl2(-/-)/beta5(-/-) double-knockout mice were exposed to 13,000 lux of white light for 2 hours to induce severe photoreceptor degeneration. Real-time PCR and Western blot analysis were used to analyze gene and protein expression, light- and electron microscopy to judge retinal morphology, and immunofluorescence to study retinal distribution of proteins. Individual or combined deletion of beta3 and beta5 integrin subunits did not affect the pattern of photoreceptor cell loss or the clearance of photoreceptor debris in mice compared with that in wild-type mice. Invading macrophages may contribute to efficient phagocytosis. However, ablation of the MCP-1 gene did not prevent macrophage recruitment. Several chemokines in addition to MCP-1 were induced after light-induced damage that may have compensated for the deletion of MCP-1. Acute clearance of a large amount of cellular debris from the subretinal space involves invading macrophages and does not depend on beta3 and beta5 integrins.

Na+/H+ exchanger isoform 1-induced osteopontin expression facilitates cardiac hypertrophy through p90 ribosomal S6 kinase.

PubMed

Abdulrahman, Nabeel; Jaspard-Vinassa, Beatrice; Fliegel, Larry; Jabeen, Aayesha; Riaz, Sadaf; Gadeau, Alain-Pierre; Mraiche, Fatima

2018-05-01

Cardiovascular diseases are the leading cause of death worldwide. One in three cases of heart failure is due to dilated cardiomyopathy. The Na + /H + exchanger isoform 1 (NHE1), a multifunctional protein and the key pH regulator in the heart, has been demonstrated to be increased in this condition. We have previously demonstrated that elevated NHE1 activity induced cardiac hypertrophy in vivo. Furthermore, the overexpression of active NHE1 elicited modulation of gene expression in cardiomyocytes including an upregulation of myocardial osteopontin (OPN) expression. To determine the role of OPN in inducing NHE1-mediated cardiomyocyte hypertrophy, double transgenic mice expressing active NHE1 and OPN knockout were generated and assessed by echocardiography and the cardiac phenotype. Our studies showed that hearts expressing active NHE1 exhibited cardiac remodeling indicated by increased systolic and diastolic left ventricular internal diameter and increased ventricular volume. Moreover, these hearts demonstrated impaired function with decreased fractional shortening and ejection fraction. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA was upregulated, and there was an increase in heart cell cross-sectional area confirming the cardiac hypertrophic effect. Moreover, NHE1 transgenic mice also showed increased collagen deposition, upregulation of CD44 and phosphorylation of p90 ribosomal s6 kinase (RSK), effects that were regressed in OPN knockout mice. In conclusion, we developed an interesting comparative model of active NHE1 transgenic mouse lines which express a dilated hypertrophic phenotype expressing CD44 and phosphorylated RSK, effects which were regressed in absence of OPN.

Age-dependent Changes of Cerebral Copper Metabolism in Atp7b−/− Knockout Mouse Model of Wilson’s Disease by [64Cu]CuCl2-PET/CT

PubMed Central

Xie, Fang; Xi, Yin; Pascual, Juan M.; Muzik, Otto; Peng, Fangyu

2017-01-01

Copper is a nutritional metal required for brain development and function. Wilson’s disease (WD), or hepatolenticular degeneration, is an inherited human copper metabolism disorder caused by mutation of ATP7B gene. Many WD patients present with variable neurological and psychiatric symptoms, which may be related to neurodegeneration secondary to copper metabolism imbalance. The objective of this study is to explore feasibility and use of copper-64 chloride ([64C]CuCl2) as a tracer for noninvasive assessment of age-dependence changes of cerebral copper metabolism in WD using an Atp7b−/− knockout mouse model of WD and a positron emission tomography/computed tomography (PET/CT) scanner. Continuing from recent study of biodistribution and radiation dosimetry of [64C]CuCl2 in Atp7b−/− knockout mice, PET quantitative analysis revealed low 64Cu radioactivity in the brains of Atp7b−/− knockout mice at 7th week of age, compared with the 64Cu radioactivity in the brains of age and gender-matched wild type C57BL/6 mice, at 24 hour (h) post intravenous injection of [64C]CuCl2 as a tracer. Furthermore, age-dependent increase of 64Cu radioactivity was detected in the brains of Atp7b−/− knockout mice from 13th to 21th week of age, using the data derived from a longitudinal [64C]CuCl2-PET/CT study of Atp7b−/− knockout mice with orally administered [64Cu]CuCl2 as a tracer. The findings of this study support the use of [64Cu]CuCl2-PET/CT as a tool for noninvasive assessment of age-dependent changes of cerebral copper metabolism in WD patients presenting with variable neurological and psychiatric symptoms. PMID:28130615

Age-dependent changes of cerebral copper metabolism in Atp7b -/- knockout mouse model of Wilson's disease by [64Cu]CuCl2-PET/CT.

PubMed

Xie, Fang; Xi, Yin; Pascual, Juan M; Muzik, Otto; Peng, Fangyu

2017-06-01

Copper is a nutritional metal required for brain development and function. Wilson's disease (WD), or hepatolenticular degeneration, is an inherited human copper metabolism disorder caused by a mutation of the ATP7B gene. Many WD patients present with variable neurological and psychiatric symptoms, which may be related to neurodegeneration secondary to copper metabolism imbalance. The objective of this study was to explore the feasibility and use of copper-64 chloride ([ 64 C]CuCl 2 ) as a tracer for noninvasive assessment of age-dependent changes of cerebral copper metabolism in WD using an Atp7b -/- knockout mouse model of WD and positron emission tomography/computed tomography (PET/CT) imaging. Continuing from our recent study of biodistribution and radiation dosimetry of [ 64 C]CuCl 2 in Atp7b -/- knockout mice, PET quantitative analysis revealed low 64 Cu radioactivity in the brains of Atp7b -/- knockout mice at 7th weeks of age, compared with 64 Cu radioactivity in the brains of age- and gender-matched wild type C57BL/6 mice, at 24 h (h) post intravenous injection of [ 64 C]CuCl 2 as a tracer. Furthermore, age-dependent increase of 64 Cu radioactivity was detected in the brains of Atp7b -/- knockout mice from the 13th to 21th weeks of age, based on the data derived from a longitudinal [ 64 C]CuCl 2 -PET/CT study of Atp7b -/- knockout mice with orally administered [ 64 Cu]CuCl 2 as a tracer. The findings of this study support clinical use of [ 64 Cu]CuCl 2 -PET/CT imaging as a tool for noninvasive assessment of age-dependent changes of cerebral copper metabolism in WD patients presenting with variable neurological and psychiatric symptoms.

The guinea pig ileum lacks the direct, high-potency, M(2)-muscarinic, contractile mechanism characteristic of the mouse ileum.

PubMed

Griffin, Michael T; Matsui, Minoru; Ostrom, Rennolds S; Ehlert, Frederick J

2009-10-01

We explored whether the M(2) muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M(3) muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M(3) selectivity. Then, we used 4-DAMP mustard to inactivate M(3) responses in the guinea pig ileum to attempt to reveal direct, M(2) receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M(2) receptor knockout, and M(3) receptor knockout mice characterized by negative log EC(50) (pEC (50)) values +/- SEM of 6.75 +/- 0.03, 6.26 +/- 0.05, and 6.99 +/- 0.08, respectively. The corresponding E (max) values in wild-type and M(2) receptor knockout mice were approximately the same, but that in the M(3) receptor knockout mouse was only 36% of wild type. Following 4-DAMP mustard treatment, the concentration-response curve of oxotremorine-M in wild-type ileum resembled that of the M(3) knockout mouse in terms of its pEC (50), E (max), and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M(3) responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M(3) knockout mouse. Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M(3) response. The guinea pig ileum, therefore, lacks a direct, highly potent, M(2)-contractile component but may have a direct, lower potency M(2) component.

Integrin beta 1 inhibition alleviates the chronic hyperproliferative dermatitis phenotype of SHARPIN-deficient mice.

PubMed

Peuhu, Emilia; Salomaa, Siiri I; De Franceschi, Nicola; Potter, Christopher S; Sundberg, John P; Pouwels, Jeroen

2017-01-01

SHARPIN (Shank-Associated RH Domain-Interacting Protein) is a component of the linear ubiquitin chain assembly complex (LUBAC), which enhances TNF-induced NF-κB activity. SHARPIN-deficient (Sharpincpdm/cpdm) mice display multi-organ inflammation and chronic proliferative dermatitis (cpdm) due to TNF-induced keratinocyte apoptosis. In cells, SHARPIN also inhibits integrins independently of LUBAC, but it has remained enigmatic whether elevated integrin activity levels in the dermis of Sharpincpdm/cpdm mice is due to increased integrin activity or is secondary to inflammation. In addition, the functional contribution of increased integrin activation to the Sharpincpdm/cpdm phenotype has not been investigated. Here, we find increased integrin activity in keratinocytes from Tnfr1-/- Sharpincpdm/cpdm double knockout mice, which do not display chronic inflammation or proliferative dermatitis, thus suggesting that SHARPIN indeed acts as an integrin inhibitor in vivo. In addition, we present evidence for a functional contribution of integrin activity to the Sharpincpdm/cpdm skin phenotype. Treatment with an integrin beta 1 function blocking antibody reduced epidermal hyperproliferation and epidermal thickness in Sharpincpdm/cpdm mice. Our data indicate that, while TNF-induced cell death triggers the chronic inflammation and proliferative dermatitis, absence of SHARPIN-dependent integrin inhibition exacerbates the epidermal hyperproliferation in Sharpincpdm/cpdm mice.

Targeted disruption of glutathione peroxidase 4 (GPx4) in mouse skin epithelial cells impairs postnatal hair follicle morphogenesis that is partially rescued through inhibition of COX-2

PubMed Central

Sengupta, Aniruddha; Lichti, Ulrike F.; Carlson, Bradley A.; Cataisson, Christophe; Ryscavage, Andrew O.; Mikulec, Carol; Conrad, Marcus; Fischer, Susan M.; Hatfield, Dolph L.; Yuspa, Stuart H.

2013-01-01

Selenoproteins are essential molecules for the mammalian antioxidant network. We previously demonstrated that targeted loss of all selenoproteins in mouse epidermis disrupted skin and hair development and caused premature death. In the current study we targeted specific selenoproteins for epidermal deletion to determine whether similar phenotypes developed. Keratinocyte-specific knockout mice lacking either the glutathione peroxidase 4 (GPx4) or thioredoxin reductase 1 (TR1) gene were generated by cre-lox technology using K14-cre. TR1 knockout mice had a normal phenotype in resting skin while GPx4 loss in epidermis caused epidermal hyperplasia, dermal inflammatory infiltrate, dysmorphic hair follicles and alopecia in perinatal mice. Unlike epidermal ablation of all selenoproteins, mice ablated for GPx4 recovered after 5 weeks and had a normal lifespan. GPx1 and TR1 were upregulated in the skin and keratinocytes of GPx4 knockout mice. GPx4 deletion reduces keratinocyte adhesion in culture and increases lipid peroxidation and COX-2 levels in cultured keratinocytes and whole skin. Feeding a COX-2 inhibitor to nursing mothers partially prevents development of the abnormal skin phenotype in knockout pups. These data link the activity of cutaneous GPx4 to the regulation of COX-2 and hair follicle morphogenesis and provide insight into the function of individual selenoprotein activity in maintaining cutaneous homeostasis. PMID:23364477

Generation and phenotypic analysis of mice lacking all urea transporters.

PubMed

Jiang, Tao; Li, Yingjie; Layton, Anita T; Wang, Weiling; Sun, Yi; Li, Min; Zhou, Hong; Yang, Baoxue

2017-02-01

Urea transporters (UT) are a family of transmembrane urea-selective channel proteins expressed in multiple tissues and play an important role in the urine concentrating mechanism of the mammalian kidney. UT inhibitors have diuretic activity and could be developed as novel diuretics. To determine if functional deficiency of all UTs in all tissues causes physiological abnormality, we established a novel mouse model in which all UTs were knocked out by deleting an 87 kb of DNA fragment containing most parts of Slc14a1 and Slc14a2 genes. Western blot analysis and immunofluorescence confirmed that there is no expression of urea transporter in these all-UT-knockout mice. Daily urine output was nearly 3.5-fold higher, with significantly lower urine osmolality in all-UT-knockout mice than that in wild-type mice. All-UT-knockout mice were not able to increase urinary urea concentration and osmolality after water deprivation, acute urea loading, or high protein intake. A computational model that simulated UT-knockout mouse models identified the individual contribution of each UT in urine concentrating mechanism. Knocking out all UTs also decreased the blood pressure and promoted the maturation of the male reproductive system. Thus, functional deficiency of all UTs caused a urea-selective urine-concentrating defect with little physiological abnormality in extrarenal organs. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Generation and phenotypic analysis of mice lacking all urea transporters

PubMed Central

Jiang, Tao; Li, Yingjie; Layton, Anita T.; Wang, Weiling; Sun, Yi; Li, Min; Zhou, Hong; Yang, Baoxue

2017-01-01

Urea transporters (UT) are a family of transmembrane urea-selective channel proteins expressed in multiple tissues and play an important role in the urine concentrating mechanism of the mammalian kidney. UT inhibitors have been identified to have diuretic activity and might be developed as novel diuretics. To determine if functional deficiency of all UTs in all tissues causes physiological abnormality, we established a novel mouse model in which all UTs were knocked out by deleting an 87 kb of DNA fragment containing most parts of Slc14a1 and Slc14a2 genes. Western blot analysis and immunofluorescence confirmed that there is no expression of urea transporter in all-UT-knockout mice. Daily urine output was nearly 3.5-fold higher, with significantly lower urine osmolality, in all-UT-knockout-mice than that in wild-type mice, and urine osmolality was significantly lower. All-UT-knockout mice were not able to increase urinary urea concentration and osmolality after water deprivation, acute urea loading or high protein intake. A computational model that simulated UT knockout mouse models identified the individual contribution of each UT in urine concentrating mechanism. Knocking out all UTs also decreased the blood pressure and promoted the maturation of the male reproductive system. These results revealed that functional deficiency of all UTs caused urea selective urine concentrating defect with little physiological abnormality in extrarenal organs. PMID:27914708

A High Serum Iron Level Causes Mouse Retinal Iron Accumulation Despite an Intact Blood-Retinal Barrier

PubMed Central

Zhao, Liangliang; Li, Yafeng; Song, Delu; Song, Ying; Theurl, Milan; Wang, Chenguang; Cwanger, Alyssa; Su, Guanfang; Dunaief, Joshua L.

2015-01-01

The retina can be shielded by the blood-retinal barrier. Because photoreceptors are damaged by excess iron, it is important to understand whether the blood-retinal barrier protects against high serum iron levels. Bone morphogenic protein 6 (Bmp6) knockout mice have serum iron overload. Herein, we tested whether the previously documented retinal iron accumulation in Bmp6 knockout mice might result from the high serum iron levels or, alternatively, low levels of retinal hepcidin, an iron regulatory hormone whose transcription can be up-regulated by Bmp6. Furthermore, to determine whether increases in serum iron can elevate retinal iron levels, we i.v. injected iron into wild-type mice. Retinas were analyzed by real-time quantitative PCR and immunofluorescence to assess the levels of iron-regulated genes/proteins and oxidative stress. Retinal hepcidin mRNA levels in Bmp6 knockout retinas were the same as, or greater than, those in age-matched wild-type retinas, indicating that Bmp6 knockout does not cause retinal hepcidin deficiency. Changes in mRNA levels of L ferritin and transferrin receptor indicated increased retinal iron levels in i.v. iron-injected wild-type mice. Oxidative stress markers were elevated in photoreceptors of mice receiving i.v. iron. These findings suggest that elevated serum iron levels can overwhelm local retinal iron regulatory mechanisms. PMID:25174877

High-sensitivity O-glycomic analysis of mice deficient in core 2 β1,6-N-acetylglucosaminyltransferases

PubMed Central

Ismail, Mohd Nazri; Stone, Erica L; Panico, Maria; Lee, Seung Ho; Luu, Ying; Ramirez, Kevin; Ho, Samuel B; Fukuda, Minoru; Marth, Jamey D; Haslam, Stuart M; Dell, Anne

2011-01-01

Core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT), which exists in three isoforms, C2GnT1, C2GnT2 and C2GnT3, is one of the key enzymes in the O-glycan biosynthetic pathway. These isoenzymes produce core 2 O-glycans and have been correlated with the biosynthesis of core 4 O-glycans and I-branches. Previously, we have reported mice with single and multiple deficiencies of C2GnT isoenzyme(s) and have evaluated the biological and structural consequences of the loss of core 2 function. We now present more comprehensive O-glycomic analyses of neutral and sialylated glycans expressed in the colon, small intestine, stomach, kidney, thyroid/trachea and thymus of wild-type, C2GnT2 and C2GnT3 single knockouts and the C2GnT1–3 triple knockout mice. Very high-quality data have emerged from our mass spectrometry techniques with the capability of detecting O-glycans up to at least 3500 Da. We were able to unambiguously elucidate the types of O-glycan core, branching location and residue linkages, which allowed us to exhaustively characterize structural changes in the knockout tissues. The C2GnT2 knockout mice suffered a major loss of core 2 O-glycans as well as glycans with I-branches on core 1 antennae especially in the stomach and the colon. In contrast, core 2 O-glycans still dominated the O-glycomic profile of most tissues in the C2GnT3 knockout mice. Analysis of the C2GnT triple knockout mice revealed a complete loss of both core 2 O-glycans and branched core 1 antennae, confirming that the three known isoenzymes are entirely responsible for producing these structures. Unexpectedly, O-linked mannosyl glycans are upregulated in the triple deficient stomach. In addition, our studies have revealed an interesting terminal structure detected on O-glycans of the colon tissues that is similar to the RM2 antigen from glycolipids. PMID:20855471

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution.

PubMed

Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

2015-10-01

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53(-/-) mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy.

Isolation, characterization, and molecular regulation of muscle stem cells

PubMed Central

Fukada, So-ichiro; Ma, Yuran; Ohtani, Takuji; Watanabe, Yoko; Murakami, Satoshi; Yamaguchi, Masahiko

2013-01-01

Skeletal muscle has great regenerative capacity which is dependent on muscle stem cells, also known as satellite cells. A loss of satellite cells and/or their function impairs skeletal muscle regeneration and leads to a loss of skeletal muscle power; therefore, the molecular mechanisms for maintaining satellite cells in a quiescent and undifferentiated state are of great interest in skeletal muscle biology. Many studies have demonstrated proteins expressed by satellite cells, including Pax7, M-cadherin, Cxcr4, syndecan3/4, and c-met. To further characterize satellite cells, we established a method to directly isolate satellite cells using a monoclonal antibody, SM/C-2.6. Using SM/C-2.6 and microarrays, we measured the genes expressed in quiescent satellite cells and demonstrated that Hesr3 may complement Hesr1 in generating quiescent satellite cells. Although Hesr1- or Hesr3-single knockout mice show a normal skeletal muscle phenotype, including satellite cells, Hesr1/Hesr3-double knockout mice show a gradual decrease in the number of satellite cells and increase in regenerative defects dependent on satellite cell numbers. We also observed that a mouse's genetic background affects the regenerative capacity of its skeletal muscle and have established a line of DBA/2-background mdx mice that has a much more severe phenotype than the frequently used C57BL/10-mdx mice. The phenotype of DBA/2-mdx mice also seems to depend on the function of satellite cells. In this review, we summarize the methodology of direct isolation, characterization, and molecular regulation of satellite cells based on our results. The relationship between the regenerative capacity of satellite cells and progression of muscular disorders is also summarized. In the last part, we discuss application of the accumulating scientific information on satellite cells to treatment of patients with muscular disorders. PMID:24273513

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

PubMed Central

Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

2015-01-01

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53−/− mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy. PMID:25656653

Combined Allogeneic Tumor Cell Vaccination and Systemic Interleukin 12 Prevents Mammary Carcinogenesis in HER-2/neu Transgenic Mice

PubMed Central

Nanni, Patrizia; Nicoletti, Giordano; De Giovanni, Carla; Landuzzi, Lorena; Di Carlo, Emma; Cavallo, Federica; Pupa, Serenella M.; Rossi, Ilaria; Colombo, Mario P.; Ricci, Cinzia; Astolfi, Annalisa; Musiani, Piero; Forni, Guido; Lollini, Pier-Luigi

2001-01-01

Transgenic Balb/c mice expressing the transforming rat HER-2/neu oncogene develop early and multifocal mammary carcinomas. Within the first 5 months of life the tissue-specific expression of HER-2/neu causes a progression in all their 10 mammary glands from atypical hyperplasia to invasive carcinoma. It was previously observed that chronic administration of interleukin (IL)-12 increased tumor latency, but every mouse eventually succumbed to multiple carcinomas. A significant improvement in tumor prevention was sought by administering allogeneic mammary carcinoma cells expressing HER-2/neu combined with systemic IL-12. This treatment reduced tumor incidence by 90% and more than doubled mouse lifetime. For the maximum prevention p185neu antigen must be expressed by allogeneic cells. IL-12 treatment strongly increased the cell vaccine efficacy. The mammary glands of mice receiving the combined treatment displayed a markedly reduced epithelial cell proliferation, angiogenesis, and HER-2/neu expression, while the few hyperplastic foci were heavily infiltrated by granulocytes, macrophages, and CD8+ lymphocytes. Specific anti–HER-2/neu antibodies were produced and a nonpolarized activation of CD4+ and CD8+ cells secreting IL-4 and interferon (IFN)-γ were evident. A central role for IFN-γ in the preventive effect was proven by the lack of efficacy of vaccination in IFN-γ gene knockout HER-2/neu transgenic Balb/c mice. A possible requirement for IFN-γ is related to its effect on antibody production, in particular on IgG2a and IgG2b subclasses, that were not induced in IFN-γ knockout HER-2/neu mice. In conclusion, our data show that an allogeneic HER-2/neu–expressing cell vaccine combined with IL-12 systemic treatment can prevent the onset of genetically determined tumors. PMID:11696586

Absence of renal enlargement in fructose-fed proximal-tubule-select insulin receptor (IR), insulin-like-growth factor receptor (IGF1R) double knockout mice.

PubMed

Li, Lijun; Byrd, Marcus; Doh, Kwame; Dixon, Patrice D; Lee, Hwal; Tiwari, Swasti; Ecelbarger, Carolyn M

2016-12-01

The major site of fructose metabolism in the kidney is the proximal tubule (PT). To test whether insulin and/or IGF1 signaling in the PT is involved in renal structural/functional responses to dietary fructose, we bred mice with dual knockout (KO) of the insulin receptor (IR) and the IGF1 receptor (IGF1R) in PT by Cre-lox recombination, using a γ-glutamyl transferase promoter. KO mice had slightly (~10%) reduced body and kidney weights, as well as, a reduction in mean protein-to-DNA ratio in kidney cortex suggesting smaller cell size. Under control diet, IR and IGF1R protein band densities were 30-50% (P < 0.05) lower than WT, and the relative difference was greater in male animals. Male, but not female KO, also had significantly reduced band densities for Akt (protein kinase B), phosphorylated Akt T308 and IR Y 1162/1163 A high-fructose diet (1-month) led to a significant increase in kidney weight in WT males (12%), but not in KO males or in either genotype of female mice. Kidney enlargement in the WT males was accompanied by a small, insignificant fall in protein-to-DNA ratio, supporting hyperplasia rather than hypertrophy. Fructose feeding of male WT mice led to significantly higher sodium bicarbonate exchanger (NBCe1), sodium hydrogen exchanger (NHE3), sodium phosphate co-transporter (NaPi-2), and transforming growth factor-β (TGF-β) abundances, as compared to male KO, suggesting elevated transport capacity and an early feature of fibrosis may have accompanied the renal enlargement. Overall, IR and/or IGF1R appear to have a role in PT cell size and enlargement in response to high-fructose diet. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Enhanced effects of amphetamine but reduced effects of the hallucinogen, 5-MeO-DMT, on locomotor activity in 5-HT(1A) receptor knockout mice: implications for schizophrenia.

PubMed

van den Buuse, Maarten; Ruimschotel, Emma; Martin, Sally; Risbrough, Victoria B; Halberstadt, Adam L

2011-01-01

Serotonin-1A (5-HT(1A)) receptors may play a role in schizophrenia and the effects of certain antipsychotic drugs. However, the mechanism of interaction of 5-HT(1A) receptors with brain systems involved in schizophrenia, remains unclear. Here we show that 5-HT(1A) receptor knockout mice display enhanced locomotor hyperactivity to acute treatment with amphetamine, a widely used animal model of hyperdopaminergic mechanisms in psychosis. In contrast, the effect of MK-801 on locomotor activity, modeling NMDA receptor hypoactivity, was unchanged in the knockouts. The effect of the hallucinogen 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) was markedly reduced in 5-HT(1A) receptor knockout mice. There were no changes in apomorphine-induced disruption of PPI, a model of sensory gating deficits seen in schizophrenia. Similarly, there were no major changes in density of dopamine transporters (DAT) or dopamine D(1) or D(2) receptors which could explain the behavioural changes observed in 5-HT(1A) receptor knockout mice. These results extend our insight into the possible role of these receptors in aspects of schizophrenia. As also suggested by previous studies using agonist and antagonist drugs, 5-HT(1A) receptors may play an important role in hallucinations and to modulate dopaminergic activity in the brain. Copyright © 2011 Elsevier Ltd. All rights reserved.

UHRF2 regulates local 5-methylcytosine and suppresses spontaneous seizures

PubMed Central

Liu, Yidan; Zhang, Bin; Meng, Xiaoyu; Korn, Matthew J.; Parent, Jack M.; Lu, Lin-Yu; Yu, Xiaochun

2017-01-01

ABSTRACT The 5-methylcytosine (5mC) modification regulates multiple cellular processes and is faithfully maintained following DNA replication. In addition to DNA methyltransferase (DNMT) family proteins, ubiquitin-like PHD and ring finger domain-containing protein 1 (UHRF1) plays an important role in the maintenance of 5mC levels. Loss of UHRF1 abolishes 5mC in cells and leads to embryonic lethality in mice. Interestingly, UHRF1 has a paralog, UHRF2, that has similar sequence and domain architecture, but its biologic function is not clear. Here, we have generated Uhrf2 knockout mice and characterized the role of UHRF2 in vivo. Uhrf2 knockout mice are viable, but the adult mice develop frequent spontaneous seizures and display abnormal electrical activities in brain. Despite no global DNA methylation changes, 5mC levels are decreased at certain genomic loci in the brains of Uhrf2 knockout mice. Therefore, our study has revealed a unique role of UHRF2 in the maintenance of local 5mC levels in brain that is distinct from that of its paralog UHRF1. PMID:28402695

Suppression of Autophagy in Osteocytes Mimics Skeletal Aging*

PubMed Central

Onal, Melda; Piemontese, Marilina; Xiong, Jinhu; Wang, Yiying; Han, Li; Ye, Shiqiao; Komatsu, Masaaki; Selig, Martin; Weinstein, Robert S.; Zhao, Haibo; Jilka, Robert L.; Almeida, Maria; Manolagas, Stavros C.; O'Brien, Charles A.

2013-01-01

Bone mass declines with age but the mechanisms responsible remain unclear. Here we demonstrate that deletion of a conditional allele for Atg7, a gene essential for autophagy, from osteocytes caused low bone mass in 6-month-old male and female mice. Cancellous bone volume and cortical thickness were decreased, and cortical porosity increased, in conditional knock-out mice compared with control littermates. These changes were associated with low osteoclast number, osteoblast number, bone formation rate, and wall width in the cancellous bone of conditional knock-out mice. In addition, oxidative stress was higher in the bones of conditional knock-out mice as measured by reactive oxygen species levels in the bone marrow and by p66shc phosphorylation in L6 vertebra. Each of these changes has been previously demonstrated in the bones of old versus young adult mice. Thus, these results demonstrate that suppression of autophagy in osteocytes mimics, in many aspects, the impact of aging on the skeleton and suggest that a decline in autophagy with age may contribute to the low bone mass associated with aging. PMID:23645674

Male and Female Mice Lacking Neuroligin-3 Modify the Behavior of Their Wild-Type Littermates.

PubMed

Kalbassi, Shireene; Bachmann, Sven O; Cross, Ellen; Roberton, Victoria H; Baudouin, Stéphane J

2017-01-01

In most mammals, including humans, the postnatal acquisition of normal social and nonsocial behavior critically depends on interactions with peers. Here we explore the possibility that mixed-group housing of mice carrying a deletion of Nlgn3 , a gene associated with autism spectrum disorders, and their wild-type littermates induces changes in each other's behavior. We have found that, when raised together, male Nlgn3 knockout mice and their wild-type littermates displayed deficits in sociability. Moreover, social submission in adult male Nlgn3 knockout mice correlated with an increase in their anxiety. Re-expression of Nlgn3 in parvalbumin-expressing cells in transgenic animals rescued their social behavior and alleviated the phenotype of their wild-type littermates, further indicating that the social behavior of Nlgn3 knockout mice has a direct and measurable impact on wild-type animals' behavior. Finally, we showed that, unlike male mice, female mice lacking Nlgn3 were insensitive to their peers' behavior but modified the social behavior of their littermates. Altogether, our findings show that the environment is a critical factor in the development of behavioral phenotypes in transgenic and wild-type mice. In addition, these results reveal that the social environment has a sexually dimorphic effect on the behavior of mice lacking Nlgn3 , being more influential in males than females.

Male and Female Mice Lacking Neuroligin-3 Modify the Behavior of Their Wild-Type Littermates

PubMed Central

Kalbassi, Shireene; Cross, Ellen

2017-01-01

Abstract In most mammals, including humans, the postnatal acquisition of normal social and nonsocial behavior critically depends on interactions with peers. Here we explore the possibility that mixed-group housing of mice carrying a deletion of Nlgn3, a gene associated with autism spectrum disorders, and their wild-type littermates induces changes in each other’s behavior. We have found that, when raised together, male Nlgn3 knockout mice and their wild-type littermates displayed deficits in sociability. Moreover, social submission in adult male Nlgn3 knockout mice correlated with an increase in their anxiety. Re-expression of Nlgn3 in parvalbumin-expressing cells in transgenic animals rescued their social behavior and alleviated the phenotype of their wild-type littermates, further indicating that the social behavior of Nlgn3 knockout mice has a direct and measurable impact on wild-type animals’ behavior. Finally, we showed that, unlike male mice, female mice lacking Nlgn3 were insensitive to their peers’ behavior but modified the social behavior of their littermates. Altogether, our findings show that the environment is a critical factor in the development of behavioral phenotypes in transgenic and wild-type mice. In addition, these results reveal that the social environment has a sexually dimorphic effect on the behavior of mice lacking Nlgn3, being more influential in males than females. PMID:28795135

Analysis of knockout mice suggests a role for VGF in the control of fat storage and energy expenditure

PubMed Central

Watson, Elizabeth; Fargali, Samira; Okamoto, Haruka; Sadahiro, Masato; Gordon, Ronald E; Chakraborty, Tandra; Sleeman, Mark W; Salton, Stephen R

2009-01-01

Background Previous studies of mixed background mice have demonstrated that targeted deletion of Vgf produces a lean, hypermetabolic mouse that is resistant to diet-, lesion-, and genetically-induced obesity. To investigate potential mechanism(s) and site(s) of action of VGF, a neuronal and endocrine secreted protein and neuropeptide precursor, we further analyzed the metabolic phenotypes of two independent VGF knockout lines on C57Bl6 backgrounds. Results Unlike hyperactive VGF knockout mice on a mixed C57Bl6-129/SvJ background, homozygous mutant mice on a C57Bl6 background were hypermetabolic with similar locomotor activity levels to Vgf+/Vgf+ mice, during day and night cycles, indicating that mechanism(s) other than hyperactivity were responsible for their increased energy expenditure. In Vgf-/Vgf- knockout mice, morphological analysis of brown and white adipose tissues (BAT and WAT) indicated decreased fat storage in both tissues, and decreased adipocyte perimeter and area in WAT. Changes in gene expression measured by real-time RT-PCR were consistent with increased fatty acid oxidation and uptake in BAT, and increased lipolysis, decreased lipogenesis, and brown adipocyte differentiation in WAT, suggesting that increased sympathetic nervous system activity in Vgf-/Vgf- mice may be associated with or responsible for alterations in energy expenditure and fat storage. In addition, uncoupling protein 1 (UCP1) and UCP2 protein levels, mitochondrial number, and mitochondrial cristae density were upregulated in Vgf-/Vgf- BAT. Using immunohistochemical and histochemical techniques, we detected VGF in nerve fibers innervating BAT and Vgf promoter-driven reporter expression in cervical and thoracic spinal ganglia that project to and innervate the chest wall and tissues including BAT. Moreover, VGF peptide levels were quantified by radioimmunoassay in BAT, and were found to be down-regulated by a high fat diet. Lastly, despite being hypermetabolic, VGF knockout mice were cold intolerant. Conclusion We propose that VGF and/or VGF-derived peptides modulate sympathetic outflow pathways to regulate fat storage and energy expenditure. PMID:19863797

Differential expression of glutamate transporters EAAT1 and EAAT2 in mice deficient for PACAP-type I receptor.

PubMed

Zink, M; Schmitt, A; Henn, F A; Gass, P

2004-12-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) modulates glutamatergic neurotransmission and induces the expression of glutamate transporters EAAT1 and EAAT2 in newborn mouse astroglial cell cultures. Since nanomolar concentrations of PACAP exert this effect, signal transduction via the high affinity PACAP-type I-receptor PAC1 was assumed. To test this hypothesis and to assess the importance of PAC1-signalling in vivo, we analyzed glutamate transporter expression in mice with a PAC1 knockout. EAAT1 and EAAT2 expression was investigated in the hippocampus and the cerebral cortex of PAC1 mutant mice and wildtype littermates by semiquantitative in-situ-hybridization. PAC1-knockout mice show a subtle but significant reduction of EAAT1 expression in the dentate gyrus. In contrast, reduced expression levels of EAAT1 in the cerebral cortex did not reach statistical significance and EAAT2 expression was unchanged in CA3 and cerebral cortex of PAC1 mutant mice. Our data confirm the previously reported in-vitro-regulation of EAAT1 in the adult nervous system in vivo. EAAT2 expression, however, is unchanged in PAC1 knockout mice, most likely due to counterbalancing factors.

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

PubMed

Kuwahara, Go; Hashimoto, Takuya; Tsuneki, Masayuki; Yamamoto, Kota; Assi, Roland; Foster, Trenton R; Hanisch, Jesse J; Bai, Hualong; Hu, Haidi; Protack, Clinton D; Hall, Michael R; Schardt, John S; Jay, Steven M; Madri, Joseph A; Kodama, Shohta; Dardik, Alan

2017-06-01

Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation. AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice. CD44 mRNA and protein expression was increased in wild-type AVF. CD44 knockout mice showed no increase in AVF wall thickness (8.9 versus 26.8 μm; P =0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared with control AVF. CD44 knockout mice also showed no increase in vascular cell adhesion molecule-1 expression, intercellular adhesion molecule-1 expression, and monocyte chemoattractant protein-1 expression in the AVF compared with controls; there were also no increased M2 macrophage markers (transglutaminase-2: 81.5-fold, P =0.0015; interleukin-10: 7.6-fold, P =0.0450) in CD44 knockout mice. Delivery of monocyte chemoattractant protein-1 to CD44 knockout mice rescued the phenotype with thicker AVF walls (27.2 versus 14.7 μm; P =0.0306), increased collagen density (2.4-fold; P =0.0432), and increased number of M2 macrophages (2.1-fold; P =0.0335). CD44 promotes accumulation of M2 macrophages, extracellular matrix deposition, and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation. © 2017 American Heart Association, Inc.

Slc25a12 disruption alters myelination and neurofilaments: a model for a hypomyelination syndrome and childhood neurodevelopmental disorders.

PubMed

Sakurai, Takeshi; Ramoz, Nicolas; Barreto, Marta; Gazdoiu, Mihaela; Takahashi, Nagahide; Gertner, Michael; Dorr, Nathan; Gama Sosa, Miguel A; De Gasperi, Rita; Perez, Gissel; Schmeidler, James; Mitropoulou, Vivian; Le, H Carl; Lupu, Mihaela; Hof, Patrick R; Elder, Gregory A; Buxbaum, Joseph D

2010-05-01

SLC25A12, a susceptibility gene for autism spectrum disorders that is mutated in a neurodevelopmental syndrome, encodes a mitochondrial aspartate-glutamate carrier (aspartate-glutamate carrier isoform 1 [AGC1]). AGC1 is an important component of the malate/aspartate shuttle, a crucial system supporting oxidative phosphorylation and adenosine triphosphate production. We characterized mice with a disruption of the Slc25a12 gene, followed by confirmatory in vitro studies. Slc25a12-knockout mice, which showed no AGC1 by immunoblotting, were born normally but displayed delayed development and died around 3 weeks after birth. In postnatal day 13 to 14 knockout brains, the brains were smaller with no obvious alteration in gross structure. However, we found a reduction in myelin basic protein (MBP)-positive fibers, consistent with a previous report. Furthermore, the neocortex of knockout mice contained abnormal neurofilamentous accumulations in neurons, suggesting defective axonal transport and/or neurodegeneration. Slice cultures prepared from knockout mice also showed a myelination defect, and reduction of Slc25a12 in rat primary oligodendrocytes led to a cell-autonomous reduction in MBP expression. Myelin deficits in slice cultures from knockout mice could be reversed by administration of pyruvate, indicating that reduction in AGC1 activity leads to reduced production of aspartate/N-acetylaspartate and/or alterations in the dihydronicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide(+) ratio, resulting in myelin defects. Our data implicate AGC1 activity in myelination and in neuronal structure and indicate that while loss of AGC1 leads to hypomyelination and neuronal changes, subtle alterations in AGC1 expression could affect brain development, contributing to increased autism susceptibility. Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Muscarinic receptor subtypes involved in carbachol-induced contraction of mouse uterine smooth muscle.

PubMed

Kitazawa, Takio; Hirama, Ryuichi; Masunaga, Kozue; Nakamura, Tatsuro; Asakawa, Koichi; Cao, Jinshan; Teraoka, Hiroki; Unno, Toshihiro; Komori, Sei-ichi; Yamada, Masahisa; Wess, Jürgen; Taneike, Tetsuro

2008-06-01

Functional muscarinic acetylcholine receptors present in the mouse uterus were characterized by pharmacological and molecular biological studies using control (DDY and wild-type) mice, muscarinic M2 or M3 single receptor knockout (M2KO, M3KO), and M2 and M3 receptor double knockout mice (M2/M3KO). Carbachol (10 nM-100 microM) increased muscle tonus and phasic contractile activity of uterine strips of control mice in a concentration-dependent manner. The maximum carbachol-induced contractions (Emax) differed between cervical and ovarian regions of the uterus. The stage of the estrous cycle had no significant effect on carbachol concentration-response relationships. Tetrodotoxin did not decrease carbachol-induced contractions, but the muscarinic receptor antagonists (11-[[2-[(diethylaminomethyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b[2,3-b][1,4]benzodiazepin6-one (AF-DX116), N-[2-[2-[(dipropylamino)methyl]-1-piperidinyl]ethyl]-5,6-dihydro-6-oxo-11H-pyrido[2,3-b][1,4] benzodiazepine-11-carboxamide (AF-DX384), 4-diphenylacetoxy-N-methyl-piperidine(4-DAMP), para-fluoro-hexa hydro-sila-diphenidol (p-F-HHSiD), himbacine, methoctramine, pirenzepine, and tropicamide) inhibited carbachol-induced contractions in a competitive fashion. The pKb values for these muscarinic receptor antagonists correlated well with the known pKi values of these antagonists for the M3 muscarinic receptor. In uterine strips isolated from mice treated with pertussis toxin (100 microg/kg, i.p. for 96 h), Emax values for carbachol were significantly decreased, but effective concentration that caused 50% of Emax values (EC50) remained unchanged. In uterine strips treated with 4-DAMP mustard (30 nM) and AF-DX116 (1 microM), followed by subsequent washout of AF-DX116, neither carbachol nor N,N,N,-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium iodide (oxotremorine-M) caused any contractile responses. Both M2 and M3 muscarinic receptor messenger RNAs were detected in the mouse uterus via reverse transcription polymerase chain reaction. Carbachol also caused contraction of uterine strips isolated from M2KO mice, but the concentration-response curve was shifted to the right and downward compared with that for the corresponding wild-type mice. On the other hand, uterine strips isolated from M3KO and M2/M3 double KO mice were virtually insensitive to carbachol. In conclusion, although both M2 and M3 muscarinic receptors were expressed in the mouse uterus, carbachol-induced contractile responses were predominantly mediated by the M3 receptor. Activation of M2 receptors alone did not cause uterine contractions; however, M2 receptor activation enhanced M3 receptor-mediated contractions in the mouse uterus.

Role of P-glycoprotein and breast cancer resistance protein-1 in the brain penetration and brain pharmacodynamic activity of the novel phosphatidylinositol 3-kinase inhibitor GDC-0941.

PubMed

Salphati, Laurent; Lee, Leslie B; Pang, Jodie; Plise, Emile G; Zhang, Xiaolin

2010-09-01

2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) is a novel small molecule inhibitor of the phosphatidylinositol 3-kinase (PI3K) pathway currently evaluated in the clinic as an anticancer agent. The objectives of this study were to determine in vitro whether GDC-0941 was a substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp1) and to investigate the impact of these transporters on the pharmacokinetics, brain penetration, and activity of GDC-0941 in FVBn mice (wild-type) and Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)/Bcrp1(-/-) knockout mice. Studies with Madin-Darby canine kidney cells transfected with P-gp or Bcrp1 established that this compound was a substrate of both transporters. After administrations to mice, GDC-0941 brain-to-plasma ratio ranged from 0.02 to 0.06 in the wild-type and Bcrp1(-/-) mice and was modestly higher in the Mdr1a/b(-/-) mice, ranging from 0.08 to 0.11. In contrast, GDC-0941 brain-to-plasma ratio in Mdr1a/b(-/-)/Bcrp1(-/-) triple knockout mice was 30-fold higher than in the wild-type mice. The plasma clearance of GDC-0941 was similar in wild-type and all knockout mice, ranging from 15 to 25 ml/(min . kg) in the wild-type mice and from 18 to 35 ml/(min . kg) in the knockout mice. Exposure after oral administration was comparable in the four strains of mice. The PI3K pathway was markedly inhibited in the brain of Mdr1a/b(-/-)/Bcrp1(-/-) mice for up to 6 h postdose, as evidenced by a 60% suppression of the phosphorylated Akt signal, whereas no inhibition was detected in the brain of wild-type mice. The concerted effects of P-gp and Bcrp1 in restricting GDC-0941 access and pathway modulation in mouse brain may have implications for the treatment of patients with brain tumors.

Lynx1 Limits Dendritic Spine Turnover in the Adult Visual Cortex

PubMed Central

Sajo, Mari

2016-01-01

Dendritic spine turnover becomes limited in the adult cerebral cortex. Identification of specific aspects of spine dynamics that can be unmasked in adulthood and its regulatory molecular mechanisms could provide novel therapeutic targets for inducing plasticity at both the functional and structural levels for robust recovery from brain disorders and injuries in adults. Lynx1, an endogenous inhibitor of nicotinic acetylcholine receptors, was previously shown to increase its expression in adulthood and thus to limit functional ocular dominance plasticity in adult primary visual cortex (V1). However, the role of this “brake” on spine dynamics is not known. We examined the contribution of Lynx1 on dendritic spine turnover before and after monocular deprivation (MD) in adult V1 with chronic in vivo imaging using two-photon microscopy and determined the spine turnover rate of apical dendrites of layer 5 (L5) and L2/3 pyramidal neurons in adult V1 of Lynx1 knock-out (KO) mice. We found that the deletion of Lynx1 doubled the baseline spine turnover rate, suggesting that the spine dynamics in the adult cortex is actively limited by the presence of Lynx1. After MD, adult Lynx1-KO mice selectively exhibit higher rate of spine loss with no difference in gain rate in L5 neurons compared with control wild-type counterparts, revealing a key signature of spine dynamics associated with robust functional plasticity in adult V1. Overall, Lynx1 could be a promising therapeutic target to induce not only functional, but also structural plasticity at the level of spine dynamics in the adult brain. SIGNIFICANCE STATEMENT Dendritic spine turnover becomes limited in the adult cortex. In mouse visual cortex, a premier model of experience-dependent plasticity, we found that the deletion of Lynx1, a nicotinic “brake” for functional plasticity, doubled the baseline spine turnover in adulthood, suggesting that the spine dynamics in the adult cortex is actively limited by Lynx1. After visual deprivation, spine loss, but not gain rate, remains higher in adult Lynx1 knock-out mice than in control wild-type mice, revealing a key signature of spine dynamics associated with robust functional plasticity. Lynx1 would be a promising target to induce not only functional, but also structural plasticity at the level of spine dynamics in adulthood. PMID:27605620

Hypotension, lipodystrophy, and insulin resistance in generalized PPARγ-deficient mice rescued from embryonic lethality

PubMed Central

Duan, Sheng Zhong; Ivashchenko, Christine Y.; Whitesall, Steven E.; D’Alecy, Louis G.; Duquaine, Damon C.; Brosius, Frank C.; Gonzalez, Frank J.; Vinson, Charles; Pierre, Melissa A.; Milstone, David S.; Mortensen, Richard M.

2007-01-01

We rescued the embryonic lethality of global PPARγ knockout by breeding Mox2-Cre (MORE) mice with floxed PPARγ mice to inactivate PPARγ in the embryo but not in trophoblasts and created a generalized PPARγ knockout mouse model, MORE-PPARγ knockout (MORE-PGKO) mice. PPARγ inactivation caused severe lipodystrophy and insulin resistance; surprisingly, it also caused hypotension. Paradoxically, PPARγ agonists had the same effect. We showed that another mouse model of lipodystrophy was hypertensive, ruling out the lipodystrophy as a cause. Further, high salt loading did not correct the hypotension in MORE-PGKO mice. In vitro studies showed that the vasculature from MORE-PGKO mice was more sensitive to endothelial-dependent relaxation caused by muscarinic stimulation, but was not associated with changes in eNOS expression or phosphorylation. In addition, vascular smooth muscle had impaired contraction in response to α-adrenergic agents. The renin-angiotensin-aldosterone system was mildly activated, consistent with increased vascular capacitance or decreased volume. These effects are likely mechanisms contributing to the hypotension. Our results demonstrated that PPARγ is required to maintain normal adiposity and insulin sensitivity in adult mice. Surprisingly, genetic loss of PPARγ function, like activation by agonists, lowered blood pressure, likely through a mechanism involving increased vascular relaxation. PMID:17304352

Adenosine A3 receptors regulate heart rate, motor activity and body temperature

PubMed Central

Yang, Jiangning; Wang, Yingqing; Garcia-Roves, Pablo; Björnholm, Marie; Fredholm, Bertil B.

2010-01-01

Aim We wanted to examine the phenotype of mice that lack the adenosine A3 receptor (A3R). Methods We examined the heart rate, body temperature and locomotion continuously by telemetry over several days. In addition the effect of the adenosine analogue R - N6- phenylisopropyl-adenosine (R-PIA) was examined. In addition, we examined heat production and food intake. Results We found that the marked diurnal variation in activity, heart rate and body temperature, with markedly higher values at night than during day time, was reduced in the A3R knockout mice. Surprisingly, the reduction in heart rate, activity and body temperature seen after injection of R-PIA in wild type mice was virtually eliminated in the A3R knock-out mice. The marked reduction in activity was associated with a decreased heat production, as expected. However, the A3R knock-out mice, surprisingly, had a higher food intake but no difference in body weight compared to wild type mice. Conclusions The mice lacking adenosine A3 receptors exhibit a surprisingly clear phenotype with changes in e.g. diurnal rhythm and temperature regulation. Whether these effects are due to a physiological role of A3 receptors in these processes or if they represent a role in development remains to be elucidated. PMID:20121716

PLAG1 deficiency impairs spermatogenesis and sperm motility in mice.

PubMed

Juma, Almas R; Grommen, Sylvia V H; O'Bryan, Moira K; O'Connor, Anne E; Merriner, D Jo; Hall, Nathan E; Doyle, Stephen R; Damdimopoulou, Pauliina E; Barriga, Daniel; Hart, Adam H; Van de Ven, Wim J M; De Groef, Bert

2017-07-13

Deficiency in pleomorphic adenoma gene 1 (PLAG1) leads to reduced fertility in male mice, but the mechanism by which PLAG1 contributes to reproduction is unknown. To investigate the involvement of PLAG1 in testicular function, we determined (i) the spatial distribution of PLAG1 in the testis using X-gal staining; (ii) transcriptomic consequences of PLAG1 deficiency in knock-out and heterozygous mice compared to wild-type mice using RNA-seq; and (iii) morphological and functional consequences of PLAG1 deficiency by determining testicular histology, daily sperm production and sperm motility in knock-out and wild-type mice. PLAG1 was sparsely expressed in germ cells and in Sertoli cells. Genes known to be involved in spermatogenesis were downregulated in the testes of knock-out mice, as well as Hsd17b3, which encodes a key enzyme in androgen biosynthesis. In the absence of Plag1, a number of genes involved in immune processes and epididymis-specific genes were upregulated in the testes. Finally, loss of PLAG1 resulted in significantly lowered daily sperm production, in reduced sperm motility, and in several animals, in sloughing of the germinal epithelium. Our results demonstrate that the subfertility seen in male PLAG1-deficient mice is, at least in part, the result of significantly reduced sperm output and sperm motility.

The Modified Selenenyl Amide, M-hydroxy Ebselen, Attenuates Diabetic Nephropathy and Diabetes-Associated Atherosclerosis in ApoE/GPx1 Double Knockout Mice

PubMed Central

Tan, Sih Min; Sharma, Arpeeta; Yuen, Derek Y. C.; Stefanovic, Nada; Krippner, Guy; Mugesh, Govindasamy; Chai, Zhonglin; de Haan, Judy B.

2013-01-01

Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced pro-inflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-β (TGF-β) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-β-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation. PMID:23874911

Deletion of the Inflammasome Sensor Aim2 Mitigates Aβ Deposition and Microglial Activation but Increases Inflammatory Cytokine Expression in an Alzheimer Disease Mouse Model.

PubMed

Wu, Pei-Jung; Hung, Yun-Fen; Liu, Hsin-Yu; Hsueh, Yi-Ping

2017-01-01

Inflammation is clearly associated with Alzheimer disease (AD). Knockout of Nlrp3, a gene encoding an inflammasome sensor, has been shown to ameliorate AD pathology in a mouse model. Because AIM2 is the most dominant inflammasome sensor expressed in mouse brains, here we investigate whether Aim2 deletion also influences the phenotype of a 5XFAD AD mouse model. Quantitative RT-PCR, immunostaining, immunoblotting, and behavioral analyses were applied to compare wild-type, Aim2-/-, 5XFAD, and Aim2-/-;5XFAD mice. We found that Aim2 knockout mitigates Aβ deposition in the cerebral cortex and hippocampus of 5XFAD mice. The activation of microglial cells is also reduced in Aim2-/-;5XFAD brains compared with 5XFAD brains. However, Aim2 knockout does not improve memory and anxiety phenotypes of 5XFAD mice in an open field, cued Y-maze, or Barnes maze. Compared with 5XFAD mice, Il-1 expression levels are not reduced in Aim2-/-;5XFAD mice. Unexpectedly, Il-6 and Il-18 expression levels in 5XFAD brains were further increased when Aim2 was deleted. Thus, inflammatory cytokine expression in 5XFAD brains is upregulated by Aim2 deletion through an unknown mechanism. Although Aim2 knockout mitigates Aβ deposition and microglial activation, Aim2 deletion does not have a beneficial effect on the spatial memory or cytokine expression of 5XFAD mice. Our findings suggest that Aβ aggregation and microglial activation may not always be correlated with the expression of inflammatory cytokines or cognitive function of 5XFAD mice. Our study also implies that different inflammasomes likely perform distinct roles in different physiological and/or pathological events. © 2017 S. Karger AG, Basel.

Distribution of Nidogen in the Murine Eye and Ocular Phenotype of the Nidogen-1 Knockout Mouse

PubMed Central

May, Christian Albrecht

2012-01-01

Distribution and lack of nidogen-1, part of numerous basement membranes, were studied in the mouse eye. For that purpose, eyes of C57BL/6 and nidogen-1 knockout mice were stained immunohistochemically for nidogen-1, and intraocular pressure measurements and light- and electron microscopy were used to study the nidogen-1 knockout animals. In normal mice, nidogen-1 was present in many basement membranes, but showed irregularities underneath the corneal epithelium, in Bruch's membrane and in the iris. Homozygous knockout of nidogen-1 in the mouse showed only mild pathological changes. In the anterior eye segment, small interruptions were noted in the nonpigmented ciliary epithelium without further consequences. In the posterior eye segment, interruptions of the inner limiting membrane led to small retinal ectopias and subsequent changes in the optic nerve. In summary, the knockout of nidogen-1 showed mild but significant morphological changes pointing to the importance of this protein which can in part, but not completely; be replaced by nidogen-2. PMID:24555126

Cardiomyopathy and response to enzyme replacement therapy in a male mouse model for Fabry disease.

PubMed

Nguyen Dinh Cat, Aurelie; Escoubet, Brigitte; Agrapart, Vincent; Griol-Charhbili, Violaine; Schoeb, Trenton; Feng, Wenguang; Jaimes, Edgar; Warnock, David G; Jaisser, Frederic

2012-01-01

Fabry disease is an X-linked disorder of glycosphingolipid metabolism that results in progressive accumulation of neutral glycosphingolipids, (predominately globotriaosylceramide; GL-3) in lysosomes, as well as other cellular compartments and the extracellular space. Our aim was to characterize the cardiac phenotype of male knock-out mice that are deficient in alpha-galactosidase A activity, as a model for Fabry disease and test the efficacy of Enzyme Replacement Therapy with agalsidase-beta. Male mice (3-4 months of age) were characterized with awake blood pressure and heart rate measurements, cardiac echocardiography and electrocardiography measurements under light anesthesia, histological studies and molecular studies with real-time polymerase chain reaction. The Fabry knock-out mouse has bradycardia and lower blood pressure than control wild type (CB7BL/6J) mice. In Fabry knock-out mice, the cardiomyopathy associated mild hypertrophy at echography with normal systolic LV function and mild diastolic dysfunction. Premature atrial contractions were more frequent in without conduction defect. Heart weight normalized to tibial length was increased in Fabry knock-out mice. Ascending aorta dilatation was observed. Molecular studies were consistent with early stages of cardiac remodeling. A single dose of agalsidase-beta (3 mg/kg) did not affect the LV hypertrophy, function or heart rate, but did improve the mRNA signals of early cardiac remodeling. In conclusion, the alpha-galactosidase A deficient mice at 3 to 4 months of age have cardiac and vascular alterations similar to that described in early clinical stage of Fabry disease in children and adolescents. Enzyme replacement therapy affects cardiac molecular remodeling after a single dose.

TASK channel deletion reduces sensitivity to local anesthetic-induced seizures

PubMed Central

Du, Guizhi; Chen, Xiangdong; Todorovic, Marko S.; Shu, Shaofang; Kapur, Jaideep; Bayliss, Douglas A.

2011-01-01

Background Local anesthetics (LAs) are typically used for regional anesthesia but can be given systemically to mitigate postoperative pain, supplement general anesthesia or prevent cardiac arrhythmias. However, systemic application or inadvertent intravenous injection can be associated with substantial toxicity, including seizure induction. The molecular basis for this toxic action remains unclear. Methods We characterized effects of different LAs on homomeric and heteromeric K+ channels containing TASK-1 (K2P3.1, KCNK3) and TASK-3 (K2P9.1, KCNK9) subunits in a mammalian expression system. In addition, we used TASK-1/TASK-3 knockout mice to test the possibility that TASK channels contribute to LA-evoked seizures. Results LAs inhibited homomeric and heteromeric TASK channels in a range relevant for seizure induction; channels containing TASK-1 subunits were most sensitive and IC50 values indicated a rank order potency of bupivacaine > ropivacaine ⟫ lidocaine. LAs induced tonic-clonic seizures in mice with the same rank order potency, but higher LA doses were required to evoke seizures in TASK knockout mice. For bupivacaine, which produced the longest seizure times, seizure duration was significantly shorter in TASK knockout mice; bupivacaine-induced seizures were associated with an increase in electroencephalogram power at frequencies <5 Hz in both wild type and TASK knockout mice. Conclusions These data suggest that increased neuronal excitability associated with TASK channel inhibition by LAs contributes to seizure induction. Since all LAs were capable of evoking seizures in TASK channel deleted mice, albeit at higher doses, the results imply that other molecular targets must also be involved in this toxic action. PMID:21946151

Fabp4-Cre-mediated Sirt6 deletion impairs adipose tissue function and metabolic homeostasis in mice.

PubMed

Xiong, Xiwen; Zhang, Cuicui; Zhang, Yang; Fan, Rui; Qian, Xinlai; Dong, X Charlie

2017-06-01

SIRT6 is a member of sirtuin family of deacetylases involved in diverse processes including genome stability, metabolic homeostasis and anti-inflammation. However, its function in the adipose tissue is not well understood. To examine the metabolic function of SIRT6 in the adipose tissue, we generated two mouse models that are deficient in Sirt6 using the Cre-lox approach. Two commonly used Cre lines that are driven by either the mouse Fabp4 or Adipoq gene promoter were chosen for this study. The Sirt6- knockout mice generated by the Fabp4-Cre line ( Sirt6 f/f : Fabp4-Cre) had a significant increase in both body weight and fat mass and exhibited glucose intolerance and insulin resistance as compared with the control wild-type mice. At the molecular levels, the Sirt6 f/f :Fabp4-Cre-knockout mice had increased expression of inflammatory genes including F4/80, TNFα, IL-6 and MCP-1 in both white and brown adipose tissues. Moreover, the knockout mice showed decreased expression of the adiponectin gene in the white adipose tissue and UCP1 in the brown adipose tissue, respectively. In contrast, the Sirt6 knockout mice generated by the Adipoq-Cre line ( Sirt6 f/f :Adipoq-Cre) only had modest insulin resistance. In conclusion, our data suggest that the function of SIRT6 in the Fabp4-Cre-expressing cells in addition to mature adipocytes plays a critical role in body weight maintenance and metabolic homeostasis. © 2017 Society for Endocrinology.

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.

[Quantitative changes of main components of erythrocyte membranes which define architectonics of cells under pttg gene knockout].

PubMed

Kaniuka, O P; Filiak, Ie Z; Kulachkovs'kyĭ, O R; Osyp, Iu L; Sybirna, N O

2014-01-01

A pttg gene knockout affects the functional state of erythron in mice which could be associated with structural changes in the structure of erythrocyte membranes. The pttg gene knockout causes a significant modification of fatty acids composition of erythrocyte membrane lipids by reducing the content of palmitic acid and increasing of polyunsaturated fatty acids amount by 18%. Analyzing the erythrocyte surface architectonics of mice under pttg gene knockout, it was found that on the background of reduction of the functionally complete biconcave discs population one could observe an increase of the number of transformed cells at different degeneration stages. Researches have shown that in mice with a pttg gene knockout compared with a control group of animals cytoskeletal protein--beta-spectrin was reduced by 17.03%. However, there is a reduction of membrane protein band 3 by 33.04%, simultaneously the content of anion transport protein band 4.5 increases by 35.2% and protein band 4.2 by 32.1%. The lectin blot analysis has helped to reveal changes in the structure of the carbohydrate determinants of erythrocyte membrane glycoproteins under conditions of directed pttg gene inactivation, accompanied by changes in the type of communication, which joins the terminal residue in carbohydrate determinant of glycoproteins. Thus, a significant redistribution of protein and fatty acids contents in erythrocyte membranes that manifested in the increase of the deformed shape of red blood cells is observed underpttg gene knockout.

Characterization of nasal potential difference in cftr knockout and F508del-CFTR mice.

PubMed

Saussereau, Emilie Lyne; Roussel, Delphine; Diallo, Siradiou; Debarbieux, Laurent; Edelman, Aleksander; Sermet-Gaudelus, Isabelle

2013-01-01

Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR) defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF). Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V(TE)) to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V(TE) in CF mice must be well characterized for correct interpretation. We performed V(TE) measurements in large-scale studies of two mouse models of CF--B6;129 cftr knockout and FVB F508del-CFTR--and their respective wild-type (WT) littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice. We determined the typical V(TE) values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl(-) solution was considered to indicate a normal response. These data will make it possible to interpret changes in nasal V(TE) in mouse models of CF, in future preclinical studies.

Cancer resistance of SR/CR mice in the genetic knockout backgrounds of leukocyte effector mechanisms: determinations for functional requirements.

PubMed

Sanders, Anne M; Stehle, John R; Blanks, Michael J; Riedlinger, Gregory; Kim-Shapiro, Jung W; Monjazeb, Arta M; Adams, Jonathan M; Willingham, Mark C; Cui, Zheng

2010-03-31

Spontaneous Regression/Complete Resistant (SR/CR) mice are a colony of cancer-resistant mice that can detect and rapidly destroy malignant cells with innate cellular immunity, predominately mediated by granulocytes. Our previous studies suggest that several effector mechanisms, such as perforin, granzymes, or complements, may be involved in the killing of cancer cells. However, none of these effector mechanisms is known as critical for granulocytes. Additionally, it is unclear which effector mechanisms are required for the cancer killing activity of specific leukocyte populations and the survival of SR/CR mice against the challenges of lethal cancer cells. We hypothesized that if any of these effector mechanisms was required for the resistance to cancer cells, its functional knockout in SR/CR mice should render them sensitive to cancer challenges. This was tested by cross breeding SR/CR mice into the individual genetic knockout backgrounds of perforin (Prf-/-), superoxide (Cybb-/), or inducible nitric oxide (Nos2-/). SR/CR mice were bred into individual Prf-/-, Cybb-/-, or Nos2-/- genetic backgrounds and then challenged with sarcoma 180 (S180). Their overall survival was compared to controls. The cancer killing efficiency of purified populations of macrophages and neutrophils from these immunodeficient mice was also examined. When these genetically engineered mice were challenged with cancer cells, the knockout backgrounds of Prf-/-, Cybb-/-, or Nos2-/- did not completely abolish the SR/CR cancer resistant phenotype. However, the Nos2-/- background did appear to weaken the resistance. Incidentally, it was also observed that the male mice in these immunocompromised backgrounds tended to be less cancer-resistant than SR/CR controls. Despite the previously known roles of perforin, superoxide or nitric oxide in the effector mechanisms of innate immune responses, these effector mechanisms were not required for cancer-resistance in SR/CR mice. The resistance was functional when any one of these effector mechanisms was completely absent, except some noticeably reduced penetrance, but not abolishment, of the phenotype in the male background in comparison to female background. These results also indicate that some other effector mechanism(s) of granulocytes may be involved in the killing of cancer cells in SR/CR mice.

Apolipoprotein E-knockout mice show increased titers of serum anti-nuclear and anti-dsDNA antibodies

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

Wang, Yuehai; Huang, Ziyang, E-mail: huangziyang666@126.com; Lu, Huixia

2012-07-13

Highlights: Black-Right-Pointing-Pointer Titers of ANA and anti-dsDNA antibodies were higher in ApoE{sup -/-} than C57B6/L mice. Black-Right-Pointing-Pointer Spleen was greater and splenocyte apoptosis lower in ApoE{sup -/-} than B6 mice. Black-Right-Pointing-Pointer Level of TLR4 was lower in spleen tissue of ApoE{sup -/-} than B6 mice. Black-Right-Pointing-Pointer The TLR4 pathway may participate in maintaining the balance of splenocyte apoptosis. Black-Right-Pointing-Pointer The TLR4 pathway may participate in antibody production in spleen tissue. -- Abstract: Apolipoprotein E-knockout (ApoE{sup -/-}) mice, atherosclerosis-prone mice, show an autoimmune response, but the pathogenesis is not fully understood. We investigated the pathogenesis in female and male ApoE{sup -/-}more » mice. The spleens of all ApoE{sup -/-} and C57BL/6 (B6) mice were weighed. The serum IgG level and titers of anti-nuclear antibody (ANA) and anti-double-stranded DNA (anti-dsDNA) antibody were assayed by ELISA. Apoptosis of spleen tissue was evaluated by TUNEL. TLR4 level in spleen tissue was tested by immunohistochemistry and Western blot analysis. Levels of MyD88, p38, phosphorylated p38 (pp38), interferon regulatory factor 3 (IRF3) and Bcl-2-associated X protein (Bax) in spleen tissue were detected by Western blot analysis. We also survey the changes of serum autoantibodies, spleen weight, splenocyte apoptosis and the expressions of TLR4, MyD88, pp38, IRF3 and Bax in spleen tissue in male ApoE{sup -/-} mice after 4 weeks of lipopolysaccharide (LPS), Toll-like receptor 4 ligand, administration. ApoE{sup -/-} mice showed splenomegaly and significantly increased serum level of IgG and titers of ANA and anti-dsDNA antibody as compared with B6 mice. Splenocyte apoptosis and the expression of TLR4, MyD88, pp38, IRF3 and Bax in spleen tissue were significantly lower in ApoE{sup -/-} than B6 mice. The expression of TLR4, MyD88, IRF3, pp38, and Bax differed by sex in ApoE{sup -/-} spleen tissue. The down-regulation of TLR4 signal molecules induced by LPS led to decreased expression of Bax and increased serum titers of ANA and anti-dsDNA antibody. Therefore, the TLR4 signal pathway may participate in maintaining the balance of splenocyte apoptosis and autoantibody production in ApoE{sup -/-} mice.« less

Early Social Enrichment Improves Social Motivation and Skills in a Monogenic Mouse Model of Autism, the Oprm1 (-/-) Mouse.

PubMed

Garbugino, Luciana; Centofante, Eleonora; D'Amato, Francesca R

2016-01-01

Environmental enrichment has been proven to have positive effects on both behavioral and physiological phenotypes in rodent models of mental and neurodevelopmental disorders. In this study, we used mice lacking the µ-opioid receptor gene (Oprm1 (-/-)), which has been shown to have deficits in social competence and communication, to assess the hypothesis that early enrichment can ameliorate sociability during development and adulthood. Due to the immaturity of sensory-motor capabilities of young pups, we chose as environmental stimulation a second lactating female, who provided extra maternal care and stimulation from birth. The results show that double mothering normalized the abnormal response to maternal separation in Oprm1 (-/-) pups and increased social motivation in juveniles and adult knockout mice. Additionally, we observed that Oprm1 (-/-) mice act as less attractive social partners than wild types, which suggests that social motivation can be modulated by the stimulus employed. This experiment supports previous findings suggesting that early social environmental stimulation has profound and long-term beneficial effects, encouraging the use of nonpharmacological interventions for the treatment of social defects in neurodevelopmental diseases.

Deletion of the GluA1 AMPA receptor subunit impairs recency-dependent object recognition memory

PubMed Central

Sanderson, David J.; Hindley, Emma; Smeaton, Emily; Denny, Nick; Taylor, Amy; Barkus, Chris; Sprengel, Rolf; Seeburg, Peter H.; Bannerman, David M.

2011-01-01

Deletion of the GluA1 AMPA receptor subunit impairs short-term spatial recognition memory. It has been suggested that short-term recognition depends upon memory caused by the recent presentation of a stimulus that is independent of contextual–retrieval processes. The aim of the present set of experiments was to test whether the role of GluA1 extends to nonspatial recognition memory. Wild-type and GluA1 knockout mice were tested on the standard object recognition task and a context-independent recognition task that required recency-dependent memory. In a first set of experiments it was found that GluA1 deletion failed to impair performance on either of the object recognition or recency-dependent tasks. However, GluA1 knockout mice displayed increased levels of exploration of the objects in both the sample and test phases compared to controls. In contrast, when the time that GluA1 knockout mice spent exploring the objects was yoked to control mice during the sample phase, it was found that GluA1 deletion now impaired performance on both the object recognition and the recency-dependent tasks. GluA1 deletion failed to impair performance on a context-dependent recognition task regardless of whether object exposure in knockout mice was yoked to controls or not. These results demonstrate that GluA1 is necessary for nonspatial as well as spatial recognition memory and plays an important role in recency-dependent memory processes. PMID:21378100

Knocking-out matrix metalloproteinase-13 exacerbates rotator cuff muscle fatty infiltration.

PubMed

Liu, Xuhui; Ravishankar, Bharat; Ning, Anne; Liu, Mengyao; Kim, Hubert T; Feeley, Brian T

2017-01-01

Rotator cuff (RC) tears are common tendon injuries. Clinically, both muscle atrophy and fatty infiltration have generally been attributed to poor functional outcomes. Matrix metalloproteinase-13 plays a crucial role in extracellular matrix remodeling in many physiological and pathological processes. Nevertheless, its role in rotator cuff muscle atrophy and fatty infiltration remains unknown. The purpose of this study is to define the functional role of MMP-13 in rotator cuff muscle atrophy and fatty infiltration using a mouse RC tears model. Unilateral complete supraspinatus and infraspinatus tendon transection and suprascapular nerve transection was performed on nine of MMP-13 (-/-) knockout and nine of MMP-13 (+/+) wildtype mice at 3 months old. Mice were sacrificed 6 weeks after surgery. Supraspinatus (SS) and infraspinatus (IS) muscles were harvested for histology and gene expression analysis with RT-PCR. Six weeks after RC surgery, no significant difference in muscle atrophy and fibrosis between MMP-13 knockout and wild type mice was observed. However, there was a significant increase in the amount of fatty infiltration in MMP-13 knockout mice compared to the wild types. Muscles from MMP-13 knockout mice have significantly higher expression of fatty infiltration related genes. Results from this study suggest that MMP-13 plays a crucial role in rotator cuff muscle fatty degeneration. This novel finding suggests a new molecular mechanism that governs RC muscle FI and MMP-13 may serve as a target for therapeutics to treat muscle FI after RC tears.

Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety

PubMed Central

Chiu, Gabriel S.; Darmody, Patrick T.; Walsh, John P.; Moon, Morgan L.; Kwakwa, Kristin A.; Bray, Julie K.; McCusker, Robert H.; Freund, Gregory G.

2014-01-01

Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by two-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain. PMID:24907587

Knockout of Foxp2 disrupts vocal development in mice.

PubMed

Castellucci, Gregg A; McGinley, Matthew J; McCormick, David A

2016-03-16

The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/-) mice and their wildtype (WT) littermates from juvenile to adult ages, and observe severe abnormalities in the courtship song of Foxp2+/- mice. In comparison to their WT littermates, Foxp2+/- mice vocalized less, produced shorter syllable sequences, and possessed an abnormal syllable inventory. In addition, Foxp2+/- song also exhibited irregular rhythmic structure, and its development did not follow the consistent trajectories observed in WT vocalizations. These results demonstrate that the Foxp2 gene is critical for normal vocal behavior in juvenile and adult mice, and that Foxp2 mutant mice may provide a tractable model system for the study of the gene's role in general vocal motor control.

2,3,7, 8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)-MEDIATED OXIDATIVE STRESS IN FEMALE CYP1A-2 KNOCKOUT (CYP1A2-/-) MICE

EPA Science Inventory

2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD)-Mediated Oxidative Stress in Female CYP1A2 Knockout (CYP1A2-/-) Mice

Deborah Burgin1, Janet Diliberto2, Linda Birnbaum2
1UNC Toxicology; 2USEPA/ORD/NHEERL, RTP, NC

Most of the effects due to TCDD exposure are mediated via...

Beta-arrestin-1 protein represses diet-induced obesity.

PubMed

Zhuang, Le-nan; Hu, Wen-xiang; Zhang, Ming-liang; Xin, Shun-mei; Jia, Wei-ping; Zhao, Jian; Pei, Gang

2011-08-12

Diet-related obesity is a major metabolic disorder. Excessive fat mass is associated with type 2 diabetes, hepatic steatosis, and arteriosclerosis. Dysregulation of lipid metabolism and adipose tissue function contributes to diet-induced obesity. Here, we report that β-arrestin-1 knock-out mice are susceptible to diet-induced obesity. Knock-out of the gene encoding β-arrestin-1 caused increased fat mass accumulation and decreased whole-body insulin sensitivity in mice fed a high-fat diet. In β-arrestin-1 knock-out mice, we observed disrupted food intake and energy expenditure and increased macrophage infiltration in white adipose tissue. At the molecular level, β-arrestin-1 deficiency affected the expression of many lipid metabolic genes and inflammatory genes in adipose tissue. Consistently, transgenic overexpression of β-arrestin-1 repressed diet-induced obesity and improved glucose tolerance and systemic insulin sensitivity. Thus, our findings reveal that β-arrestin-1 plays a role in metabolism regulation.

ROLE OF ESTROGEN RECEPTOR-α ON FOOD DEMAND ELASTICITY

PubMed Central

Minervini, Vanessa; Rowland, Neil E.; Robertson, Kimberly L.; Foster, Thomas C.

2016-01-01

Estrogens have been shown to have an inhibitory effect on food intake under free-feeding conditions, yet the effects of estrogens on food-maintained operant responding have been studied to a much lesser extent and, thus, are not well understood. Therefore, the purpose of the present experiment was to use a behavioral economics paradigm to assess differences in demand elasticity between mice with knockout of the estrogen receptor subtype α, knockout of subtype β, and their wild type controls. The mice responded in a closed economy, and the price of food was increased by increasing the fixed-ratio response requirement every four sessions. Overall, we found that mice with the knockout of receptor subtype α had the most elastic demand functions. Therefore, under these conditions, estrogens increased food seeking via activation of the receptor subtype α. The results were inconsistent with those reported by previous studies that employed free-feeding conditions. PMID:25869426

Critical period plasticity is disrupted in the barrel cortex of Fmr1 knockout mice

PubMed Central

Harlow, Emily G.; Till, Sally M.; Russell, Theron A.; Wijetunge, Lasani S.; Kind, Peter; Contractor, Anis

2010-01-01

Summary Alterations in sensory processing constitute prominent symptoms of Fragile X syndrome; however, little is known about how disrupted synaptic and circuit development in sensory cortex contributes to these deficits. To investigate how the loss of fragile X mental retardation protein (FMRP) impacts the development of cortical synapses, we examined excitatory thalamocortical synapses in somatosensory cortex during the perinatal critical period in Fmr1 knockout mice. FMRP ablation resulted in dysregulation of glutamatergic signaling maturation. The fraction of silent synapses persisting to later developmental times was increased, there was a temporal delay in the window for synaptic plasticity, while other forms of developmental plasticity were not altered in Fmr1 knockout mice. Our results indicate that FMRP is required for the normal developmental progression of synaptic maturation, and loss of this important RNA binding protein impacts the timing of the critical period for layer IV synaptic plasticity. PMID:20159451

DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids.

PubMed

Ahmed, Emad A; Scherthan, Harry; de Rooij, Dirk G

2015-12-16

Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

Innate immunity of surfactant proteins A and D in urinary tract infection with uropathogenic Escherichia coli.

PubMed

Hu, Fengqi; Ding, Guohua; Zhang, Zhiyong; Gatto, Louis A; Hawgood, Samuel; Poulain, Francis R; Cooney, Robert N; Wang, Guirong

2016-01-01

To investigate the effects of surfactant proteins A and D (SP-A and SP-D, respectively) in urinary tract infection (UTI), SP-A and SP-D double knockout (SP-A/D KO) and wild type (WT) C57BL/6 female mice were infected with uropathogenic Escherichia coli by intravesical inoculation. Compared with WT mice SP-A/D KO mice showed increased susceptibility to UTI, as evidenced by higher bacterial CFU, more infiltrating neutrophils and severe pathological changes. Keratinocyte-derived chemokine increased in the kidney of WT mice but not in SP-A/D KO mice 24 h post-infection. Compared with control, the level of IL-17 was elevated in the kidney of infected WT and SP-A/D KO mice and the level of IL-17 was higher in the infected SP-A/D KO mice than in infected WT mice 24 and 48 h post-infection. The basal level of p38 MAPK phosphorylation in SP-A/D KO mice was higher than in WT mice. The phosphorylated p38 level was elevated in the kidney of WT mice post infection but not in SP-A/D KO mice. Furthermore, in vitro growth of uropathogenic E. coli was inhibited by SP-A and SP-D. We conclude that SP-A and SP-D function as mediators of innate immunity by inhibiting bacterial growth and modulating renal inflammation in part by regulating p38 MAPK-related pathway in murine UTI. © The Author(s) 2015.

Food restriction by intermittent fasting induces diabetes and obesity and aggravates spontaneous atherosclerosis development in hypercholesterolaemic mice.

PubMed

Dorighello, Gabriel G; Rovani, Juliana C; Luhman, Christopher J F; Paim, Bruno A; Raposo, Helena F; Vercesi, Anibal E; Oliveira, Helena C F

2014-03-28

Different regimens of food restriction have been associated with protection against obesity, diabetes and CVD. In the present study, we hypothesised that food restriction would bring benefits to atherosclerosis- and diabetes-prone hypercholesterolaemic LDL-receptor knockout mice. For this purpose, 2-month-old mice were submitted to an intermittent fasting (IF) regimen (fasting every other day) over a 3-month period, which resulted in an overall 20 % reduction in food intake. Contrary to our expectation, epididymal and carcass fat depots and adipocyte size were significantly enlarged by 15, 72 and 68 %, respectively, in the IF mice compared with the ad libitum-fed mice. Accordingly, plasma levels of leptin were 50 % higher in the IF mice than in the ad libitum-fed mice. In addition, the IF mice showed increased plasma levels of total cholesterol (37 %), VLDL-cholesterol (195 %) and LDL-cholesterol (50 %). As expected, in wild-type mice, the IF regimen decreased plasma cholesterol levels and epididymal fat mass. Glucose homeostasis was also disturbed by the IF regimen in LDL-receptor knockout mice. Elevated levels of glycaemia (40 %), insulinaemia (50 %), glucose intolerance and insulin resistance were observed in the IF mice. Systemic inflammatory markers, TNF-α and C-reactive protein, were significantly increased and spontaneous atherosclerosis development were markedly increased (3-fold) in the IF mice. In conclusion, the IF regimen induced obesity and diabetes and worsened the development of spontaneous atherosclerosis in LDL-receptor knockout mice. Although being efficient in a wild-type background, this type of food restriction is not beneficial in the context of genetic hypercholesterolaemia.

Antagonism of scavenger receptor CD36 by 5A peptide prevents chronic kidney disease progression in mice independent of blood pressure regulation

PubMed Central

Souza, Ana Carolina P.; Bocharov, Alexander V.; Baranova, Irina; Vishnyakova, Tatyana; Huang, Yuning G.; Wilkins, Kenneth J.; Hu, Xuzhen; Street, Jonathan M.; Alvarez-Prats, Alejandro; Mullick, Adam E.; Patterson, Amy P.; Remaley, Alan; Eggerman, Thomas L.; Yuen, Peter S.T.; Star, Robert A.

2016-01-01

Scavenger receptor CD36 participates in lipid metabolism and inflammatory pathways important for cardiovascular disease and chronic kidney disease (CKD). Few pharmacological agents are available to slow the progression of CKD. However, apolipoprotein AI-mimetic peptide 5A antagonizes CD36 in vitro. To test the efficacy of 5A, and to test the role of CD36 during CKD, we compared wild type to CD36 knockout mice and wild type mice treated with 5A, in a progressive CKD model that resembles human disease. Knockout and 5A-treated wild type mice were protected from CKD progression without changes in blood pressure and had reductions in cardiovascular risk surrogate markers that are associated with CKD. Treatment with 5A did not further protect CD36 knockout mice from CKD progression, implicating CD36 as its main site of action. In a separate model of kidney fibrosis, 5A-treated wild type mice had less macrophage infiltration and interstitial fibrosis. Peptide 5A exerted anti-inflammatory effects in the kidney and decreases renal expression of inflammasome genes. Thus, CD36 is a new therapeutic target for CKD and its associated cardiovascular risk factors. Peptide 5A may be a promising new agent to slow CKD progression. PMID:26994575

Transcriptional and phenotypic comparisons of Ppara knockout and siRNA knockdown mice

PubMed Central

De Souza, Angus T.; Dai, Xudong; Spencer, Andrew G.; Reppen, Tom; Menzie, Ann; Roesch, Paula L.; He, Yudong; Caguyong, Michelle J.; Bloomer, Sherri; Herweijer, Hans; Wolff, Jon A.; Hagstrom, James E.; Lewis, David L.; Linsley, Peter S.; Ulrich, Roger G.

2006-01-01

RNA interference (RNAi) has great potential as a tool for studying gene function in mammals. However, the specificity and magnitude of the in vivo response to RNAi remains to be fully characterized. A molecular and phenotypic comparison of a genetic knockout mouse and the corresponding knockdown version would help clarify the utility of the RNAi approach. Here, we used hydrodynamic delivery of small interfering RNA (siRNA) to knockdown peroxisome proliferator activated receptor alpha (Ppara), a gene that is central to the regulation of fatty acid metabolism. We found that Ppara knockdown in the liver results in a transcript profile and metabolic phenotype that is comparable to those of Ppara−/− mice. Combining the profiles from mice treated with the PPARα agonist fenofibrate, we confirmed the specificity of the RNAi response and identified candidate genes proximal to PPARα regulation. Ppara knockdown animals developed hypoglycemia and hypertriglyceridemia, phenotypes observed in Ppara−/− mice. In contrast to Ppara−/− mice, fasting was not required to uncover these phenotypes. Together, these data validate the utility of the RNAi approach and suggest that siRNA can be used as a complement to classical knockout technology in gene function studies. PMID:16945951

Genetic Interactions Between the Meiosis-Specific Cohesin Components, STAG3, REC8, and RAD21L.

PubMed

Ward, Ayobami; Hopkins, Jessica; Mckay, Matthew; Murray, Steve; Jordan, Philip W

2016-06-01

Cohesin is an essential structural component of chromosomes that ensures accurate chromosome segregation during mitosis and meiosis. Previous studies have shown that there are cohesin complexes specific to meiosis, required to mediate homologous chromosome pairing, synapsis, recombination, and segregation. Meiosis-specific cohesin complexes consist of two structural maintenance of chromosomes proteins (SMC1α/SMC1β and SMC3), an α-kleisin protein (RAD21, RAD21L, or REC8), and a stromal antigen protein (STAG1, 2, or 3). STAG3 is exclusively expressed during meiosis, and is the predominant STAG protein component of cohesin complexes in primary spermatocytes from mouse, interacting directly with each α-kleisin subunit. REC8 and RAD21L are also meiosis-specific cohesin components. Stag3 mutant spermatocytes arrest in early prophase ("zygotene-like" stage), displaying failed homolog synapsis and persistent DNA damage, as a result of unstable loading of cohesin onto the chromosome axes. Interestingly, Rec8, Rad21L double mutants resulted in an earlier "leptotene-like" arrest, accompanied by complete absence of STAG3 loading. To assess genetic interactions between STAG3 and α-kleisin subunits RAD21L and REC8, our lab generated Stag3, Rad21L, and Stag3, Rec8 double knockout mice, and compared them to the Rec8, Rad21L double mutant. These double mutants are phenotypically distinct from one another, and more severe than each single knockout mutant with regards to chromosome axis formation, cohesin loading, and sister chromatid cohesion. The Stag3, Rad21L, and Stag3, Rec8 double mutants both progress further into prophase I than the Rec8, Rad21L double mutant. Our genetic analysis demonstrates that cohesins containing STAG3 and REC8 are the main complex required for centromeric cohesion, and RAD21L cohesins are required for normal clustering of pericentromeric heterochromatin. Furthermore, the STAG3/REC8 and STAG3/RAD21L cohesins are the primary cohesins required for axis formation. Copyright © 2016 Ward et al.

Beta2-adrenergic activity modulates vascular tone regulation in lecithin:cholesterol acyltransferase knockout mice.

PubMed

Manzini, S; Pinna, C; Busnelli, M; Cinquanta, P; Rigamonti, E; Ganzetti, G S; Dellera, F; Sala, A; Calabresi, L; Franceschini, G; Parolini, C; Chiesa, G

2015-11-01

Lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with hypoalphalipoproteinemia, generally a predisposing factor for premature coronary heart disease. The evidence of accelerated atherosclerosis in LCAT-deficient subjects is however controversial. In this study, the effect of LCAT deficiency on vascular tone and endothelial function was investigated in LCAT knockout mice, which reproduce the human lipoprotein phenotype. Aortas from wild-type (Lcat(wt)) and LCAT knockout (Lcat(KO)) mice exposed to noradrenaline showed reduced contractility in Lcat(KO) mice (P<0.005), whereas acetylcholine exposure showed a lower NO-dependent relaxation in Lcat(KO) mice (P<0.05). Quantitative PCR and Western blotting analyses suggested an adequate eNOS expression in Lcat(KO) mouse aortas. Real-time PCR analysis indicated increased expression of β2-adrenergic receptors vs wild-type mice. Aorta stimulation with noradrenaline in the presence of propranolol, to abolish the β-mediated relaxation, showed the same contractile response in the two mouse lines. Furthermore, propranolol pretreatment of mouse aortas exposed to L-NAME prevented the difference in responses between Lcat(wt) and Lcat(KO) mice. The results indicate that LCAT deficiency leads to increased β2-adrenergic relaxation and to a consequently decreased NO-mediated vasodilation that can be reversed to guarantee a correct vascular tone. The present study suggests that LCAT deficiency is not associated with an impaired vascular reactivity. Copyright © 2015. Published by Elsevier Inc.

Beta2-adrenergic activity modulates vascular tone regulation in lecithin:cholesterol acyltransferase knockout mice

PubMed Central

Manzini, S.; Pinna, C.; Busnelli, M.; Cinquanta, P.; Rigamonti, E.; Ganzetti, G.S.; Dellera, F.; Sala, A.; Calabresi, L.; Franceschini, G.; Parolini, C.; Chiesa, G.

2015-01-01

Lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with hypoalphalipoproteinemia, generally a predisposing factor for premature coronary heart disease. The evidence of accelerated atherosclerosis in LCAT-deficient subjects is however controversial. In this study, the effect of LCAT deficiency on vascular tone and endothelial function was investigated in LCAT knockout mice, which reproduce the human lipoprotein phenotype. Aortas from wild-type (Lcatwt) and LCAT knockout (LcatKO) mice exposed to noradrenaline showed reduced contractility in LcatKO mice (P < 0.005), whereas acetylcholine exposure showed a lower NO-dependent relaxation in LcatKO mice (P < 0.05). Quantitative PCR and Western blotting analyses suggested an adequate eNOS expression in LcatKO mouse aortas. Real-time PCR analysis indicated increased expression of β2-adrenergic receptors vs wild-type mice. Aorta stimulation with noradrenaline in the presence of propranolol, to abolish the β-mediated relaxation, showed the same contractile response in the two mouse lines. Furthermore, propranolol pretreatment of mouse aortas exposed to L-NAME prevented the difference in responses between Lcatwt and LcatKO mice. The results indicate that LCAT deficiency leads to increased β2-adrenergic relaxation and to a consequently decreased NO-mediated vasodilation that can be reversed to guarantee a correct vascular tone. The present study suggests that LCAT deficiency is not associated with an impaired vascular reactivity. PMID:26254103

CaSR-mediated interactions between calcium and magnesium homeostasis in mice.

PubMed

Quinn, Stephen J; Thomsen, Alex R B; Egbuna, Ogo; Pang, Jian; Baxi, Khanjan; Goltzman, David; Pollak, Martin; Brown, Edward M

2013-04-01

Calcium (Ca) and magnesium (Mg) homeostasis are interrelated and share common regulatory hormones, including parathyroid hormone (PTH) and vitamin D. However, the role of the calcium-sensing receptor (CaSR) in Mg homeostasis in vivo is not well understood. We sought to investigate the interactions between Mg and Ca homeostasis using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR) (double knockout, DKO). Serum Mg is lower in PTH KO and DKO mice than in WT mice on standard chow, whereas supplemental dietary Ca leads to equivalent Mg levels for all three genotypes. Mg loading increases serum Mg in all genotypes; however, the increase in serum Mg is most pronounced in the DKO mice. Serum Ca is increased with Mg loading in the PTH KO and DKO mice but not in the WT mice. Here, too, the hypercalcemia is much greater in the DKO mice. Serum and especially urinary phosphate are reduced during Mg loading, which is likely due to intestinal chelation of phosphate by Mg. Mg loading decreases serum PTH in WT mice and increases serum calcitonin in both WT and PTH KO mice but not DKO mice. Furthermore, Mg loading elevates serum 1,25-dihydroxyvitamin D in all genotypes, with greater effects in PTH KO and DKO mice, possibly due to reduced levels of serum phosphorus and FGF23. These hormonal responses to Mg loading and the CaSR's role in regulating renal function may help to explain changes in serum Mg and Ca found during Mg loading.

In vivo imaging of the mouse model of X-linked juvenile retinoschisis with fourier domain optical coherence tomography.

PubMed

Xu, Jing; Molday, Laurie L; Molday, Robert S; Sarunic, Marinko V

2009-06-01

The purpose of this study was to investigate Fourier domain optical coherence tomography (FD OCT) as a noninvasive tool for retinal imaging in the Rs1h-knockout mouse (model for X-linked juvenile retinoschisis). A prototype spectrometer-based FD OCT system was used in combination with a custom optical beam-scanning platform. Images of the retinas from wild-type and Rs1h-knockout mice were acquired noninvasively with FD OCT with the specimen anesthetized. At the completion of the noninvasive FD OCT imaging, invasive retinal cross-sectional images (histology) were acquired from a nearby region for comparison to the FD OCT images. The retinal layers were identifiable in the FD OCT images, permitting delineation and thickness measurement of the outer nuclear layer (ONL). During FD OCT in vivo imaging of the Rs1h-knockout mouse, holes were observed in the inner nuclear layer (INL), and retinal cell disorganization was observed as a change in the backscattering intensity profile. Comparison of the ONL measurements acquired noninvasively with FD OCT to measurements taken using histology at nearby locations showed a degeneration of roughly 30% of the ONL by the age of 2 months in Rs1h-knockout mice relative to wild-type. FD OCT was demonstrated to be effective for noninvasive imaging of retinal degeneration and observation of retinal holes in Rs1h-knockout mice.

Immunoproteasome in animal models of Duchenne muscular dystrophy.

PubMed

Chen, Chiao-Nan Joyce; Graber, Ted G; Bratten, Wendy M; Ferrington, Deborah A; Thompson, LaDora V

2014-04-01

Increased proteasome activity has been implicated in the atrophy and deterioration associated with dystrophic muscles of Duchenne muscular dystrophy (DMD). While proteasome inhibitors show promise in the attenuation of muscle degeneration, proteasome inhibition-induced toxicity was a major drawback of this therapeutic strategy. Inhibitors that selectively target the proteasome subtype that is responsible for the loss in muscle mass and quality would reduce side effects and be less toxic. This study examined proteasome activity and subtype populations, along with muscle function, morphology and damage in wild-type (WT) mice and two murine models of DMD, dystrophin-deficient (MDX) and dystrophin- and utrophin-double-knockout (DKO) mice. We found that immunoproteasome content was increased in dystrophic muscles while the total proteasome content was unchanged among the three genotypes of mice. Proteasome proteolytic activity was elevated in dystrophic muscles, especially in DKO mice. These mice also exhibited more severe muscle atrophy than either WT or MDX mice. Muscle damage and regeneration, characterized by the activity of muscle creatine kinase in the blood and the percentage of central nuclei were equally increased in dystrophic mice. Accordingly, the overall muscle function was similarly reduced in both dystrophic mice compared with WT. These data demonstrated that there was transformation of standard proteasomes to immunoproteasomes in dystrophic muscles. In addition, DKO that showed greatest increase in proteasome activities also demonstrated more severe atrophy compared with MDX and WT. These results suggest a putative role for the immunoproteasome in muscle deterioration associated with DMD and provide a potential target for therapeutic intervention.

Normal development of mice lacking PAXX, the paralogue of XRCC4 and XLF.

PubMed

Gago-Fuentes, Raquel; Xing, Mengtan; Sæterstad, Siri; Sarno, Antonio; Dewan, Alisa; Beck, Carole; Bradamante, Stefano; Bjørås, Magnar; Oksenych, Valentyn

2018-03-01

DNA repair consists of several cellular pathways which recognize and repair damaged DNA. The classical nonhomologous DNA end-joining (NHEJ) pathway repairs double-strand breaks in DNA. It is required for maturation of both B and T lymphocytes by supporting V(D)J recombination as well as B-cell differentiation during class switch recombination (CSR). Inactivation of NHEJ factors Ku70, Ku80, XRCC4, DNA ligase 4, DNA-PKcs, and Artemis impairs V(D)J recombination and blocks lymphocyte development. Paralogue of XRCC4 and XLF (PAXX) is an accessory NHEJ factor that has a significant impact on the repair of DNA lesions induced by ionizing radiation in human, murine, and chicken cells. However, the role of PAXX during development is poorly understood. To determine the physiological role of PAXX, we deleted part of the Paxx promoter and the first two exons in mice. Further, we compared Paxx -knockout mice with wild-type (WT) and NHEJ-deficient controls including Ku80- and Dna-pkcs -null and severe combined immunodeficiency mice. Surprisingly, Paxx -deficient mice were not distinguishable from the WT littermates; they were the same weight and size, fertility status, had normal spleen, thymus, and bone marrow. Paxx -deficient mice had the same number of chromosomal and chromatid breaks as WT mice. Moreover, Paxx -deficient primary B lymphocytes had the same level of CSR as lymphocytes isolated from WT mice. We concluded that PAXX is dispensable for normal mouse development.

Role of the ectonucleotidase NTPDase2 in taste bud function

PubMed Central

Vandenbeuch, Aurelie; Anderson, Catherine B.; Parnes, Jason; Enjyoji, Keiichi; Robson, Simon C.; Finger, Thomas E.; Kinnamon, Sue C.

2013-01-01

Taste buds are unusual in requiring ATP as a transmitter to activate sensory nerve fibers. In response to taste stimuli, taste cells release ATP, activating purinergic receptors containing the P2X2 and P2X3 subunits on taste nerves. In turn, the released ATP is hydrolyzed to ADP by a plasma membrane nucleoside triphosphate previously identified as nucleoside triphosphate diphosphohydrolase-2 (NTPDase2). In this paper we investigate the role of this ectonucleotidase in the function of taste buds by examining gene-targeted Entpd2-null mice globally lacking NTPDase2. RT-PCR confirmed the absence of NTPDase2, and ATPase enzyme histochemistry reveals no reaction product in taste buds of knockout mice, suggesting that NTPDase2 is the dominant form in taste buds. RT-PCR and immunocytochemistry demonstrated that in knockout mice all cell types are present in taste buds, even those cells normally expressing NTPDase2. In addition, the overall number and size of taste buds are normal in Entpd2-null mice. Luciferin/luciferase assays of circumvallate tissue of knockout mice detected elevated levels of extracellular ATP. Electrophysiological recordings from two taste nerves, the chorda tympani and glossopharyngeal, revealed depressed responses to all taste stimuli in Entpd2-null mice. Responses were more depressed in the glossopharyngeal nerve than in the chorda tympani nerve and involved all taste qualities; responses in the chorda tympani were more depressed to sweet and umami stimuli than to other qualities. We suggest that the excessive levels of extracellular ATP in the Entpd2-knockout animals desensitize the P2X receptors associated with nerve fibers, thereby depressing taste responses. PMID:23959882

Pharmacokinetic Assessment of Cooperative Efflux of the Multitargeted Kinase Inhibitor Ponatinib Across the Blood-Brain Barrier.

PubMed

Laramy, Janice K; Kim, Minjee; Parrish, Karen E; Sarkaria, Jann N; Elmquist, William F

2018-05-01

A compartmental blood-brain barrier (BBB) model describing drug transport across the BBB was implemented to evaluate the influence of efflux transporters on the rate and extent of the multikinase inhibitor ponatinib penetration across the BBB. In vivo pharmacokinetic studies in wild-type and transporter knockout mice showed that two major BBB efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), cooperate to modulate the brain exposure of ponatinib. The total and unbound (free) brain-to-plasma ratios were approximately 15-fold higher in the triple knockout mice lacking both P-gp and Bcrp [ Mdr1a/b(-/-)Bcrp1(-/-) ] compared with the wild-type mice. The triple knockout mice had a greater than an additive increase in the brain exposure of ponatinib when compared with single knockout mice [ Bcrp1(-/-) or Mdr1a/b(-/-) ], suggesting functional compensation of transporter-mediated drug efflux. Based on the BBB model characterizing the observed brain and plasma concentration-time profiles, the brain exit rate constant and clearance out of the brain were approximately 15-fold higher in the wild-type compared with Mdr1a/b(-/-)Bcrp1(-/-) mice, resulting in a significant increase in the mean transit time (the average time spent by ponatinib in the brain in a single passage) in the absence of efflux transporters (P-gp and Bcrp). This study characterized transporter-mediated drug efflux from the brain, a process that reduces the duration and extent of ponatinib exposure in the brain and has critical implications for the use of targeted drug delivery for brain tumors. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Contributions of the three CYP1 monooxygenases to pro-inflammatory and inflammation-resolution lipid mediator pathways.

PubMed

Divanovic, Senad; Dalli, Jesmond; Jorge-Nebert, Lucia F; Flick, Leah M; Gálvez-Peralta, Marina; Boespflug, Nicholas D; Stankiewicz, Traci E; Fitzgerald, Jonathan M; Somarathna, Maheshika; Karp, Christopher L; Serhan, Charles N; Nebert, Daniel W

2013-09-15

All three cytochrome P450 1 (CYP1) monooxygenases are believed to participate in lipid mediator biosynthesis and/or their local inactivation; however, distinct metabolic steps are unknown. We used multiple-reaction monitoring and liquid chromatography-UV coupled with tandem mass spectrometry-based lipid-mediator metabololipidomics to identify and quantify three lipid-mediator metabolomes in basal peritoneal and zymosan-stimulated inflammatory exudates, comparing Cyp1a1/1a2/1b1(⁻/⁻) C57BL/6J-background triple-knockout mice with C57BL/6J wild-type mice. Significant differences between untreated triple-knockout and wild-type mice were not found for peritoneal cell number or type or for basal CYP1 activities involving 11 identified metabolic steps. Following zymosan-initiated inflammation, 18 lipid mediators were identified, including members of the eicosanoids and specialized proresolving mediators (i.e., resolvins and protectins). Compared with wild-type mice, Cyp1 triple-knockout mice exhibited increased neutrophil recruitment in zymosan-treated peritoneal exudates. Zymosan stimulation was associated with eight statistically significantly altered metabolic steps: increased arachidonic acid-derived leukotriene B₄ (LTB₄) and decreased 5S-hydroxyeicosatetraenoic acid; decreased docosahexaenoic acid-derived neuroprotectin D1/protectin D1, 17S-hydroxydocosahexaenoic acid, and 14S-hydroxydocosahexaenoic acid; and decreased eicosapentaenoic acid-derived 18R-hydroxyeicosapentaenoic acid (HEPE), 15S-HEPE, and 12S-HEPE. In neutrophils analyzed ex vivo, elevated LTB₄ levels were shown to parallel increased neutrophil numbers, and 20-hydroxy-LTB₄ formation was found to be deficient in Cyp1 triple-knockout mice. Together, these results demonstrate novel contributions of CYP1 enzymes to the local metabolite profile of lipid mediators that regulate neutrophilic inflammation.

Role of the ectonucleotidase NTPDase2 in taste bud function.

PubMed

Vandenbeuch, Aurelie; Anderson, Catherine B; Parnes, Jason; Enjyoji, Keiichi; Robson, Simon C; Finger, Thomas E; Kinnamon, Sue C

2013-09-03

Taste buds are unusual in requiring ATP as a transmitter to activate sensory nerve fibers. In response to taste stimuli, taste cells release ATP, activating purinergic receptors containing the P2X2 and P2X3 subunits on taste nerves. In turn, the released ATP is hydrolyzed to ADP by a plasma membrane nucleoside triphosphate previously identified as nucleoside triphosphate diphosphohydrolase-2 (NTPDase2). In this paper we investigate the role of this ectonucleotidase in the function of taste buds by examining gene-targeted Entpd2-null mice globally lacking NTPDase2. RT-PCR confirmed the absence of NTPDase2, and ATPase enzyme histochemistry reveals no reaction product in taste buds of knockout mice, suggesting that NTPDase2 is the dominant form in taste buds. RT-PCR and immunocytochemistry demonstrated that in knockout mice all cell types are present in taste buds, even those cells normally expressing NTPDase2. In addition, the overall number and size of taste buds are normal in Entpd2-null mice. Luciferin/luciferase assays of circumvallate tissue of knockout mice detected elevated levels of extracellular ATP. Electrophysiological recordings from two taste nerves, the chorda tympani and glossopharyngeal, revealed depressed responses to all taste stimuli in Entpd2-null mice. Responses were more depressed in the glossopharyngeal nerve than in the chorda tympani nerve and involved all taste qualities; responses in the chorda tympani were more depressed to sweet and umami stimuli than to other qualities. We suggest that the excessive levels of extracellular ATP in the Entpd2-knockout animals desensitize the P2X receptors associated with nerve fibers, thereby depressing taste responses.

Cardiomyopathy and Response to Enzyme Replacement Therapy in a Male Mouse Model for Fabry Disease

PubMed Central

Nguyen Dinh Cat, Aurelie; Escoubet, Brigitte; Agrapart, Vincent; Griol-Charhbili, Violaine; Schoeb, Trenton; Feng, Wenguang; Jaimes, Edgar; Warnock, David G.; Jaisser, Frederic

2012-01-01

Fabry disease is an X-linked disorder of glycosphingolipid metabolism that results in progressive accumulation of neutral glycosphingolipids, (predominately globotriaosylceramide; GL-3) in lysosomes, as well as other cellular compartments and the extracellular space. Our aim was to characterize the cardiac phenotype of male knock-out mice that are deficient in alpha-galactosidase A activity, as a model for Fabry disease and test the efficacy of Enzyme Replacement Therapy with agalsidase-beta. Male mice (3–4 months of age) were characterized with awake blood pressure and heart rate measurements, cardiac echocardiography and electrocardiography measurements under light anesthesia, histological studies and molecular studies with real-time polymerase chain reaction. The Fabry knock-out mouse has bradycardia and lower blood pressure than control wild type (CB7BL/6J) mice. In Fabry knock-out mice, the cardiomyopathy associated mild hypertrophy at echography with normal systolic LV function and mild diastolic dysfunction. Premature atrial contractions were more frequent in without conduction defect. Heart weight normalized to tibial length was increased in Fabry knock-out mice. Ascending aorta dilatation was observed. Molecular studies were consistent with early stages of cardiac remodeling. A single dose of agalsidase-beta (3 mg/kg) did not affect the LV hypertrophy, function or heart rate, but did improve the mRNA signals of early cardiac remodeling. In conclusion, the alpha-galactosidase A deficient mice at 3 to 4 months of age have cardiac and vascular alterations similar to that described in early clinical stage of Fabry disease in children and adolescents. Enzyme replacement therapy affects cardiac molecular remodeling after a single dose. PMID:22574107

Overlapping functions of bone sialoprotein and pyrophosphate regulators in directing cementogenesis.

PubMed

Ao, M; Chavez, M B; Chu, E Y; Hemstreet, K C; Yin, Y; Yadav, M C; Millán, J L; Fisher, L W; Goldberg, H A; Somerman, M J; Foster, B L

2017-12-01

Although acellular cementum is essential for tooth attachment, factors directing its development and regeneration remain poorly understood. Inorganic pyrophosphate (PP i ), a mineralization inhibitor, is a key regulator of cementum formation: tissue-nonspecific alkaline phosphatase (Alpl/TNAP) null mice (increased PP i ) feature deficient cementum, while progressive ankylosis protein (Ank/ANK) null mice (decreased PP i ) feature increased cementum. Bone sialoprotein (Bsp/BSP) and osteopontin (Spp1/OPN) are multifunctional extracellular matrix components of cementum proposed to have direct and indirect effects on cell activities and mineralization. Studies on dentoalveolar development of Bsp knockout (Bsp -/- ) mice revealed severely reduced acellular cementum, however underlying mechanisms remain unclear. The similarity in defective cementum phenotypes between Bsp -/- mice and Alpl -/- mice (the latter featuring elevated PP i and OPN), prompted us to examine whether BSP is operating by modulating PP i -associated genes. Genetic ablation of Bsp caused a 2-fold increase in circulating PP i , altered mRNA expression of Alpl, Spp1, and Ank, and increased OPN protein in the periodontia. Generation of a Bsp knock-out (KO) cementoblast cell line revealed significantly decreased mineralization capacity, 50% increased PP i in culture media, and increased Spp1 and Ank mRNA expression. While addition of 2μg/ml recombinant BSP altered Spp1, Ank, and Enpp1 expression in cementoblasts, changes resulting from this dose were not dependent on the integrin-binding RGD motif or MAPK/ERK signaling pathway. Decreasing PP i by genetic ablation of Ank on the Bsp -/- mouse background reestablished cementum formation, allowing >3-fold increased acellular cementum volume compared to wild-type (WT). However, deleting Ank did not fully compensate for the absence of BSP. Bsp -/- ; Ank -/- double-deficient mice exhibited mean 20-27% reduced cementum thickness and volume compared to Ank -/- mice. From these data, we conclude that the perturbations in PP i metabolism are not solely driving the cementum pathology in Bsp -/- mice, and that PP i is more potent than BSP as a cementum regulator, as shown by the ability to override loss of BSP by lowering PP i . We propose that BSP and PP i work in concert to direct mineralization in cementum and likely other mineralized tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanistic Links between PARP, NAD, and Brain Inflammation after TBI

DTIC Science & Technology

2014-10-01

metabolite which we have in prior studies shown to also suppress poly(ADP-ribose) polymerase activity and inflammatory responses) and ketogenic diet . CtBP1/2...knockout mice will be generated to test a specific mechanisms by which ketogenic diet can have anti-inflammatory effects. For all studies, outcome...inflammatory responses. (3) Ketogenic diet , begun 12 hours after TBI. CtBP1/2 knockout mice will be generated to test a specific mechanisms by which

COMPARISON OF OVERALL METABOLISM OF 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIOXIN IN CYP1A2(-/-) KNOCKOUT AND C57BL/6N PARENTAL STRAINS OF MICE

EPA Science Inventory

Comparison of Overall Metabolism of 2,3,7,8-TCDD
in CYP1A2 (-/-) Knockout and C57BL/6N Parental Strains of Mice

Heldur Hakk* and Janet J. Diliberto**

* USDA-ARS Biosciences Research Laboratory, P.O. Box 5674, Fargo, ND, USA
** US-EPA ORD, National Health Eff...

Rapid multislice T1 mapping of mouse myocardium: Application to quantification of manganese uptake in α-Dystrobrevin knockout mice.

PubMed

Jiang, Kai; Li, Wen; Li, Wei; Jiao, Sen; Castel, Laurie; Van Wagoner, David R; Yu, Xin

2015-11-01

The aim of this study was to develop a rapid, multislice cardiac T1 mapping method in mice and to apply the method to quantify manganese (Mn(2+)) uptake in a mouse model with altered Ca(2+) channel activity. An electrocardiography-triggered multislice saturation-recovery Look-Locker method was developed and validated both in vitro and in vivo. A two-dose study was performed to investigate the kinetics of T1 shortening, Mn(2+) relaxivity in myocardium, and the impact of Mn(2+) on cardiac function. The sensitivity of Mn(2+)-enhanced MRI in detecting subtle changes in altered Ca(2+) channel activity was evaluated in a mouse model with α-dystrobrevin knockout. Validation studies showed strong agreement between the current method and an established method. High Mn(2+) dose led to significantly accelerated T1 shortening. Heart rate decreased during Mn(2+) infusion, while ejection ratio increased slightly at the end of imaging protocol. No statistical difference in cardiac function was detected between the two dose groups. Mice with α-dystrobrevin knockout showed enhanced Mn(2+) uptake in vivo. In vitro patch-clamp study showed increased Ca(2+) channel activity. The saturation recovery method provides rapid T1 mapping in mouse hearts, which allowed sensitive detection of subtle changes in Mn(2+) uptake in α-dystrobrevin knockout mice. © 2014 Wiley Periodicals, Inc.

PAR-2 mediates increased inflammatory cell adhesion and neointima formation following vascular injury in the mouse.

PubMed

Tennant, Gail M; Wadsworth, Roger M; Kennedy, Simon

2008-05-01

Activation of PAR-2 in the vasculature affects vascular tone and adhesion of leukocytes to the endothelium. Since adhesion of leukocytes is increased following vascular injury and is important in determining the extent of neointima formation, we hypothesised that mice lacking PAR-2 may have reduced neointima formation following vascular injury. PAR-2 activating peptides and trypsin induced endothelium-dependent relaxation of mouse carotid artery which was absent in the knockout mouse. Lack of a PAR-2 receptor did not affect lymphocyte adhesion under basal conditions, but reduced the contractile response produced by lymphocytes. Twenty-eight days after denuding injury, vessel contraction to lymphocytes was reduced in both strains while lymphocyte adhesion was significantly greater in PAR-2(+/+) mice compared to the PAR-2 knockout mice. Neointimal area was markedly reduced in the PAR-2 knockout mouse. Our data show that PAR-2 modulates inflammatory cell adhesion when stimulated and in mice lacking the PAR-2 receptor, adhesion to injured vessels is reduced with a consequent reduction in neointima formation.

Deletion of H-Ras decreases renal fibrosis and myofibroblast activation following ureteral obstruction in mice.

PubMed

Grande, M Teresa; Fuentes-Calvo, Isabel; Arévalo, Miguel; Heredia, Fabiana; Santos, Eugenio; Martínez-Salgado, Carlos; Rodríguez-Puyol, Diego; Nieto, M Angela; López-Novoa, José M

2010-03-01

Tubulointerstitial fibrosis is characterized by the presence of myofibroblasts that contribute to extracellular matrix accumulation. These cells may originate from resident fibroblasts, bone-marrow-derived cells, or renal epithelial cells converting to a mesenchymal phenotype. Ras GTPases are activated during renal fibrosis and play crucial roles in regulating both cell proliferation and TGF-beta-induced epithelial-mesenchymal transition. Here we set out to assess the contribution of Ras to experimental renal fibrosis using the well-established model of unilateral ureteral obstruction. Fifteen days after obstruction, both fibroblast proliferation and inducers of epithelial-mesenchymal transition were lower in obstructed kidneys of H-ras knockout mice and in fibroblast cell lines derived from these mice. Interestingly, fibronectin, collagen I accumulation, overall interstitial fibrosis, and the myofibroblast population were also lower in the knockout than in the wild-type mice. As expected, we found lower levels of activated Akt in the kidneys and cultured fibroblasts of the knockout. Whether Ras inhibition will turn out to prevent progression of renal fibrosis will require more direct studies.

Essential Role of NMDA Receptor Channel ε4 Subunit (GluN2D) in the Effects of Phencyclidine, but Not Methamphetamine

PubMed Central

Hagino, Yoko; Kasai, Shinya; Han, Wenhua; Yamamoto, Hideko; Nabeshima, Toshitaka; Mishina, Masayoshi; Ikeda, Kazutaka

2010-01-01

Phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, increases locomotor activity in rodents and causes schizophrenia-like symptoms in humans. Although activation of the dopamine (DA) pathway is hypothesized to mediate these effects of PCP, the precise mechanisms by which PCP induces its effects remain to be elucidated. The present study investigated the effect of PCP on extracellular levels of DA (DAex) in the striatum and prefrontal cortex (PFC) using in vivo microdialysis in mice lacking the NMDA receptor channel ε1 or ε4 subunit (GluRε1 [GluN2A] or GluRε4 [GluN2D]) and locomotor activity. PCP significantly increased DAex in wildtype and GluRε1 knockout mice, but not in GluRε4 knockout mice, in the striatum and PFC. Acute and repeated administration of PCP did not increase locomotor activity in GluRε4 knockout mice. The present results suggest that PCP enhances dopaminergic transmission and increases locomotor activity by acting at GluRε4. PMID:21060893

Oral chromium picolinate impedes hyperglycemia-induced atherosclerosis and inhibits proatherogenic protein TSP-1 expression in STZ-induced type 1 diabetic ApoE-/- mice.

PubMed

Ganguly, Rituparna; Sahu, Soumyadip; Ohanyan, Vahagn; Haney, Rebecca; Chavez, Ronaldo J; Shah, Shivani; Yalamanchili, Siri; Raman, Priya

2017-03-27

Increasing evidence suggests thrombospondin-1 (TSP-1), a potent proatherogenic matricellular protein, as a putative link between hyperglycemia and atherosclerotic complications in diabetes. We previously reported that the micronutrient chromium picolinate (CrP), with long-standing cardiovascular benefits, inhibits TSP-1 expression in glucose-stimulated human aortic smooth muscle cells in vitro. Here, we investigated the atheroprotective action of orally administered CrP in type 1 diabetic apolipoprotein E-deficient (ApoE -/- ) mice and elucidated the role of TSP-1 in this process. CrP decreased lipid burden and neointimal thickness in aortic root lesions of hyperglycemic ApoE -/- mice; also, smooth muscle cell (SMC), macrophage and leukocyte abundance was prevented coupled with reduced cell proliferation. Attenuated lesion progression was accompanied with inhibition of hyperglycemia-induced TSP-1 expression and reduced protein O-glycosylation following CrP treatment; also, PCNA and vimentin (SMC synthetic marker) expression were reduced while SM-MHC (SMC contractile marker) levels were increased. To confirm a direct role of TSP-1 in diabetic atherosclerosis, hyperglycemic TSP-1 -/- /ApoE -/- double knockout mice were compared with age-matched hyperglycemic ApoE -/- littermates. Lack of TSP-1 prevented lesion formation in hyperglycemic ApoE -/- mice, mimicking the atheroprotective phenotype of CrP-treated mice. These results suggest that therapeutic TSP-1 inhibition may have important atheroprotective potential in diabetic vascular disease.

Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism

PubMed Central

Pan, Youdong; Tian, Tian; Park, Chang Ook; Lofftus, Serena Y.; Mei, Shenglin; Liu, Xing; Luo, Chi; O’Malley, John T.; Gehad, Ahmed; Teague, Jessica E.; Divito, Sherrie J.; Fuhlbrigge, Robert; Puigserver, Pere; Krueger, James G.; Hotamisligil, Gökhan S.; Clark, Rachael A.; Kupper, Thomas S.

2017-01-01

Tissue-resident memory T (TRM) cells persist indefinitely in epithelial barrier tissues and protect the host against pathogens1–4. However, the biological pathways that enable the long-term survival of TRM cells are obscure4,5. Here we show that mouse CD8+ TRM cells generated by viral infection of the skin differentially express high levels of several molecules that mediate lipid uptake and intracellular transport, including fatty-acid-binding proteins 4 and 5 (FABP4 and FABP5). We further show that T-cell-specific deficiency of Fabp4 and Fabp5 (Fabp4/Fabp5) impairs exogenous free fatty acid (FFA) uptake by CD8+ TRM cells and greatly reduces their long-term survival in vivo, while having no effect on the survival of central memory T (TCM) cells in lymph nodes. In vitro, CD8+ TRM cells, but not CD8+ TCM, demonstrated increased mitochondrial oxidative metabolism in the presence of exogenous FFAs; this increase was not seen in Fabp4/Fabp5 double-knockout CD8+ TRM cells. The persistence of CD8+ TRM cells in the skin was strongly diminished by inhibition of mitochondrial FFA β-oxidation in vivo. Moreover, skin CD8+ TRM cells that lacked Fabp4/Fabp5 were less effective at protecting mice from cutaneous viral infection, and lung Fabp4/Fabp5 double-knockout CD8+ TRM cells generated by skin vaccinia virus (VACV) infection were less effective at protecting mice from a lethal pulmonary challenge with VACV. Consistent with the mouse data, increased FABP4 and FABP5 expression and enhanced extracellular FFA uptake were also demonstrated in human CD8+ TRM cells in normal and psoriatic skin. These results suggest that FABP4 and FABP5 have a critical role in the maintenance, longevity and function of CD8+ TRM cells, and suggest that CD8+ TRM cells use exogenous FFAs and their oxidative metabolism to persist in tissue and to mediate protective immunity. PMID:28219080

Global Nav1.7 Knockout Mice Recapitulate the Phenotype of Human Congenital Indifference to Pain

PubMed Central

Gingras, Jacinthe; Smith, Sarah; Matson, David J.; Johnson, Danielle; Nye, Kim; Couture, Lauren; Feric, Elma; Yin, Ruoyuan; Moyer, Bryan D.; Peterson, Matthew L.; Rottman, James B.; Beiler, Rudolph J.; Malmberg, Annika B.; McDonough, Stefan I.

2014-01-01

Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund’s adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain. PMID:25188265

Genotype identification of Math1/LacZ knockout mice based on real-time PCR with SYBR Green I dye.

PubMed

Krizhanovsky, Valery; Golenser, Esther; Ben-Arie, Nissim

2004-07-30

Knockout mice are widely used in all fields of biomedical research. Determining the genotype of every newborn mouse is a tedious task, usually performed by Southern blot hybridization or Polymerase Chain Reaction (PCR). We describe here a quick and simple genotype identification assay based on real-time PCR and SYBR Green I dye, without using fluorescent primers. The discrimination between the wild type and targeted alleles is based on a PCR design that leads to a different melting temperature for each product. The identification of the genotype is obvious immediately after amplification, and no post-PCR manipulations are needed, reducing cost and time. Therefore, while the real-time PCR amplification increases the sensitivity, the fact that the reactions tubes are never opened after amplification, reduces the risk of contamination and eliminates errors, which are common during the repeated handling of dozens of samples from the same mouse line. The protocol we provide was tested on Math1 knockout mice, but is general, and may be utilized for any knockout line and real-time thermocycler, without any further modification, accessories or special reagents. Copyright 2004 Elsevier B.V.

Macrophage migration inhibitory factor (MIF) knockout preserves cardiac homeostasis through alleviating Akt-mediated myocardial autophagy suppression in high-fat diet-induced obesity.

PubMed

Xu, X; Ren, J

2015-03-01

Macrophage migration inhibitory factor (MIF) has a role in the development of obesity and diabetes. However, whether MIF has a role in fat diet-induced obesity and associated cardiac anomalies still remains unknown. The aim of this study was to examine the impact of MIF knockout on high-fat diet-induced obesity, obesity-associated cardiac anomalies and the underlying mechanisms involved with a focus on Akt-mediated autophagy. Adult male wild-type (WT) and MIF knockout (MIF(-/-)) mice were placed on 45% high-fat diet for 5 months. Oxygen consumption, CO2 production, respiratory exchange ratio, locomotor activity and heat generation were measured using energy calorimeter. Echocardiographic, cardiomyocyte mechanical and intracellular Ca2+ properties were assessed. Apoptosis was examined using terminal dUTP nick end labeling staining and western blot analysis. Akt signaling pathway and autophagy markers were evaluated. Cardiomyocytes isolated from WT and MIF(-/-) mice were treated with recombinant mouse MIF (rmMIF). High-fat diet feeding elicited increased body weight gain, insulin resistance and caloric disturbance in WT and MIF(-/-) mice. High-fat diet induced unfavorable geometric, contractile and histological changes in the heart, the effects of which were alleviated by MIF knockout. In addition, fat diet-induced cardiac anomalies were associated with Akt activation and autophagy suppression, which were nullified by MIF deficiency. In cardiomyocytes from WT mice, autophagy was inhibited by exogenous rmMIF through Akt activation. In addition, MIF knockout rescued palmitic acid-induced suppression of cardiomyocyte autophagy, the effect of which was nullified by rmMIF. These results indicate that MIF knockout preserved obesity-associated cardiac anomalies without affecting fat diet-induced obesity, probably through restoring myocardial autophagy in an Akt-dependent manner. Our findings provide new insights for the role of MIF in obesity and associated cardiac anomalies.

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.

PubMed

Gao, Hanchao; Zhao, Chengjiang; Xiang, Xi; Li, Yong; Zhao, Yanli; Li, Zesong; Pan, Dengke; Dai, Yifan; Hara, Hidetaka; Cooper, David K C; Cai, Zhiming; Mou, Lisha

2017-02-16

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

PubMed

Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

2015-08-01

We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Ca2+-activated Cl- currents in the murine vomeronasal organ enhance neuronal spiking but are dispensable for male-male aggression.

PubMed

Münch, Jonas; Billig, Gwendolyn; Huebner, Christian A; Leinders-Zufall, Trese; Zufall, Frank; Jentsch, Thomas J

2018-05-16

Ca2+-activated Cl- currents have been observed in many physiological processes, including sensory transduction in mammalian olfaction. The olfactory vomeronasal (or Jacobson's) organ (VNO) detects molecular cues originating from animals of the same species or from predators. It then triggers innate behaviors such as aggression, mating, or flight. In the VNO, Ca2+-activated Cl- channels (CaCCs) are thought to amplify the initial pheromone-evoked receptor potential by mediating a depolarizing Cl- efflux. Here, we confirmed the co-localization of the Ca2+-activated Cl- channels anoctamin 1 (Ano1, also called TMEM16A) and Ano2 (TMEM16B) in microvilli of apically and basally located vomeronasal sensory neurons (VSNs) and their absence in supporting cells of the VNO. Both channels were expressed as functional isoforms capable of giving rise to Ca2+-activated Cl- currents. While these currents persisted in the VNOs of mice lacking Ano2, they were undetectable in olfactory neuron-specific Ano1 knock-out mice irrespective of the presence of Ano2. The loss of Ca2+-activated Cl- currents resulted in diminished spontaneous and drastically reduced pheromone-evoked spiking of VSNs. Although this indicated an important role of anoctamin channels in VNO signal amplification, the lack of this amplification did not alter VNO-dependent male-male territorial aggression in olfactory Ano1/Ano2 double knock-out mice. We conclude that Ano1 mediates the bulk of Ca2+-activated Cl- currents in the VNO and that Ano2 plays only a minor role. Furthermore, vomeronasal signal amplification by CaCCs appears to be dispensable for the detection of male-specific pheromones and for near-normal aggressive behavior in mice. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Scavenger receptor function of mouse Fcγ receptor III contributes to progression of atherosclerosis in apolipoprotein E hyperlipidemic mice.

PubMed

Zhu, Xinmei; Ng, Hang Pong; Lai, Yen-Chun; Craigo, Jodi K; Nagilla, Pruthvi S; Raghani, Pooja; Nagarajan, Shanmugam

2014-09-01

Recent studies showed loss of CD36 or scavenger receptor-AI/II (SR-A) does not ameliorate atherosclerosis in a hyperlipidemic mouse model, suggesting receptors other than CD36 and SR-A may also contribute to atherosclerosis. In this report, we show that apolipoprotein E (apoE)-CD16 double knockout (DKO; apoE-CD16 DKO) mice have reduced atherosclerotic lesions compared with apoE knockout mice. In vivo and in vitro foam cell analyses showed apoE-CD16 DKO macrophages accumulated less neutral lipids. Reduced foam cell formation in apoE-CD16 DKO mice is not due to change in expression of CD36, SR-A, and LOX-1. This led to a hypothesis that CD16 may have scavenger receptor activity. We presented evidence that a soluble form of recombinant mouse CD16 (sCD16) bound to malondialdehyde-modified low-density lipoprotein (MDALDL), and this binding is blocked by molar excess of MDA- modified BSA and anti-MDA mAbs, suggesting CD16 specifically recognizes MDA epitopes. Interestingly, sCD16 inhibited MDALDL binding to macrophage cell line, as well as soluble forms of recombinant mouse CD36, SR-A, and LOX-1, indicating CD16 can cross-block MDALDL binding to other scavenger receptors. Anti-CD16 mAb inhibited immune complex binding to sCD16, whereas it partially inhibited MDALDL binding to sCD16, suggesting MDALDL binding site may be in close proximity to the immune complex binding site in CD16. Loss of CD16 expression resulted in reduced levels of MDALDL-induced proinflammatory cytokine expression. Finally, CD16-deficient macrophages showed reduced MDALDL-induced Syk phosphorylation. Collectively, our findings suggest scavenger receptor activity of CD16 may, in part, contribute to the progression of atherosclerosis. Copyright © 2014 by The American Association of Immunologists, Inc.

Hepatocyte specific deletion of c-Met leads to the development of severe non-alcoholic steatohepatitis in mice.

PubMed

Kroy, Daniela C; Schumacher, Fabienne; Ramadori, Pierluigi; Hatting, Maximilian; Bergheim, Ina; Gassler, Nikolaus; Boekschoten, Mark V; Müller, Michael; Streetz, Konrad L; Trautwein, Christian

2014-10-01

Non-alcoholic-fatty-liver disease (NAFLD) is part of the metabolic syndrome. The spectrum of NAFLD includes NASH (non-alcoholic steatohepatitis), which is characterised by progressive inflammation associated with oxidative stress and apoptosis, finally triggering liver cirrhosis and hepatocellular carcinoma. HGF (hepatocyte growth factor)/mesenchymal-epithelial transition factor (c-Met) receptor signalling is known to activate distinct intracellular pathways mediating among others anti-apoptotic properties to hepatocytes. Therefore, the aim was to characterise the role of c-Met during NASH development. Hepatocyte specific c-Met knockout mice (c-MetΔ(hepa)) using the cre-loxP system and wild type controls (c-Met(loxP/loxP)) were fed a methionine-choline deficient (MCD) diet. MCD feeding triggered massive steatosis, decreased survival and higher transaminases in c-MetΔ(hepa) livers compared to c-Met(loxP/loxP). Gene array analysis demonstrated that genes involved in fatty acid metabolism were strongly upregulated in c-MetΔ(hepa) livers correlating with higher amounts of hepatic free fatty acids. Consequently, c-MetΔ(hepa) mice showed significantly more TUNEL positive cells and more superoxide anion production than c-Met(loxPloxP) animals. Additionally, c-MetΔ(hepa) livers showed significantly larger fractions of infiltrating neutrophils, macrophages, and cytotoxic T cells. These changes correlated with an enhanced progression of liver fibrosis as evidenced by higher collagen deposition in c-MetΔ(hepa) livers. As increased apoptosis was a prominent feature in c-MetΔ(hepa) livers, we generated c-Met/Casp8Δ(hepa) double knockout mice. In these animals compared to c-MetΔ(hepa) animals the increase in apoptosis could be reverted. c-Met deletion in hepatocytes triggers NASH progression. A prominent mechanism is higher fatty acid accumulation and increased apoptosis, which in part can be reverted by blocking caspase 8. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Single and Compound Knock-outs of MicroRNA (miRNA)-155 and Its Angiogenic Gene Target CCN1 in Mice Alter Vascular and Neovascular Growth in the Retina via Resident Microglia.

PubMed

Yan, Lulu; Lee, Sangmi; Lazzaro, Douglas R; Aranda, Jacob; Grant, Maria B; Chaqour, Brahim

2015-09-18

The response of the retina to ischemic insult typically leads to aberrant retinal neovascularization, a major cause of blindness. The epigenetic regulation of angiogenic gene expression by miRNAs provides new prospects for their therapeutic utility in retinal neovascularization. Here, we focus on miR-155, a microRNA functionally important in inflammation, which is of paramount importance in the pathogenesis of retinal neovascularization. Whereas constitutive miR-155-deficiency in mice results in mild vascular defects, forced expression of miR-155 causes endothelial hyperplasia and increases microglia count and activation. The mouse model of oxygen-induced retinopathy, which recapitulates ischemia-induced aberrant neovessel growth, is characterized by increased expression of miR-155 and localized areas of microglia activation. Interestingly, miR-155 deficiency in mice reduces microglial activation, curtails abnormal vessel growth, and allows for rapid normalization of the retinal vasculature following ischemic insult. miR-155 binds to the 3'-UTR and represses the expression of the CCN1 gene, which encodes an extracellular matrix-associated integrin-binding protein that both promotes physiological angiogenesis and harnesses growth factor-induced abnormal angiogenic responses. Single CCN1 deficiency or double CCN1 and miR-155 knock-out in mice causes retinal vascular malformations typical of faulty maturation, mimicking the vascular alterations of miR-155 gain of function. During development, the miR-155/CCN1 regulatory axis balances the proangiogenic and proinflammatory activities of microglia to allow for their function as guideposts for sprout fusion and anastomosis. Under ischemic conditions, dysregulated miR-155 and CCN1 expression increases the inflammatory load and microglial activation, prompting aberrant angiogenic responses. Thus, miR-155 functions in tandem with CCN1 to modulate inflammation-induced vascular homeostasis and repair. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Single and Compound Knock-outs of MicroRNA (miRNA)-155 and Its Angiogenic Gene Target CCN1 in Mice Alter Vascular and Neovascular Growth in the Retina via Resident Microglia*

PubMed Central

Yan, Lulu; Lee, Sangmi; Lazzaro, Douglas R.; Aranda, Jacob; Grant, Maria B.; Chaqour, Brahim

2015-01-01

The response of the retina to ischemic insult typically leads to aberrant retinal neovascularization, a major cause of blindness. The epigenetic regulation of angiogenic gene expression by miRNAs provides new prospects for their therapeutic utility in retinal neovascularization. Here, we focus on miR-155, a microRNA functionally important in inflammation, which is of paramount importance in the pathogenesis of retinal neovascularization. Whereas constitutive miR-155-deficiency in mice results in mild vascular defects, forced expression of miR-155 causes endothelial hyperplasia and increases microglia count and activation. The mouse model of oxygen-induced retinopathy, which recapitulates ischemia-induced aberrant neovessel growth, is characterized by increased expression of miR-155 and localized areas of microglia activation. Interestingly, miR-155 deficiency in mice reduces microglial activation, curtails abnormal vessel growth, and allows for rapid normalization of the retinal vasculature following ischemic insult. miR-155 binds to the 3′-UTR and represses the expression of the CCN1 gene, which encodes an extracellular matrix-associated integrin-binding protein that both promotes physiological angiogenesis and harnesses growth factor-induced abnormal angiogenic responses. Single CCN1 deficiency or double CCN1 and miR-155 knock-out in mice causes retinal vascular malformations typical of faulty maturation, mimicking the vascular alterations of miR-155 gain of function. During development, the miR-155/CCN1 regulatory axis balances the proangiogenic and proinflammatory activities of microglia to allow for their function as guideposts for sprout fusion and anastomosis. Under ischemic conditions, dysregulated miR-155 and CCN1 expression increases the inflammatory load and microglial activation, prompting aberrant angiogenic responses. Thus, miR-155 functions in tandem with CCN1 to modulate inflammation-induced vascular homeostasis and repair. PMID:26242736

The bcl-2 knockout mouse exhibits marked changes in osteoblast phenotype and collagen deposition in bone as well as a mild growth plate phenotype

PubMed Central

BOOT-HANDFORD, R. P.; MICHAELIDIS, T. M.; HILLARBY, M. C.; ZAMBELLI, A.; DENTON, J.; HOYLAND, J. A.; FREEMONT, A. J.; GRANT, M. E.; WALLIS, G. A.

1998-01-01

Histological examination of long bones from 1-day-old bcl-2 knockout and age-matched control mice revealed no obvious differences in length of bone, growth plate architecture or stage of endochondral ossification. In 35-day-old bcl-2 knockout mice that are growth retarded or ‘dwarfed’, the proliferative zone of the growth plate appeared slightly thinner and the secondary centres of ossification less well developed than their age-matched wild-type controls. The most marked histological effects of bcl-2 ablation were on osteoblasts and bone. 35-day-old knockout mouse bones exhibited far greater numbers of osteoblasts than controls and the osteoblasts had a cuboidal phenotype in comparison with the normal flattened cell appearance. In addition, the collagen deposited by the osteoblasts in the bcl-2 knockout mouse bone was disorganized in comparison with control tissue and had a pseudo-woven appearance. The results suggest an important role for Bcl-2 in controlling osteoblast phenotype and bone deposition in vivo. PMID:10193316

Mice Deficient in NF-κB p50 and p52 or RANK Have Defective Growth Plate Formation and Post-natal Dwarfism.

PubMed

Xing, Lianping; Chen, Di; Boyce, Brendan F

2013-12-01

NF-κBp50/p52 double knockout (dKO) and RANK KO mice have no osteoclasts and develop severe osteopetrosis associated with dwarfism. In contrast, Op/Op mice, which form few osteoclasts, and Src KO mice, which have osteoclasts with defective resorptive function, are osteopetrotic, but they are not dwarfed. Here, we compared the morphologic features of long bones from p50/p52 dKO, RANK KO, Op/Op and Src KO mice to attempt to explain the differences in their long bone lengths. We found that growth plates in p50/p52 dKO and RANK KO mice are significantly thicker than those in WT mice due to a 2-3-fold increase in the hypertrophic chondrocyte zone associated with normal a proliferative chondrocyte zone. This growth plate abnormality disappears when animals become older, but their dwarfism persists. Op/Op or Src KO mice have relatively normal growth plate morphology. In-situ hybridization study of long bones from p50/p52 dKO mice showed marked thickening of the growth plate region containing type 10 collagen-expressing chondrocytes. Treatment of micro-mass chondrocyte cultures with RANKL did not affect expression levels of type 2 collagen and Sox9, markers for proliferative chondrocytes, but RANKL reduced the number of type 10 collagen-expressing hypertrophic chondrocytes. Thus, RANK/NF-κB signaling plays a regulatory role in post-natal endochondral ossification that maintains hypertrophic conversion and prevents dwarfism in normal mice.

Deletion of Dual Specificity Phosphatase 1 Does Not Predispose Mice to Increased Spontaneous Osteoarthritis

PubMed Central

Pest, Michael Andrew; Pest, Courtney Alice; Bellini, Melina Rodrigues; Feng, Qingping; Beier, Frank

2015-01-01

Background Osteoarthritis (OA) is a degenerative joint disease with poorly understood etiology and pathobiology. Mitogen activated protein kinases (MAPKs) including ERK and p38 play important roles in the mediation of downstream pathways involved in cartilage degenerative processes. Dual specificity phosphatase 1 (DUSP1) dephosphorylates the threonine/serine and tyrosine sites on ERK and p38, causing deactivation of downstream signalling. In this study we examined the role of DUSP1 in spontaneous OA development at 21 months of age using a genetically modified mouse model deficient in Dusp1 (DUSP1 knockout mouse). Results Utilizing histochemical stains of paraffin embedded knee joint sections in DUSP1 knockout and wild type female and male mice, we showed similar structural progression of cartilage degeneration associated with OA at 21 months of age. A semi-quantitative cartilage degeneration scoring system also demonstrated similar scores in the various aspects of the knee joint articular cartilage in DUSP1 knockout and control mice. Examination of overall articular cartilage thickness in the knee joint demonstrated similar results between DUSP1 knockout and wild type mice. Immunostaining for cartilage neoepitopes DIPEN, TEGE and C1,2C was similar in the cartilage lesion sites and chondrocyte pericellular matrix of both experimental groups. Likewise, immunostaining for phosphoERK and MMP13 showed similar intensity and localization between groups. SOX9 immunostaining demonstrated a decreased number of positive cells in DUSP1 knockout mice, with correspondingly decreased staining intensity. Analysis of animal walking patterns (gait) did not show a discernable difference between groups. Conclusion Loss of DUSP1 does not cause changes in cartilage degeneration and gait in a mouse model of spontaneous OA at 21 months of age. Altered staining was observed in SOX9 immunostaining which may prove promising for future studies examining the role of DUSPs in cartilage and OA, as well as models of post-traumatic OA. PMID:26562438

Knocking-out matrix metalloproteinase-13 exacerbates rotator cuff muscle fatty infiltration

PubMed Central

Liu, Xuhui; Ravishankar, Bharat; Ning, Anne; Liu, Mengyao; Kim, Hubert T.; Feeley, Brian T.

2017-01-01

Summary Introduction Rotator cuff (RC) tears are common tendon injuries. Clinically, both muscle atrophy and fatty infiltration have generally been attributed to poor functional outcomes. Matrix metalloproteinase-13 plays a crucial role in extracellular matrix remodeling in many physiological and pathological processes. Nevertheless, its role in rotator cuff muscle atrophy and fatty infiltration remains unknown. The purpose of this study is to define the functional role of MMP-13 in rotator cuff muscle atrophy and fatty infiltration using a mouse RC tears model. Materials and methods Unilateral complete supraspinatus and infraspinatus tendon transection and suprascapular nerve transection was performed on nine of MMP-13 (−/−) knockout and nine of MMP-13 (+/+) wildtype mice at 3 months old. Mice were sacrificed 6 weeks after surgery. Supraspinatus (SS) and infraspinatus (IS) muscles were harvested for histology and gene expression analysis with RT-PCR. Results Six weeks after RC surgery, no significant difference in muscle atrophy and fibrosis between MMP-13 knockout and wild type mice was observed. However, there was a significant increase in the amount of fatty infiltration in MMP-13 knockout mice compared to the wild types. Muscles from MMP-13 knockout mice have significantly higher expression of fatty infiltration related genes. Discussion Results from this study suggest that MMP-13 plays a crucial role in rotator cuff muscle fatty degeneration. This novel finding suggests a new molecular mechanism that governs RC muscle FI and MMP-13 may serve as a target for therapeutics to treat muscle FI after RC tears. PMID:29264329

The β3-adrenergic receptor is dispensable for browning of adipose tissues.

PubMed

de Jong, Jasper M A; Wouters, René T F; Boulet, Nathalie; Cannon, Barbara; Nedergaard, Jan; Petrovic, Natasa

2017-06-01

Brown and brite/beige adipocytes are attractive therapeutic targets to treat metabolic diseases. To maximally utilize their functional potential, further understanding is required about their identities and their functional differences. Recent studies with β 3 -adrenergic receptor knockout mice reported that brite/beige adipocytes, but not classical brown adipocytes, require the β 3 -adrenergic receptor for cold-induced transcriptional activation of thermogenic genes. We aimed to further characterize this requirement of the β 3 -adrenergic receptor as a functional distinction between classical brown and brite/beige adipocytes. However, when comparing wild-type and β 3 -adrenergic receptor knockout mice, we observed no differences in cold-induced thermogenic gene expression ( Ucp1 , Pgc1a , Dio2 , and Cidea ) in brown or white (brite/beige) adipose tissues. Irrespective of the duration of the cold exposure or the sex of the mice, we observed no effect of the absence of the β 3 -adrenergic receptor. Experiments with the β 3 -adrenergic receptor agonist CL-316,243 verified the functional absence of β 3 -adrenergic signaling in these knockout mice. The β 3 -adrenergic receptor knockout model in the present study was maintained on a FVB/N background, whereas earlier reports used C57BL/6 and 129Sv mice. Thus our data imply background-dependent differences in adrenergic signaling mechanisms in response to cold exposure. Nonetheless, the present data indicate that the β 3 -adrenergic receptor is dispensable for cold-induced transcriptional activation in both classical brown and, as opposed to earlier studies, brite/beige cells. Copyright © 2017 the American Physiological Society.

Motor Deficits and Decreased Striatal Dopamine Receptor 2 Binding Activity in the Striatum-Specific Dyt1 Conditional Knockout Mice

PubMed Central

Yokoi, Fumiaki; Dang, Mai Tu; Li, Jianyong; Standaert, David G.; Li, Yuqing

2011-01-01

DYT1 early-onset generalized dystonia is a hyperkinetic movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Recently, significant progress has been made in studying pathophysiology of DYT1 dystonia using targeted mouse models. Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 knock-down (KD) mice exhibit motor deficits and alterations of striatal dopamine metabolisms, while Dyt1 knockout (KO) and Dyt1 ΔGAG homozygous KI mice show abnormal nuclear envelopes and neonatal lethality. However, it has not been clear whether motor deficits and striatal abnormality are caused by Dyt1 mutation in the striatum itself or the end results of abnormal signals from other brain regions. To identify the brain region that contributes to these phenotypes, we made a striatum-specific Dyt1 conditional knockout (Dyt1 sKO) mouse. Dyt1 sKO mice exhibited motor deficits and reduced striatal dopamine receptor 2 (D2R) binding activity, whereas they did not exhibit significant alteration of striatal monoamine contents. Furthermore, we also found normal nuclear envelope structure in striatal medium spiny neurons (MSNs) of an adult Dyt1 sKO mouse and cerebral cortical neurons in cerebral cortex-specific Dyt1 conditional knockout (Dyt1 cKO) mice. The results suggest that the loss of striatal torsinA alone is sufficient to produce motor deficits, and that this effect may be mediated, at least in part, through changes in D2R function in the basal ganglia circuit. PMID:21931745

Examining Hippocampal Mossy Fiber Synapses by 3D Electron Microscopy in Wildtype and Kirrel3 Knockout Mice

PubMed Central

Rawson, Randi L.

2017-01-01

Neural circuits balance excitatory and inhibitory activity and disruptions in this balance are commonly found in neurodevelopmental disorders. Mice lacking the intellectual disability and autism-associated gene Kirrel3 have an excitation-inhibition imbalance in the hippocampus but the precise synaptic changes underlying this functional defect are unknown. Kirrel3 is a homophilic adhesion molecule expressed in dentate gyrus (DG) and GABA neurons. It was suggested that the excitation-inhibition imbalance of hippocampal neurons in Kirrel3 knockout mice is due to loss of mossy fiber (MF) filopodia, which are DG axon protrusions thought to excite GABA neurons and thereby provide feed-forward inhibition to CA3 pyramidal neurons. Fewer filopodial structures were observed in Kirrel3 knockout mice but neither filopodial synapses nor DG en passant synapses, which also excite GABA neurons, were examined. Here, we used serial block-face scanning electron microscopy (SBEM) with 3D reconstruction to define the precise connectivity of MF filopodia and elucidate synaptic changes induced by Kirrel3 loss. Surprisingly, we discovered wildtype MF filopodia do not synapse exclusively onto GABA neurons as previously thought, but instead synapse with similar frequency onto GABA neurons and CA3 neurons. Moreover, Kirrel3 loss selectively reduces MF filopodial synapses onto GABA neurons but not those made onto CA3 neurons or en passant synapses. In sum, the selective loss of MF filopodial synapses with GABA neurons likely underlies the hippocampal activity imbalance observed in Kirrel3 knockout mice and may impact neural function in patients with Kirrel3-dependent neurodevelopmental disorders. PMID:28670619

Dietary quercetin attenuates oxidant-induced endothelial dysfunction and atherosclerosis in apolipoprotein E knockout mice fed a high-fat diet: a critical role for heme oxygenase-1.

PubMed

Shen, Yu; Ward, Natalie C; Hodgson, Jonathan M; Puddey, Ian B; Wang, Yutang; Zhang, Di; Maghzal, Ghassan J; Stocker, Roland; Croft, Kevin D

2013-12-01

Several lines of evidence indicate that quercetin, a polyphenol derived in the diet from fruit and vegetables, contributes to cardiovascular health. We aimed to investigate the effects of dietary quercetin on endothelial function and atherosclerosis in mice fed a high-fat diet. Wild-type C57BL/6 (WT) and apolipoprotein E gene knockout (ApoE(-/-)) mice were fed: (i) a high-fat diet (HFD) or (ii) a HFD supplemented with 0.05% w/w quercetin (HFD+Q), for 14 weeks. Compared with animals fed HFD, HFD+Q attenuated atherosclerosis in ApoE(-/-) mice. Treatment with the HFD+Q significantly improved endothelium-dependent relaxation of aortic rings isolated from WT but not ApoE(-/-) mice and attenuated hypochlorous acid-induced endothelial dysfunction in aortic rings of both WT and ApoE(-/-) mice. Mechanistic studies revealed that HFD+Q significantly improved plasma F2-isoprostanes, 24h urinary nitrite, and endothelial nitric oxide synthase activity, and increased heme oxygenase-1 (HO-1) protein expression in the aortas of both WT and ApoE(-/-) mice (P<0.05). HFD+Q also resulted in small changes in plasma cholesterol (P<0.05 in WT) and plasma triacylglycerols (P<0.05 in ApoE (-/-)mice). In a separate experiment, quercetin did not protect against hypochlorite-induced endothelial dysfunction in arteries obtained from heterozygous HO-1 gene knockout mice with low expression of HO-1 protein. Quercetin protects mice fed a HFD against oxidant-induced endothelial dysfunction and ApoE(-/-) mice against atherosclerosis. These effects are associated with improvements in nitric oxide bioavailability and are critically related to arterial induction of HO-1. © 2013 Elsevier Inc. All rights reserved.

Progressive hearing loss and degeneration of hair cell stereocilia in taperin gene knockout mice

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

Chen, Mo; Wang, Qin; Zhu, Gang-Hua

The TPRN gene encodes taperin, which is prominently present at the taper region of hair cell stereocilia. Mutations in TPRN have been reported to cause autosomal recessive nonsyndromic deafness 79(DFNB 79). To investigate the role of taperin in pathogenesis of hearing loss, we generated TPRN knockout mice using TALEN technique. Sanger sequencing confirmed an 11 bp deletion at nucleotide 177–187 in exon 1 of TPRN, which results in a truncated form of taperin protein. Heterozygous TPRN{sup +/−} mice showed apparently normal auditory phenotypes to their wide-type (WT) littermates. Homozygous TPRN{sup −/−} mice exhibited progressive sensorineural hearing loss as reflected bymore » auditory brainstem response to both click and tone burst stimuli at postnatal days 15 (P15), 30 (P30), and 60 (P60). Alex Fluor-594 phalloidin labeling showed no obvious difference in hair cell numbers in the cochlea between TPRN{sup −/−} mice and WT mice under light microscope. However, scanning electronic microscopy revealed progressive degeneration of inner hair cell stereocilia, from apparently normal at postnatal days 3 (P3) to scattered absence at P15 and further to substantial loss at P30. The outer hair cell stereocilia also showed progressive degeneration, though much less severe, Collectively, we conclude that taperin plays an important role in maintenance of hair cell stereocilia. Establishment of TPRN knockout mice enables further investigation into the function of this gene. - Highlights: • TPRN{sup −/−} mice were generated using TALEN technique. • TPRN{sup −/−} mice presented progressive hearing loss. • WT and TPRN{sup −/−} mice showed no difference in hair cell numbers. • TPRN{sup −/−} mice showed progressive degeneration of hair cell stereocilia.« less

Embryonic ablation of neuronal VGF increases energy expenditure and reduces body weight

PubMed Central

Jiang, Cheng; Lin, Wei-Jye; Sadahiro, Masato; Shin, Andrew C.; Buettner, Christoph; Salton, Stephen R.

2016-01-01

Germline ablation of VGF, a secreted neuronal, neuroendocrine, and endocrine peptide precursor, results in lean, hypermetabolic, and infertile adult mice that are resistant to diet-, lesion-, and genetically-induced obesity and diabetes (Hahm et al., 1999, 2002). To assess whether this phenotype is predominantly driven by reduced VGF expression in developing and/or adult neurons, or in peripheral endocrine and neuroendocrine tissues, we generated and analyzed conditional VGF knockout mice, obtained by mating loxP-flanked (floxed) Vgf mice with either pan-neuronal Synapsin-Cre- or forebrain alpha-CaMKII-Cre-recombinase-expressing transgenic mice. Adult male and female mice, with conditional ablation of the Vgf gene in embryonic neurons had significantly reduced body weight, increased energy expenditure, and were resistant to diet-induced obesity. Conditional forebrain postnatal ablation of VGF in male mice, primarily in adult excitatory neurons, had no measurable effect on body weight nor on energy expenditure, but led to a modest increase in adiposity, partially overlapping the effect of AAV-Cre-mediated targeted ablation of VGF in the adult ventromedial hypothalamus and arcuate nucleus of floxed Vgf mice (Foglesong et al., 2016), and also consistent with results of icv delivery of the VGF-derived peptide TLQP-21 to adult mice, which resulted in increased energy expenditure and reduced adiposity (Bartolomucci et al., 2006). Because the lean, hypermetabolic phenotype of germline VGF knockout mice is to a great extent recapitulated in Syn-Cre+/−,Vgfflpflox/flpflox mice, we conclude that the metabolic profile of germline VGF knockout mice is largely the result of VGF ablation in embryonic CNS neurons, rather than peripheral endocrine and/or neuroendocrine cells, and that in forebrain structures such as hypothalamus, VGF and/or VGF-derived peptides play uniquely different roles in the developing and adult nervous system. PMID:28024880

A STAT-1 Knockout Mouse Model for Machupo Virus Pathogenesis

DTIC Science & Technology

2011-06-14

hemorrhagic fever viruses, including Ebola, Marburg, Junín, and Crimean - Congo Hemorrhagic Fever viruses [11-14...Akerstrom S, Klingstrom J, Mirazimi A: Crimean - Congo hemorrhagic fever virus infection is lethal for adult type I interferon receptor-knockout mice. J...Shieh WJ, Camus G, Stroher U, Zaki S, Jones SM: Pathogenesis and immune response of Crimean - Congo hemorrhagic fever virus in a STAT-1 knockout

Roles of α- and β-estrogen receptors in mouse social recognition memory: effects of gender and the estrous cycle.

PubMed

Sánchez-Andrade, G; Kendrick, K M

2011-01-01

Establishing clear effects of gender and natural hormonal changes during female ovarian cycles on cognitive function has often proved difficult. Here we have investigated such effects on the formation and long-term (24 h) maintenance of social recognition memory in mice together with the respective involvement of α- and β-estrogen receptors using α- and β-estrogen receptor knockout mice and wildtype controls. Results in wildtype animals showed that while females successfully formed a memory in the context of a habituation/dishabituation paradigm at all stages of their ovarian cycle, only when learning occurred during proestrus (when estrogen levels are highest) was it retained after 24 h. In α-receptor knockout mice (which showed no ovarian cycles) both formation and maintenance of this social recognition memory were impaired, whereas β-receptor knockouts showed no significant deficits and exhibited the same proestrus-dependent retention of memory at 24 h. To investigate possible sex differences, male α- and β-estrogen receptor knockout mice were also tested and showed similar effects to females excepting that α-receptor knockouts had normal memory formation and only exhibited a 24 h retention deficit. This indicates a greater dependence in females on α-receptor expression for memory formation in this task. Since non-specific motivational and attentional aspects of the task were unaffected, our findings suggest a general α-receptor dependent facilitation of memory formation by estrogen as well as an enhanced long-term retention during proestrus. Results are discussed in terms of the differential roles of the two estrogen receptors, the neural substrates involved and putative interactions with oxytocin. Copyright © 2010 Elsevier Inc. All rights reserved.

Polycystin-1 Is a Cardiomyocyte Mechanosensor That Governs L-Type Ca2+ Channel Protein Stability.

PubMed

Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K; Morales, Cyndi R; Contreras-Ferrat, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J; Somlo, Stefan; Rothermel, Beverly A; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

2015-06-16

L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress-induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction-induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. © 2015 American Heart Association, Inc.

Cardiac Ablation of Rheb1 Induces Impaired Heart Growth, Endoplasmic Reticulum-Associated Apoptosis and Heart Failure in Infant Mice

PubMed Central

Cao, Yunshan; Tao, Lichan; Shen, Shutong; Xiao, Junjie; Wu, Hang; Li, Beibei; Wu, Xiangqi; Luo, Wen; Xiao, Qi; Hu, Xiaoshan; Liu, Hailang; Nie, Junwei; Lu, Shuangshuang; Yuan, Baiyin; Han, Zhonglin; Xiao, Bo; Yang, Zhongzhou; Li, Xinli

2013-01-01

Ras homologue enriched in brain 1 (Rheb1) plays an important role in a variety of cellular processes. In this study, we investigate the role of Rheb1 in the post-natal heart. We found that deletion of the gene responsible for production of Rheb1 from cardiomyocytes of post-natal mice resulted in malignant arrhythmias, heart failure, and premature death of these mice. In addition, heart growth impairment, aberrant metabolism relative gene expression, and increased cardiomyocyte apoptosis were observed in Rheb1-knockout mice prior to the development of heart failure and arrhythmias. Also, protein kinase B (PKB/Akt) signaling was enhanced in Rheb1-knockout mice, and removal of phosphatase and tensin homolog (Pten) significantly prolonged the survival of Rheb1-knockouts. Furthermore, signaling via the mammalian target of rapamycin complex 1 (mTORC1) was abolished and C/EBP homologous protein (CHOP) and phosphorylation levels of c-Jun N-terminal kinase (JNK) were increased in Rheb1 mutant mice. In conclusion, this study demonstrates that Rheb1 is important for maintaining cardiac function in post-natal mice via regulation of mTORC1 activity and stress on the endoplasmic reticulum. Moreover, activation of Akt signaling helps to improve the survival of mice with advanced heart failure. Thus, this study provides direct evidence that Rheb1 performs multiple important functions in the heart of the post-natal mouse. Enhancing Akt activity improves the survival of infant mice with advanced heart failure. PMID:24351823

Comparison of Whole Body SOD1 Knockout with Muscle-Specific SOD1 Knockout Mice Reveals a Role for Nerve Redox Signaling in Regulation of Degenerative Pathways in Skeletal Muscle.

PubMed

Sakellariou, Giorgos K; McDonagh, Brian; Porter, Helen; Giakoumaki, Ifigeneia I; Earl, Kate E; Nye, Gareth A; Vasilaki, Aphrodite; Brooks, Susan V; Richardson, Arlan; Van Remmen, Holly; McArdle, Anne; Jackson, Malcolm J

2018-02-01

Lack of Cu,Zn-superoxide dismutase (CuZnSOD) in homozygous knockout mice (Sod1 -/- ) leads to accelerated age-related muscle loss and weakness, but specific deletion of CuZnSOD in skeletal muscle (mSod1KO mice) or neurons (nSod1KO mice) resulted in only mild muscle functional deficits and failed to recapitulate the loss of mass and function observed in Sod1 -/- mice. To dissect any underlying cross-talk between motor neurons and skeletal muscle in the degeneration in Sod1 -/- mice, we characterized neuromuscular changes in the Sod1 -/- model compared with mSod1KO mice and examined degenerative molecular mechanisms and pathways in peripheral nerve and skeletal muscle. In contrast to mSod1KO mice, myofiber atrophy in Sod1 -/- mice was associated with increased muscle oxidative damage, neuromuscular junction degeneration, denervation, nerve demyelination, and upregulation of proteins involved in maintenance of myelin sheaths. Proteomic analyses confirmed increased proteasomal activity and adaptive stress responses in muscle of Sod1 -/- mice that were absent in mSod1KO mice. Peripheral nerve from neither Sod1 -/- nor mSod1KO mice showed increased oxidative damage or molecular responses to increased oxidation compared with wild type mice. Differential cysteine (Cys) labeling revealed a specific redox shift in the catalytic Cys residue of peroxiredoxin 6 (Cys47) in the peripheral nerve from Sod1 -/- mice. Innovation and Conclusion: These findings demonstrate that neuromuscular integrity, redox mechanisms, and pathways are differentially altered in nerve and muscle of Sod1 -/- and mSod1KO mice. Results support the concept that impaired redox signaling, rather than oxidative damage, in peripheral nerve plays a key role in muscle loss in Sod1 -/- mice and potentially sarcopenia during aging. Antioxid. Redox Signal. 28, 275-295.

The preproghrelin gene is required for the normal integration of thermoregulation and sleep in mice

PubMed Central

Szentirmai, Éva; Kapás, Levente; Sun, Yuxiang; Smith, Roy G.; Krueger, James M.

2009-01-01

Peptidergic mechanisms controlling feeding, metabolism, thermoregulation, and sleep overlap in the hypothalamus. Low ambient temperatures and food restriction induce hypothermic (torpor) bouts and characteristic metabolic and sleep changes in mice. We report that mice lacking the preproghrelin gene, but not those lacking the ghrelin receptor, have impaired abilities to manifest and integrate normal sleep and thermoregulatory responses to metabolic challenges. In response to fasting at 17 °C (a subthermoneutral ambient temperature), preproghrelin knockout mice enter hypothermic bouts associated with reduced sleep, culminating in a marked drop in body temperature to near-ambient levels. Prior treatment with obestatin, another preproghrelin gene product, attenuates the hypothermic response of preproghrelin knockout mice. Results suggest that obestatin is a component in the coordinated regulation of metabolism and sleep during torpor. PMID:19666521

Acute morphine effects on respiratory activity in mice with target deletion of the tachykinin 1 gene (Tac1-/-).

PubMed

Shvarev, Yuri; Berner, Jonas; Bilkei-Gorzo, Andras; Lagercrantz, Hugo; Wickström, Ronny

2010-01-01

Search for physiological mechanisms which could antagonize the opioid-induced respiratory depression is of important clinical value. In this study, we investigated the acute effects of morphine on respiratory activity in genetically modified newborn (P2) mice with target deletion of the (Tac1 -/-) gene lacking substance P (SP) and neurokinin A (NKA). In vivo, as shown with whole-body flow barometric plethysmography technique, morphine induced significantly attenuated minute ventilation during intermittent hypoxia in control animals. In contrast, knockout mice revealed significant increase in minute ventilation. In vitro, in brainstem preparation, knockout mice demonstrated greater changes in burst frequency during intermittent anoxia challenge. The data suggest that hereditary deficiency in tachykinins, SP and NKA results in more robust hypoxic response in newborn Tac1-/- mice during respiratory depression induced by morphine.

COMPARISON OF OVERALL METABOLISM OF 1, 2, 7, 8-PECDD IN CYP1A2(-L-) KNOCKOUT AND C57BL/6N PARENTAL STRAINS OF MICE

EPA Science Inventory

COMPARISON OF OVERALL METABOLISM OF 1,2,3,7,8-PeCDD
IN CYP1A2 (-/-) KNOCKOUT AND C57BL/6N PARENTAL
STRAINS OF MICE

Heldur Hakk1 and Janet J. Diliberto2

1 USDA-ARS, Biosciences Research Laboratory, P.O. Box 5674, Fargo, ND, USA
2 US EPA, ORD, National Heal...

USE OF CYP1A2(-/-) KNOCKOUT AND CYP1A2(+/+) C57BL/6N PARENTAL STRAINS OF MICE TO COMPARE METABOLISM OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)

EPA Science Inventory

USE OF CYP1A2 (-/-) KNOCKOUT AND CYP1A2 (+/+) C57BL/6N PARENTAL STRAINS OF MICE TO COMPARE METABOLISM OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD). J J Diliberto1 and H Hakk2. 1USEPA ORD, NHEERL, ETD, PKB, Research Triangle Park, NC, USA; 2USDA-ARS, BRL, Fargo, ND, USA. Spons...

Genetic and Pharmacological Inhibition of TREM-1 Limits the Development of Experimental Atherosclerosis.

PubMed

Joffre, Jeremie; Potteaux, Stephane; Zeboudj, Lynda; Loyer, Xavier; Boufenzer, Amir; Laurans, Ludivine; Esposito, Bruno; Vandestienne, Marie; de Jager, Saskia C A; Hénique, Carole; Zlatanova, Ivana; Taleb, Soraya; Bruneval, Patrick; Tedgui, Alain; Mallat, Ziad; Gibot, Sebastien; Ait-Oufella, Hafid

2016-12-27

Innate immune responses activated through myeloid cells contribute to the initiation, progression, and complications of atherosclerosis in experimental models. However, the critical upstream pathways that link innate immune activation to foam cell formation are still poorly identified. This study sought to investigate the hypothesis that activation of the triggering receptor expressed on myeloid cells (TREM-1) plays a determinant role in macrophage atherogenic responses. After genetically invalidating Trem-1 in chimeric Ldlr -/- Trem-1 -/- mice and double knockout ApoE -/- Trem-1 -/- mice, we pharmacologically inhibited Trem-1 using LR12 peptide. Ldlr -/- mice reconstituted with bone marrow deficient for Trem-1 (Trem-1 -/- ) showed a strong reduction of atherosclerotic plaque size in both the aortic sinus and the thoracoabdominal aorta, and were less inflammatory compared to plaques of Trem-1 +/+ chimeric mice. Genetic invalidation of Trem-1 led to alteration of monocyte recruitment into atherosclerotic lesions and inhibited toll-like receptor 4 (TLR 4)-initiated proinflammatory macrophage responses. We identified a critical role for Trem-1 in the upregulation of cluster of differentiation 36 (CD36), thereby promoting the formation of inflammatory foam cells. Genetic invalidation of Trem-1 in ApoE -/- /Trem-1 -/- mice or pharmacological blockade of Trem-1 in ApoE -/- mice using LR-12 peptide also significantly reduced the development of atherosclerosis throughout the vascular tree, and lessened plaque inflammation. TREM-1 was expressed in human atherosclerotic lesions, mainly in lipid-rich areas with significantly higher levels of expression in atheromatous than in fibrous plaques. We identified TREM-1 as a major upstream proatherogenic receptor. We propose that TREM-1 activation orchestrates monocyte/macrophage proinflammatory responses and foam cell formation through coordinated and combined activation of CD36 and TLR4. Blockade of TREM-1 signaling may constitute an attractive novel and double-hit approach for the treatment of atherosclerosis. Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Mouse Cone Photoreceptors Co-express Two Functional Visual Arrestins

PubMed Central

Nikonov, Sergei S.; Brown, Bruce M.; Davis, Jason A.; Zuniga, Freddi I.; Bragin, Alvina; Pugh, Edward N.; Craft, Cheryl M.

2008-01-01

Arrestins are members of a superfamily of proteins that arrest the activity of G-protein coupled receptors. Mouse cone photoreceptors express two visual arrestins, Arr1 and Arr4 (Carr). We quantified their expression levels and subcellular distributions in mouse cones: total Arr1 was estimated to be in an ~ 6:1 ratio to cone opsin, about 50-fold higher than Arr4. Recordings from single cones of Arr1−/− and Arr4−/− mice establish that both proteins are competent to arrest the activity of photoactivated S- and M- cone opsins. Recordings from Arr1−/− , Arr4−/− double-knockout mice establish a requirement for at least one of the two visual arrestins for normal cone opsin inactivation at all flash intensities. These recordings also reveal low activity photoproducts of S- and M-opsins that are absent when Grk1 and an arrestin are co-expressed, but which decay 70-fold more rapidly than the comparable photoproducts of rhodopsin in rods. PMID:18701071

Dnmt1 and Dnmt3a are required for the maintenance of DNA methylation and synaptic function in adult forebrain neurons

PubMed Central

Feng, Jian; Zhou, Yu; Campbell, Susan L.; Le, Thuc; Li, En; Sweatt, J. David; Silva, Alcino J.; Fan, Guoping

2011-01-01

Dnmt1 and Dnmt3a, two major DNA methyltransferases, are expressed in postmitotic neurons, but their function in the central nervous system (CNS) is unclear. We generated conditional mutant mice that lack either Dnmt1, or Dnmt3a, or both exclusively in forebrain excitatory neurons and found only double knockout (DKO) mice exhibited abnormal hippocampal CA1 long-term plasticity and deficits of learning and memory. While no neuronal loss was found, the size of hippocampal neurons in DKO was smaller; furthermore, DKO neurons showed a deregulation of gene expression including class I MHC and Stat1 that are known to play a role in synaptic plasticity. In addition, we observed a significant decrease in DNA methylation in DKO neurons. We conclude that Dnmt1 and Dnmt3a are required for synaptic plasticity, learning and memory through their overlapping roles in maintaining DNA methylation and modulating neuronal gene expression in adult CNS neurons. PMID:20228804

An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells

NASA Astrophysics Data System (ADS)

Powell, Jonathan J.; Thomas-McKay, Emma; Thoree, Vinay; Robertson, Jack; Hewitt, Rachel E.; Skepper, Jeremy N.; Brown, Andy; Hernandez-Garrido, Juan Carlos; Midgley, Paul A.; Gomez-Morilla, Inmaculada; Grime, Geoffrey W.; Kirkby, Karen J.; Mabbott, Neil A.; Donaldson, David S.; Williams, Ifor R.; Rios, Daniel; Girardin, Stephen E.; Haas, Carolin T.; Bruggraber, Sylvaine F. A.; Laman, Jon D.; Tanriver, Yakup; Lombardi, Giovanna; Lechler, Robert; Thompson, Richard P. H.; Pele, Laetitia C.

2015-05-01

In humans and other mammals it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer's patches, small areas of the intestine concentrated with particle-scavenging immune cells. In wild-type mice, intestinal immune cells containing these naturally formed nanoparticles expressed the immune tolerance-associated molecule ‘programmed death-ligand 1’, whereas in NOD1/2 double knockout mice, which cannot recognize peptidoglycan, programmed death-ligand 1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and show how this helps to shape intestinal immune homeostasis.

An Endogenous Nanomineral Chaperones Luminal Antigen and Peptidoglycan to Intestinal Immune Cells

PubMed Central

Powell, Jonathan J; Thomas-McKay, Emma; Thoree, Vinay; Robertson, Jack; Hewitt, Rachel E; Skepper, Jeremy N; Brown, Andy; Hernandez-Garrido, Juan Carlos; Midgley, Paul A; Gomez-Morilla, Inmaculada; Grime, Geoffrey W; Kirkby, Karen J; Mabbott, Neil A; Donaldson, David S; Williams, Ifor R; Rios, Daniel; Girardin, Stephen E; Haas, Carolin T; Bruggraber, Sylvaine FA; Laman, Jon D; Tanriver, Yakup; Lombardi, Giovanna; Lechler, Robert; Thompson, Richard P H; Pele, Laetitia C

2015-01-01

In humans and other mammals, it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally-fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer’s patches - small areas of the intestine concentrated with particle-scavenging immune cells. In wild type mice, intestinal immune cells containing these naturally-formed nanoparticles expressed the immune tolerance-associated molecule ‘programmed death-ligand 1 (PD-L1)’, whereas in NOD1/2 double knock-out mice, which cannot recognize peptidoglycan, PD-L1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and how this helps to shape intestinal immune homeostasis. PMID:25751305

Role of Rho-mediated ROCK-Semaphorin3A signaling pathway in the pathogenesis of Parkinson's disease in a mouse model.

PubMed

Qi, Li; Tang, Yong-Gang; Wang, Lin; He, Wei; Pan, Hong-Hua; Nie, Rong-Rong; Can, Yan

2016-11-15

The present study aims to elucidate the role of Rho-mediated ROCK-Semaphorin3A signaling pathway in the pathogenesis of Parkinson's disease (PD) in a mouse model. One-hundred twelve eight-week male C57BL/6 mice were selected. The mouse model of PD was constructed by intraperitoneal injection of MPTP. All mice were divided into four groups (28 mice in each group): Blank group, Model group, Rho knockout (Rho+/-) group and ROCK knockout (ROCK+/-) group. Changes of behavior of the mice were studied through automatic moving test and rotarod test. Immunohistochemistry (IHC) was used to detect the expressions of TH, CD11b and GFAP. High performance liquid chromatograph (HPLC) was performed for detection of dopamine and its metabolic product. The mRNA and protein expressions of Rho, ROCK, Sema3A, PlexinA and NRP-1 were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Rho and ROCK knockout improved the damage caused by MPTP on the behavior of mice and protected dopaminergic neurons from injury, along with the increases of dopamine and its metabolic product. The mRNA and protein expressions of Rho, ROCK, Sema3A, PlexinA and NRP-1 were increased in PD mice in the Model group compared with those in the Blank group. Compared to the Model group, the mRNA and protein expressions of Rho, ROCK, Sema3A, PlexinA and NRP-1 were reduced in the Rho+/- and ROCK+/- groups. These findings indicate that Rho and ROCK knockout may improve the behavior of mice and prevent MPTP-induced dopaminergic neurons damage by regulating Sema3A, PlexinA and NRP-1 in a mouse model of PD. Copyright © 2016 Elsevier B.V. All rights reserved.

Behavioral and genetic investigations of low exploratory behavior in Il18r1−/− mice: We can’t always blame it on the targeted gene

PubMed Central

Eisener-Dorman, Amy F.; Lawrence, David A.; Bolivar, Valerie J.

2010-01-01

The development of gene targeting technologies has enabled research with immune system-related knockout mouse strains to advance our understanding of how cytokines and their receptors interact and influence a number of body systems, including the central nervous system. A critical issue when we are interpreting phenotypic data from these knockout strains is the potential role of genes other than the targeted one. Although many of the knockout strains have been made congenic on a C57BL/6 (B6) genetic background, there remains a certain amount of genetic material from the129 substrain that was used in the development of these strains. This genetic material could result in phenotypes incorrectly attributed to the targeted gene. We recently reported low activity behavior in Il10−/− mice that was linked to this genetic material rather than the targeted gene itself. In the current study we confirm the generalizability of those earlier findings, by assessing behavior in Il18−/− and Il18r1−/− knockout mice. We identified low activity and high anxiety-like behaviors in Il18r1−/− mice, whereas Il18−/− mice displayed little anxiety-like behavior. Although Il18r1−/− mice are considered a congenic strain, we have identified substantial regions of 129P2-derived genetic material not only flanking the ablated Il18r1 on Chromosome 1, but also on Chromosomes 4, 5, 8, 10, and 14. Our studies suggest that residual 129-derived gene(s), rather than the targeted Il18r1 gene, is/are responsible for the low level of activity seen in the Il18r1−/− mice. Mapping studies are necessary to identify the gene or genes contributing to the low activity phenotype. PMID:20580925

TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells.

PubMed

Tsagaratou, Ageliki; González-Avalos, Edahí; Rautio, Sini; Scott-Browne, James P; Togher, Susan; Pastor, William A; Rothenberg, Ellen V; Chavez, Lukas; Lähdesmäki, Harri; Rao, Anjana

2017-01-01

TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4 + CD8 + double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).

Cell surface fucosylation does not affect development of colon tumors in mice with germline Smad3 mutation

PubMed Central

Domino, Steven E.; Karnak, David M.; Hurd, Elizabeth A.

2006-01-01

Background/Aims: Neoplasia-related alterations in cell surface α(1,2)fucosylated glycans have been reported in multiple tumors including colon, pancreas, endometrium, cervix, bladder, lung, and choriocarcinoma. Spontaneous colorectal tumors from mice with a germline null mutation of transforming growth factor-β signaling gene Smad3 (Madh3) were tested for α(1,2)fucosylated glycan expression. Methods: Ulex Europaeus Agglutinin-I lectin staining, fucosyltransferase gene northern blot analysis, and a cross of mutant mice with Fut2 and Smad3 germline mutations were performed. Results: Spontaneous colorectal tumors from Smad3 (-/-) homozygous null mice were found to express α(1,2)fucosylated glycans in an abnormal pattern compared to adjacent nonneoplastic colon. Northern blot analysis of α(1,2)fucosyltransferase genes Fut1 and Fut2 revealed that Fut2, but not Fut1, steady-state mRNA levels were significantly increased in tumors relative to adjacent normal colonic mucosa. Mutant mice with a Fut2-inactivating germline mutation were crossed with Smad3 targeted mice. In Smad3 (-/-)/Fut2 (-/-) double knock-out mice, UEA-I lectin staining was eliminated from colon and colon tumors, however, the number and size of tumors present by 24 weeks of age did not vary regardless of the Fut2 genotype. Conclusions: In this model of colorectal cancer, cell surface α(1,2)fucosylation does not affect development of colon tumors. PMID:17264540

Loss of polyubiquitin gene Ubb leads to metabolic and sleep abnormalities in mice

PubMed Central

Ryu, K.-Y.; Fujiki, N.; Kazantzis, M.; Garza, J. C.; Bouley, D. M.; Stahl, A.; Lu, X.-Y.; Nishino, S.; Kopito, R. R.

2010-01-01

Aims Ubiquitin performs essential roles in a myriad of signalling pathways required for cellular function and survival. Recently, we reported that disruption of the stress-inducible ubiquitin-encoding gene Ubb reduces ubiquitin content in the hypothalamus and leads to adult-onset obesity coupled with a loss of arcuate nucleus neurones and disrupted energy homeostasis in mice. Neuropeptides expressed in the hypothalamus control both metabolic and sleep behaviours. In order to demonstrate that the loss of Ubb results in broad hypothalamic abnormalities, we attempted to determine whether metabolic and sleep behaviours were altered in Ubb knockout mice. Methods Metabolic rate and energy expenditure were measured in a metabolic chamber, and sleep stage was monitored via electroencephalographic/electromyographic recording. The presence of neurodegeneration and increased reactive gliosis in the hypothalamus were also evaluated. Results We found that Ubb disruption leads to early-onset reduced activity and metabolic rate. Additionally, we have demonstrated that sleep behaviour is altered and sleep homeostasis is disrupted in Ubb knockout mice. These early metabolic and sleep abnormalities are accompanied by persistent reactive gliosis and the loss of arcuate nucleus neurones, but are independent of neurodegeneration in the lateral hypothalamus. Conclusions Ubb knockout mice exhibit phenotypes consistent with hypothalamic dysfunction. Our data also indicate that Ubb is essential for the maintenance of the ubiquitin levels required for proper regulation of metabolic and sleep behaviours in mice. PMID:20002312