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.

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice

PubMed Central

Gerard-O'Riley, Rita L.; Acton, Dena; McQueen, Amie K.; Strobel, Isabel E.; Witcher, Phillip C.; Feng, Jian Q.; Econs, Michael J.

2017-01-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. PMID:28005411

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice.

PubMed

Ichikawa, Shoji; Gerard-O'Riley, Rita L; Acton, Dena; McQueen, Amie K; Strobel, Isabel E; Witcher, Phillip C; Feng, Jian Q; Econs, Michael J

2017-03-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. Copyright © 2017 by the Endocrine Society.

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.

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

EFFECTS OF EPIDERMAL GROWTH FACTOR (EGF), TRANSFORMING GROWTH FACTOR- (TGF), AND 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN ON FUSION OF EMBRYONIC PALATES IN SERUM-FREE ORGAN CULTURE USING WILD-TYPE, EGF KNOCKOUT, AND TGF KNOCKOUT MOUSE STRAINS

EPA Science Inventory

Backround: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing cleft palate (CP). TCDD exposure disrupts expression of epidermal growth factor (EGF) receptor, EGF, and transforming growth factor- (TGF) in the palate and affects proliferation and different...

Decreased APOE-containing HDL subfractions and cholesterol efflux capacity of serum in mice lacking Pcsk9

PubMed Central

2013-01-01

Background Studies in animals showed that PCSK9 is involved in HDL metabolism. We investigated the molecular mechanism by which PCSK9 regulates HDL cholesterol concentration and also whether Pcsk9 inactivation might affect cholesterol efflux capacity of serum and atherosclerotic fatty streak volume. Methods Mass spectrometry and western blot were used to analyze the level of apolipoprotein E (APOE) and A1 (APOA1). A mouse model overexpressing human LDLR was used to test the effect of high levels of liver LDLR on the concentration of HDL cholesterol and APOE-containing HDL subfractions. Pcsk9 knockout males lacking LDLR and APOE were used to test whether LDLR and APOE are necessary for PCSK9-mediated HDL cholesterol regulation. We also investigated the effects of Pcsk9 inactivation on cholesterol efflux capacity of serum using THP-1 and J774.A1 macrophage foam cells and atherosclerotic fatty streak volume in the aortic sinus of Pcsk9 knockout males fed an atherogenic diet. Results APOE and APOA1 were reduced in the same HDL subfractions of Pcsk9 knockout and human LDLR transgenic male mice. In Pcsk9/Ldlr double-knockout mice, HDL cholesterol concentration was lower than in Ldlr knockout mice and higher than in wild-type controls. In Pcsk9/Apoe double-knockout mice, HDL cholesterol concentration was similar to that of Apoe knockout males. In Pcsk9 knockout males, THP-1 macrophage cholesterol efflux capacity of serum was reduced and the fatty streak lesion volume was similar to wild-type controls. Conclusions In mice, LDLR and APOE are important factors for PCSK9-mediated HDL regulation. Our data suggest that, although LDLR plays a major role in PCSK9-mediated regulation of HDL cholesterol concentration, it is not the only mechanism and that, regardless of mechanism, APOE is essential. Pcsk9 inactivation decreases the HDL cholesterol concentration and cholesterol efflux capacity in serum, but does not increase atherosclerotic fatty streak volume. PMID:23883163

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.

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.

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.

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

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

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

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.

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.

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.

Differential sensitivity of Pak5, Pak6, and Pak5/Pak6 double-knockout mice to the stimulant effects of amphetamine and exercise-induced alterations in body weight.

PubMed

Furnari, Melody A; Jobes, Michelle L; Nekrasova, Tanya; Minden, Audrey; Wagner, George C

2014-04-01

PAK5 and PAK6 are protein kinases highly expressed in the brain. Previously, we observed that Pak6 knockout mice gained significantly more weight during development than Pak5 knockout mice as well as wild-type controls and double-knockout mice lacking both Pak5 and Pak6. In this study, we assessed the effects of exercise on food intake and weight gain of these mice as well as their sensitivity to the stimulant effects of amphetamine. Mice of each genotype were placed in cages with free access to run wheel exercise or in cages without run wheels for a total of 74 days. Food and fluid intake as well as body weight of each mouse were measured on a weekly basis. Finally, mice were given a high dose of amphetamine and activity levels were observed immediately thereafter for 90 minutes. Brains and testes of mice were assayed for protein levels of the estrogen alpha and progesterone receptors. While run wheel mice consumed significantly more food, they weighed less than non-run wheel mice. In addition, although Pak6 knockout mice consumed the same amount of food as wild-type mice, they were significantly heavier regardless of run wheel condition. Pak5 knockout mice were found to be more active than other genotypes after amphetamine treatment. Finally, protein levels of the progesterone and estrogen alpha receptors were altered in brain and testes of the Pak6 knockout mice. Collectively, these data suggest that PAK6 play a role in weight gain unrelated to exercise and caloric intake and that Pak5 knockout mice are more sensitive to the stimulant effects of amphetamine.

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.

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.

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.

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.

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.

An Abnormal Nitric Oxide Metabolism Contributes to Brain Oxidative Stress in the Mouse Model for the Fragile X Syndrome, a Possible Role in Intellectual Disability

PubMed Central

Lima-Cabello, Elena; Garcia-Guirado, Francisco; Calvo-Medina, Rocio; el Bekay, Rajaa; Perez-Costillas, Lucia; Quintero-Navarro, Carolina; Sanchez-Salido, Lourdes

2016-01-01

Background. Fragile X syndrome is the most common genetic cause of mental disability. Although many research has been performed, the mechanism underlying the pathogenesis is unclear and needs further investigation. Oxidative stress played major roles in the syndrome. The aim was to investigate the nitric oxide metabolism, protein nitration level, the expression of NOS isoforms, and furthermore the activation of the nuclear factor NF-κB-p65 subunit in different brain areas on the fragile X mouse model. Methods. This study involved adult male Fmr1-knockout and wild-type mice as controls. We detected nitric oxide metabolism and the activation of the nuclear factor NF-κBp65 subunit, comparing the mRNA expression and protein content of the three NOS isoforms in different brain areas. Results. Fmr1-KO mice showed an abnormal nitric oxide metabolism and increased levels of protein tyrosine nitrosylation. Besides that, nuclear factor NF-κB-p65 and inducible nitric oxide synthase appeared significantly increased in the Fmr1-knockout mice. mRNA and protein levels of the neuronal nitric oxide synthase appeared significantly decreased in the knockout mice. However, the epithelial nitric oxide synthase isoform displayed no significant changes. Conclusions. These data suggest the potential involvement of an abnormal nitric oxide metabolism in the pathogenesis of the fragile X syndrome. PMID:26788253

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.

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.

Modulation of cardiac fibrosis by Krüppel-like factor 6 through transcriptional control of thrombospondin 4 in cardiomyocytes

PubMed Central

Sawaki, Daigo; Hou, Lianguo; Tomida, Shota; Sun, Junqing; Zhan, Hong; Aizawa, Kenichi; Son, Bo-Kyung; Kariya, Taro; Takimoto, Eiki; Otsu, Kinya; Conway, Simon J.; Manabe, Ichiro; Komuro, Issei; Friedman, Scott L.; Nagai, Ryozo; Suzuki, Toru

2015-01-01

Aims Krüppel-like factors (KLFs) are a family of transcription factors which play important roles in the heart under pathological and developmental conditions. We previously identified and cloned Klf6 whose homozygous mutation in mice results in embryonic lethality suggesting a role in cardiovascular development. Effects of KLF6 on pathological regulation of the heart were investigated in the present study. Methods and results Mice heterozygous for Klf6 resulted in significantly diminished levels of cardiac fibrosis in response to angiotensin II infusion. Intriguingly, a similar phenotype was seen in cardiomyocyte-specific Klf6 knockout mice, but not in cardiac fibroblast-specific knockout mice. Microarray analysis revealed increased levels of the extracellular matrix factor, thrombospondin 4 (TSP4), in the Klf6-ablated heart. Mechanistically, KLF6 directly suppressed Tsp4 expression levels, and cardiac TSP4 regulated the activation of cardiac fibroblasts to regulate cardiac fibrosis. Conclusion Our present studies on the cardiac function of KLF6 show a new mechanism whereby cardiomyocytes regulate cardiac fibrosis through transcriptional control of the extracellular matrix factor, TSP4, which, in turn, modulates activation of cardiac fibroblasts. PMID:25987545

TERATOGENIC EFFECTS OF RETINOIC ACID ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR AND TRANSFORMING GROWTH FACTOR-ALPHA

EPA Science Inventory

Background: EGF and TGF regulate cell proliferation and differentiation in the embryo. The induction of cleft palate (CP) by all trans retinoic acid (RA) was associated with altered expression of TGF, EGF receptor and binding of EGF. The present study uses knockout (KO) mice to e...

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

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.

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

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.

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.

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.

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.

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.

Erythropoiesis and Blood Pressure Are Regulated via AT1 Receptor by Distinctive Pathways.

PubMed

Kato, Hideki; Ishida, Junji; Matsusaka, Taiji; Ishimaru, Tomohiro; Tanimoto, Keiji; Sugiyama, Fumihiro; Yagami, Ken-Ichi; Nangaku, Masaomi; Fukamizu, Akiyoshi

2015-01-01

The renin-angiotensin system (RAS) plays a central role in blood pressure regulation. Although clinical and experimental studies have suggested that inhibition of RAS is associated with progression of anemia, little evidence is available to support this claim. Here we report that knockout mice that lack angiotensin II, including angiotensinogen and renin knockout mice, exhibit anemia. The anemia of angiotensinogen knockout mice was rescued by angiotensin II infusion, and rescue was completely blocked by simultaneous administration of AT1 receptor blocker. To genetically determine the responsible receptor subtype, we examined AT1a, AT1b, and AT2 knockout mice, but did not observe anemia in any of them. To investigate whether pharmacological AT1 receptor inhibition recapitulates the anemic phenotype, we administered AT1 receptor antagonist in hypotensive AT1a receptor knockout mice to inhibit the remaining AT1b receptor. In these animals, hematocrit levels barely decreased, but blood pressure further decreased to the level observed in angiotensinogen knockout mice. We then generated AT1a and AT1b double-knockout mice to completely ablate the AT1 receptors; the mice finally exhibited the anemic phenotype. These results provide clear evidence that although erythropoiesis and blood pressure are negatively controlled through the AT1 receptor inhibition in vivo, the pathways involved are complex and distinct, because erythropoiesis is more resistant to AT1 receptor inhibition than blood pressure control.

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

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.

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.

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…

Transcription factor NF-kappaB participates in regulation of epithelial cell turnover in the colon.

PubMed

Inan, M S; Tolmacheva, V; Wang, Q S; Rosenberg, D W; Giardina, C

2000-12-01

The transcription factor nuclear factor (NF)-kappaB regulates the expression of genes that can influence cell proliferation and death. Here we analyze the contribution of NF-kappaB to the regulation of epithelial cell turnover in the colon. Immunohistochemical, immunoblot, and DNA binding analyses indicate that NF-kappaB complexes change as colonocytes mature: p65-p50 complexes predominate in proliferating epithelial cells of the colon, whereas the p50-p50 dimer is prevalent in mature epithelial cells. NF-kappaB1 (p50) knockout mice were used to study the role of NF-kappaB in regulating epithelial cell turnover. Knockout animals lacked detectable NF-kappaB DNA binding activity in isolated epithelial cells and had significantly longer crypts with a more extensive proliferative zone than their wild-type counterparts (as determined by proliferating cell nuclear antigen staining and in vivo bromodeoxyuridine labeling). Gene expression profiling reveals that the NF-kappaB1 knockout mice express the potentially growth-enhancing tumor necrosis factor (TNF)-alpha and nerve growth factor-alpha genes at elevated levels, with in situ hybridization localizing some of the TNF-alpha expression to epithelial cells. TNF-alpha is NF-kappaB regulated, and its upregulation in NF-kappaB1 knockouts may result from an alleviation of p50-p50 repression. NF-kappaB complexes may therefore influence cell proliferation in the colon through their ability to selectively activate and/or repress gene expression.

Genetic Restoration of Plasma ApoE Improves Cognition and Partially Restores Synaptic Defects in ApoE-Deficient Mice

PubMed Central

Wong, Wen Mai; Durakoglugil, Murat S.; Wasser, Catherine R.; Jiang, Shan; Xian, Xunde

2016-01-01

Alzheimer's disease (AD) is the most common form of dementia in individuals over the age of 65 years. The most prevalent genetic risk factor for AD is the ε4 allele of apolipoprotein E (ApoE4), and novel AD treatments that target ApoE are being considered. One unresolved question in ApoE biology is whether ApoE is necessary for healthy brain function. ApoE knock-out (KO) mice have synaptic loss and cognitive dysfunction; however, these findings are complicated by the fact that ApoE knock-out mice have highly elevated plasma lipid levels, which may independently affect brain function. To bypass the effect of ApoE loss on plasma lipids, we generated a novel mouse model that expresses ApoE normally in peripheral tissues, but has severely reduced ApoE in the brain, allowing us to study brain ApoE loss in the context of a normal plasma lipid profile. We found that these brain ApoE knock-out (bEKO) mice had synaptic loss and dysfunction similar to that of ApoE KO mice; however, the bEKO mice did not have the learning and memory impairment observed in ApoE KO mice. Moreover, we found that the memory deficit in the ApoE KO mice was specific to female mice and was fully rescued in female bEKO mice. Furthermore, while the AMPA/NMDA ratio was reduced in ApoE KO mice, it was unchanged in bEKO mice compared with controls. These findings suggest that plasma lipid levels can influence cognition and synaptic function independent of ApoE expression in the brain. SIGNIFICANCE STATEMENT One proposed treatment strategy for Alzheimer's disease (AD) is the reduction of ApoE, whose ε4 isoform is the most common genetic risk factor for the disease. A major concern of this strategy is that an animal model of ApoE deficiency, the ApoE knock-out (KO) mouse, has reduced synapses and cognitive impairment; however, these mice also develop dyslipidemia and severe atherosclerosis. Here, we have shown that genetic restoration of plasma ApoE to wild-type levels normalizes plasma lipids in ApoE KO mice. While this does not rescue synaptic loss, it does completely restore learning and memory in the mice, suggesting that both CNS and plasma ApoE are independent parameters that affect brain health. PMID:27683909

Regulation of bitter taste responses by tumor necrosis factor.

PubMed

Feng, Pu; Jyotaki, Masafumi; Kim, Agnes; Chai, Jinghua; Simon, Nirvine; Zhou, Minliang; Bachmanov, Alexander A; Huang, Liquan; Wang, Hong

2015-10-01

Inflammatory cytokines are important regulators of metabolism and food intake. Over production of inflammatory cytokines during bacterial and viral infections leads to anorexia and reduced food intake. However, it remains unclear whether any inflammatory cytokines are involved in the regulation of taste reception, the sensory mechanism governing food intake. Previously, we showed that tumor necrosis factor (TNF), a potent proinflammatory cytokine, is preferentially expressed in a subset of taste bud cells. The level of TNF in taste cells can be further induced by inflammatory stimuli. To investigate whether TNF plays a role in regulating taste responses, in this study, we performed taste behavioral tests and gustatory nerve recordings in TNF knockout mice. Behavioral tests showed that TNF-deficient mice are significantly less sensitive to the bitter compound quinine than wild-type mice, while their responses to sweet, umami, salty, and sour compounds are comparable to those of wild-type controls. Furthermore, nerve recording experiments showed that the chorda tympani nerve in TNF knockout mice is much less responsive to bitter compounds than that in wild-type mice. Chorda tympani nerve responses to sweet, umami, salty, and sour compounds are similar between TNF knockout and wild-type mice, consistent with the results from behavioral tests. We further showed that taste bud cells express the two known TNF receptors TNFR1 and TNFR2 and, therefore, are potential targets of TNF. Together, our results suggest that TNF signaling preferentially modulates bitter taste responses. This mechanism may contribute to taste dysfunction, particularly taste distortion, associated with infections and some chronic inflammatory diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Regulation of bitter taste responses by tumor necrosis factor

PubMed Central

Feng, Pu; Jyotaki, Masafumi; Kim, Agnes; Chai, Jinghua; Simon, Nirvine; Zhou, Minliang; Bachmanov, Alexander A.; Huang, Liquan; Wang, Hong

2015-01-01

Inflammatory cytokines are important regulators of metabolism and food intake. Over production of inflammatory cytokines during bacterial and viral infections leads to anorexia and reduced food intake. However, it remains unclear whether any inflammatory cytokines are involved in the regulation of taste reception, the sensory mechanism governing food intake. Previously, we showed that tumor necrosis factor (TNF), a potent proinflammatory cytokine, is preferentially expressed in a subset of taste bud cells. The level of TNF in taste cells can be further induced by inflammatory stimuli. To investigate whether TNF plays a role in regulating taste responses, in this study, we performed taste behavioral tests and gustatory nerve recordings in TNF knockout mice. Behavioral tests showed that TNF-deficient mice are significantly less sensitive to the bitter compound quinine than wild-type mice, while their responses to sweet, umami, salty, and sour compounds are comparable to those of wild-type controls. Furthermore, nerve recording experiments showed that the chorda tympani nerve in TNF knockout mice is much less responsive to bitter compounds than that in wild-type mice. Chorda tympani nerve responses to sweet, umami, salty, and sour compounds are similar between TNF knockout and wild-type mice, consistent with the results from behavioral tests. We further showed that taste bud cells express the two known TNF receptors TNFR1 and TNFR2 and, therefore, are potential targets of TNF. Together, our results suggest that TNF signaling preferentially modulates bitter taste responses. This mechanism may contribute to taste dysfunction, particularly taste distortion, associated with infections and some chronic inflammatory diseases. PMID:25911043

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

Takemura, Takayo; Yoshida, Yuichi; Kiso, Shinichi, E-mail: kiso@gh.med.osaka-u.ac.jp

Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibroticmore » livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.« less

[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.

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.

Genetic deletion of CB1 receptors improves non-associative learning.

PubMed

Degroot, Aldemar; Salhoff, Craig; Davis, Richard J; Nomikos, George G

2005-07-01

Habituation (a form of non-associative learning) was measured by assessing locomotion in novel activity monitors in CB1 receptor knockout mice and juxtaposed to habituation measured in muscarinic M2, M4, and double M2/M4 receptor knockout mice. M2 and M2/M4, but not M4, receptor knockout mice appeared to have an impaired ability to habituate, whereas CB1 receptor knockout mice showed enhanced habituation compared to wild-type animals. We conclude that CB1 receptor gene invalidation improves habituation tentatively through an increase in cholinergic neurotransmission.

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

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.

Macrophage Migration Inhibitory Factor (MIF) Deficiency Exacerbates Aging-Induced Cardiac Remodeling and Dysfunction Despite Improved Inflammation: Role of Autophagy Regulation.

PubMed

Xu, Xihui; Pang, Jiaojiao; Chen, Yuguo; Bucala, Richard; Zhang, Yingmei; Ren, Jun

2016-03-04

Aging leads to unfavorable geometric and functional sequelae in the heart. The proinflammatory cytokine macrophage migration inhibitory factor (MIF) plays a role in the maintenance of cardiac homeostasis under stress conditions although its impact in cardiac aging remains elusive. This study was designed to evaluate the role of MIF in aging-induced cardiac anomalies and the underlying mechanism involved. Cardiac geometry, contractile and intracellular Ca(2+) properties were examined in young (3-4 mo) or old (24 mo) wild type and MIF knockout (MIF(-/-)) mice. Our data revealed that MIF knockout exacerbated aging-induced unfavorable structural and functional changes in the heart. The detrimental effect of MIF knockout was associated with accentuated loss in cardiac autophagy with aging. Aging promoted cardiac inflammation, the effect was attenuated by MIF knockout. Intriguingly, aging-induced unfavorable responses were reversed by treatment with the autophagy inducer rapamycin, with improved myocardial ATP availability in aged WT and MIF(-/-) mice. Using an in vitro model of senescence, MIF knockdown exacerbated doxorubicin-induced premature senescence in H9C2 myoblasts, the effect was ablated by MIF replenishment. Our data indicated that MIF knockout exacerbates aging-induced cardiac remodeling and functional anomalies despite improved inflammation, probably through attenuating loss of autophagy and ATP availability in the heart.

Suppression of NLRP3 inflammasome attenuates stress-induced depression-like behavior in NLGN3-deficient mice.

PubMed

Li, Ze-Qun; Yan, Zhi-Yuan; Lan, Fu-Jun; Dong, Yi-Qun; Xiong, Ye

2018-07-02

Depression, regulated by central nervous system (CNS), is a significant inflammatory disorder. Neuroligin3 (NLGN3) has been implicated in brain functions. In the study, a chronic unpredictable mild stress (CUMS) model in wild type (WT) or NLGN3-knockout (KO) mice was established to explore the role of NLGN3 in regulating depression and to reveal the underlying molecular mechanism. The results indicated that NLGN3-knockout markedly reversed the loss of body weight, the reduction of sucrose consumption, the decrease of immobile time in the forced swimming tests (FST) and tail suspension tests (TST) induced by CUMS paradigm. CUMS up-regulated corticosterone (CORT) in serum, and down-regulated serotonin (5-HT), norepinephrine (NE) and brain-derived neurotrophic factor (BDNF) in hippocampus of mice, which were significantly reversed by NLGN3 deficiency. The results further demonstrated that NLGN3-knockout improved the degenerative neurons in cortex and hippocampus of CUMS-treated mice, accompanied with a significant decrease of ionized calciumbinding adapter molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) expressions. Additionally, NLGN3-KO mice challenged with CUMS showed a significant reduction of pro-inflammatory cytokines and chemokine, including tumor necrosis factor-alpha (TNF-α), interleukin-18 (IL-18), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), CC-chemokine ligand-1 (CCL-1) and CXC-chemokine ligand-1 (CXCL-1), in cortex, hippocampus and amygdala tissue samples. Western blot analysis suggested that NLGN3-knockout inhibited the activation of nod-like receptor protein 3 (NLRP3) inflammasome and its adaptor of apoptosis-associated speck like protein (ASC), and reduced the expression of Caspase-1, along with the inactivation of nuclear factor-κB (NF-κB) in CUMS-challenged mice. The role of NLGN3 in regulating depression in mice was confirmed in vitro using astrocytes stimulated by LPS that NLGN3 knockdown reduced LPS-induced inflammation. Importantly, the suppressive effects of NLGN3-knockdown on inflammatory response were reversed by NLRP3 or ASC over-expression in AST exposed to LPS. In sum, our findings indicated that suppressing NLGN3 played a potential antidepressant role in CUMS animal model by inactivating NLRP3 inflammasome, providing a new therapeutic avenue for depression. Copyright © 2018. Published by Elsevier Inc.

Central nervous system-specific knockout of steroidogenic factor 1 results in increased anxiety-like behavior.

PubMed

Zhao, Liping; Kim, Ki Woo; Ikeda, Yayoi; Anderson, Kimberly K; Beck, Laurel; Chase, Stephanie; Tobet, Stuart A; Parker, Keith L

2008-06-01

Steroidogenic factor 1 (SF-1) plays key roles in adrenal and gonadal development, expression of pituitary gonadotropins, and development of the ventromedial hypothalamic nucleus (VMH). If kept alive by adrenal transplants, global knockout (KO) mice lacking SF-1 exhibit delayed-onset obesity and decreased locomotor activity. To define specific roles of SF-1 in the VMH, we used the Cre-loxP system to inactivate SF-1 in a central nervous system (CNS)-specific manner. These mice largely recapitulated the VMH structural defect seen in mice lacking SF-1 in all tissues. In multiple behavioral tests, mice with CNS-specific KO of SF-1 had significantly more anxiety-like behavior than wild-type littermates. The CNS-specific SF-1 KO mice had diminished expression or altered distribution in the mediobasal hypothalamus of several genes whose expression has been linked to stress and anxiety-like behavior, including brain-derived neurotrophic factor, the type 2 receptor for CRH (Crhr2), and Ucn 3. Moreover, transfection and EMSAs support a direct role of SF-1 in Crhr2 regulation. These findings reveal important roles of SF-1 in the hypothalamic expression of key regulators of anxiety-like behavior, providing a plausible molecular basis for the behavioral effect of CNS-specific KO of this nuclear receptor.

Targeted Disruption of the Basic Krüppel-Like Factor Gene (Klf3) Reveals a Role in Adipogenesis ▿ †

PubMed Central

Sue, Nancy; Jack, Briony H. A.; Eaton, Sally A.; Pearson, Richard C. M.; Funnell, Alister P. W.; Turner, Jeremy; Czolij, Robert; Denyer, Gareth; Bao, Shisan; Molero-Navajas, Juan Carlos; Perkins, Andrew; Fujiwara, Yuko; Orkin, Stuart H.; Bell-Anderson, Kim; Crossley, Merlin

2008-01-01

Krüppel-like factors (KLFs) recognize CACCC and GC-rich sequences in gene regulatory elements. Here, we describe the disruption of the murine basic Krüppel-like factor gene (Bklf or Klf3). Klf3 knockout mice have less white adipose tissue, and their fat pads contain smaller and fewer cells. Adipocyte differentiation is altered in murine embryonic fibroblasts from Klf3 knockouts. Klf3 expression was studied in the 3T3-L1 cellular system. Adipocyte differentiation is accompanied by a decline in Klf3 expression, and forced overexpression of Klf3 blocks 3T3-L1 differentiation. Klf3 represses transcription by recruiting C-terminal binding protein (CtBP) corepressors. CtBPs bind NADH and may function as metabolic sensors. A Klf3 mutant that does not bind CtBP cannot block adipogenesis. Other KLFs, Klf2, Klf5, and Klf15, also regulate adipogenesis, and functional CACCC elements occur in key adipogenic genes, including in the C/ebpα promoter. We find that C/ebpα is derepressed in Klf3 and Ctbp knockout fibroblasts and adipocytes from Klf3 knockout mice. Chromatin immunoprecipitations confirm that Klf3 binds the C/ebpα promoter in vivo. These results implicate Klf3 and CtBP in controlling adipogenesis. PMID:18391014

Deletion of connective tissue growth factor ameliorates peritoneal fibrosis by inhibiting angiogenesis and inflammation.

PubMed

Toda, Naohiro; Mori, Kiyoshi; Kasahara, Masato; Koga, Kenichi; Ishii, Akira; Mori, Keita P; Osaki, Keisuke; Mukoyama, Masashi; Yanagita, Motoko; Yokoi, Hideki

2018-06-01

Connective tissue growth factor (CTGF/CCN2) regulates the signalling of other growth factors and promotes fibrosis. CTGF is increased in mice and humans with peritoneal fibrosis. Inhibition of CTGF has not been examined as a potential therapeutic target for peritoneal fibrosis because systemic CTGF knockout mice die at the perinatal stage. To study the role of CTGF in peritoneal fibrosis of adult mice, we generated CTGF conditional knockout (cKO) mice by crossing CTGF floxed mice with RosaCreERT2 mice. We administered tamoxifen to Rosa-CTGF cKO mice to delete the CTGF gene throughout the body. We induced peritoneal fibrosis by intraperitoneal injection of chlorhexidine gluconate (CG) in wild-type and Rosa-CTGF cKO mice. Induction of peritoneal fibrosis in wild-type mice increased CTGF expression and produced severe thickening of the peritoneum. In contrast, CG-treated Rosa-CTGF cKO mice exhibited reduced thickening of the peritoneum. Peritoneal equilibration test revealed that the excessive peritoneal small-solute transport in CG-treated wild-type mice was normalized by CTGF deletion. CG-treated Rosa-CTGF cKO mice exhibited a reduced number of αSMA-, Ki67-, CD31- and MAC-2-positive cells in the peritoneum. Analyses of peritoneal mRNA showed that CG-treated Rosa-CTGF cKO mice exhibited reduced expression of Cd68, Acta2 (αSMA), Pecam1 (CD31) and Vegfa. These results indicate that a deficiency of CTGF can reduce peritoneal thickening and help to maintain peritoneal function by reducing angiogenesis and inflammation in peritoneal fibrosis. These results suggest that CTGF plays an important role in the progression of peritoneal fibrosis.

Deletion of pro-angiogenic factor vasohibin-2 ameliorates glomerular alterations in a mouse diabetic nephropathy model

PubMed Central

Masuda, Kana; Ujike, Haruyo; Hinamoto, Norikazu; Miyake, Hiromasa; Tanimura, Satoshi; Sugiyama, Hitoshi; Sato, Yasufumi; Maeshima, Yohei; Wada, Jun

2018-01-01

Angiogenesis has been implicated in glomerular alterations in the early stage of diabetic nephropathy. We previously reported the renoprotective effects of vasohibin-1 (VASH1), which is a novel angiogenesis inhibitor derived from endothelial cells, on diabetic nephropathy progression. Vasohibin-2 (VASH2) was originally identified as a VASH1 homolog and possesses pro-angiogenic activity in contrast to VASH1. In addition, VASH2 was recently shown to promote epithelial-to-mesenchymal transition via enhanced transforming growth factor (TGF)-β signaling in cancer cells. Herein, we investigated the pathogenic roles of VASH2 in diabetic nephropathy using VAHS2-deficient mice. The type 1 diabetes model was induced by intraperitoneal injections of streptozotocin in VASH2 homozygous knockout (VASH2LacZ/LacZ) or wild-type mice. These mice were euthanized 16 weeks after inducing hyperglycemia. Increased urine albumin excretion and creatinine clearance observed in diabetic wild-type mice were significantly prevented in diabetic VASH2-deficient mice. Accordingly, diabetes-induced increase in glomerular volume and reduction in glomerular slit-diaphragm density were significantly improved in VASH2 knockout mice. Increased glomerular endothelial area was also suppressed in VASH2-deficient mice, in association with inhibition of enhanced vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2), but not VEGF level. Furthermore, glomerular accumulation of mesangial matrix, including type IV collagen, and increased expression of TGF-β were improved in diabetic VASH2 knockout mice compared with diabetic wild-type mice. Based on the immunofluorescence findings, endogenous VASH2 localization in glomeruli was consistent with mesangial cells. Human mesangial cells (HMCs) were cultured under high glucose condition in in vitro experiments. Transfection of VASH2 small interfering RNA (siRNA) into the HMCs resulted in the suppression of type IV collagen production induced by high glucose compared with control siRNA. These results indicate that VASH2 may be involved in diabetes-induced glomerular alterations, particularly impaired filtration barrier and mesangial expansion. Therefore, VASH2 is likely to represent a promising therapeutic target for diabetic nephropathy. PMID:29641565

SHH-dependent knockout of HIF-1 alpha accelerates the degenerative process in mouse intervertebral disc.

PubMed

Wu, W J; Zhang, X K; Zheng, X F; Yang, Y H; Jiang, S D; Jiang, L S

2013-01-01

Hypoxia-inducible factor-1alpha (HIF-1 alpha) has been reported to have an important role in the metabolism and synthesis of extracellular matrix of the nucleus pulposus cells (NPCs) and was assumed to be involved in the process of intervertebral disc degeneration. The objective of this study was to investigate the role of HIF-1alpha in disc degeneration in vivo using a conditional HIF-1alpha knockout (KO) mouse model. ShhCre transgenic mice were mated with HIF-1 alpha fl/fl mice to generate conditional HIF-1alpha KO mice (HIF-1alpha fl/fl-ShhCre+). Three mice of each genotype (Wide-type and HIF-1alpha KO) at the age of 3 days, 6, and 12 weeks were sacrificed after genotyping. Five lumbar disc samples were harvested from each mouse, with a total of 45 disc samples for each genotype. In situ hybridization and immunohistochemical analysis were used to check the efficacy of HIF-1alpha knockout. Histological grading of the disc degeneration was performed according to the classification system proposed by Boos et al. Picro-sirius red staining, Safranine O/fast green staining and immunohistochemical study were used to evaluate the expression of aggrecan, type-II collagen and vascular endothelial growth factor (VEGF). Histologic analysis revealed more NPC deaths and signs of degeneration in HIF-1alpha KO mice and the degeneration scores of HIF-1alpha KO mice were significantly higher than those of the Wide-type mice at the age of 6 weeks and 12 weeks. There were less expressions of aggrecan, type-II collagen and VEGF in the intervertebral discs of HIF1-alpha KO mice than in those of wild-type mice. Taken together, the results of our study indicated that HIF-1alpha is a pivotal contributor to NPC survival and the homeotasis of extracellular matrix through the HIF-1alpha/VEGF signaling pathway, and plays an important role in the development of disc degeneration.

Notch3 orchestrates epithelial and inflammatory responses to promote acute kidney injury.

PubMed

Kavvadas, Panagiotis; Keuylian, Zela; Prakoura, Niki; Placier, Sandrine; Dorison, Aude; Chadjichristos, Christos E; Dussaule, Jean-Claude; Chatziantoniou, Christos

2018-07-01

Acute kidney injury is a major risk factor for subsequent chronic renal and/or cardiovascular complications. Previous studies have shown that Notch3 was de novo expressed in the injured renal epithelium in the early phases of chronic kidney disease. Here we examined whether Notch3 is involved in the inflammatory response and the epithelial cell damage that typifies ischemic kidneys using Notch3 knockout mice and mice with short-term activated Notch3 signaling (N3ICD) in renal epithelial cells. After ischemia/reperfusion, N3ICD mice showed exacerbated infiltration of inflammatory cells and severe tubular damage compared to control mice. Inversely, Notch3 knockout mice were protected against ischemia/reperfusion injury. Renal macrophages derived from Notch3 knockout mice failed to activate proinflammatory cytokines. Chromatin immunoprecipitation analysis of the Notch3 promoter identified NF-κB as the principal inducer of Notch3 in ischemia/reperfusion. Thus, Notch3 induced by NF-κB in the injured epithelium sustains a proinflammatory environment attracting activated macrophages to the site of injury leading to a rapid deterioration of renal function and structure. Hence, targeting Notch3 may provide a novel therapeutic strategy against ischemia/reperfusion and acute kidney injury by preservation of epithelial structure and disruption of proinflammatory signaling. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Ethanol- and acetaldehyde-induced cholinergic imbalance in the hippocampus of Aldh2-knockout mice does not affect nerve growth factor or brain-derived neurotrophic factor.

PubMed

Jamal, Mostofa; Ameno, Kiyoshi; Ruby, Mostofa; Miki, Takanori; Tanaka, Naoko; Nakamura, Yu; Kinoshita, Hiroshi

2013-11-20

Neurotrophins, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), play an important role in the maintenance of cholinergic-neuron function. The objective of this study was to investigate whether ethanol (EtOH)- and acetaldehyde (AcH)- induced cholinergic effects would cause neurotrophic alterations in the hippocampus of mice. We used Aldh2 knockout (Aldh2-KO) mice, a model of aldehyde dehydrogenase 2 (ALDH2)-deficiency in humans, to examine the effects of acute administration of EtOH and the role of AcH. Hippocampal slices were collected and the mRNA and protein levels of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), NGF and BDNF were analyzed 30 min after the i.p. administration of EtOH (0.5, 1.0, or 2.0 g/kg). We show that treatment with 2.0 g/kg of EtOH decreased ChAT mRNA and protein levels in Aldh2-KO mice but not in wild-type (WT) mice, which suggests a role for AcH in the mechanism of action of EtOH. The administration of 2.0 g/kg of EtOH increased AChE mRNA in both strains of mice. EtOH failed to change the levels of NGF or BDNF at any dose. Aldh2-KO mice exhibited a distinctly lower expression of ChAT and a higher expression of NGF both at mRNA and protein levels in the hippocampus compared with WT mice. Our observations suggest that administration of EtOH and elevated AcH can alter cholinergic markers in the hippocampus of mice, and this effect did not change the levels of NGF or BDNF. © 2013 Elsevier B.V. All rights reserved.

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.

Reduced cortical BDNF expression and aberrant memory in Carf knockout mice

PubMed Central

McDowell, Kelli A.; Hutchinson, Ashley N.; Wong-Goodrich, Sarah J.E.; Presby, Matthew M.; Su, Dan; Rodriguiz, Ramona M.; Law, Krystal C.; Williams, Christina L.; Wetsel, William C.; West, Anne E.

2010-01-01

Transcription factors are a key point of convergence between the cell-intrinsic and extracellular signals that guide synaptic development and brain plasticity. Calcium-Response Factor (CaRF) is a unique transcription factor first identified as a binding protein for a calcium-response element in the gene encoding Brain-Derived Neurotrophic Factor (Bdnf). We have now generated Carf knockout (KO) mice to characterize the function of this factor in vivo. Intriguingly, Carf KO mice have selectively reduced expression of Bdnf exon IV-containing mRNA transcripts and BDNF protein in the cerebral cortex while BDNF levels in the hippocampus and striatum remain unchanged, implicating CaRF as a brain region-selective regulator of BDNF expression. At the cellular level, Carf KO mice show altered expression of GABAergic proteins at striatal synapses, raising the possibility that CaRF may contribute to aspects of inhibitory synapse development. Carf KO mice show normal spatial learning in the Morris water maze and normal context-dependent fear conditioning. However they have an enhanced ability to find a new platform location on the first day of reversal training in the water maze and they extinguish conditioned fear more slowly than their wildtype (WT) littermates. Finally, Carf KO mice show normal short-term and long-term memory in a novel object recognition task, but exhibit impairments during the remote memory phase of testing. Taken together these data reveal novel roles for CaRF in the organization and/or function of neural circuits that underlie essential aspects of learning and memory. PMID:20519520

Alimentary tract innervation deficits and dysfunction in mice lacking GDNF family receptor alpha2.

PubMed

Rossi, Jari; Herzig, Karl-Heinz; Võikar, Vootele; Hiltunen, Païvi H; Segerstråle, Mikael; Airaksinen, Matti S

2003-09-01

Subsets of parasympathetic and enteric neurons require neurturin signaling via glial cell line-derived neurotrophic factor family receptor alpha2 (GFRalpha2) for development and target innervation. Why GFRalpha2-deficient (Gfra2-/-) mice grow poorly has remained unclear. Here, we analyzed several factors that could contribute to the growth retardation. Neurturin mRNA was localized in the gut circular muscle. GFRalpha2 protein was expressed in most substance P-containing myenteric neurons, in most intrapancreatic neurons, and in surrounding glial cells. In the Gfra2-/- mice, density of substance P-containing myenteric ganglion cells and nerve bundles in the myenteric ganglion cell layer was significantly reduced, and transit of test material through small intestine was 25% slower compared to wild-type mice. Importantly, the knockout mice had approximately 80% fewer intrapancreatic neurons, severely impaired cholinergic innervation of the exocrine but not the endocrine pancreas, and increased fecal fat content. Vagally mediated stimulation of pancreatic secretion by 2-deoxy-glucose in vivo was virtually abolished. Retarded growth of the Gfra2-/- mice was accompanied by reduced fat mass and elevated basal metabolic rate. Moreover, the knockout mice drank more water than wild-type controls, and wet-mash feeding resulted in partial growth rescue. Taken together, the results suggest that the growth retardation in mice lacking GFRalpha2 is largely due to impaired salivary and pancreatic secretion and intestinal dysmotility.

Microarray analysis of retinal gene expression in Egr-1 knockout mice

PubMed Central

Schippert, Ruth; Schaeffel, Frank

2009-01-01

Purpose We found earlier that 42 day-old Egr-1 knockout mice had longer eyes and a more myopic refractive error compared to their wild-types. To identify genes that could be responsible for the temporarily enhanced axial eye growth, a microarray analysis was performed in knockout and wild-type mice at the postnatal ages of 30 and 42 days. Methods The retinas of homozygous and wild-type Egr-1 knockout mice (Taconic, Ry, Denmark) were prepared for RNA isolation (RNeasy Mini Kit, Qiagen) at the age of 30 or 42 days, respectively (n=12 each). Three retinas were pooled and labeled cRNA was made. The samples were hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Hybridization signals were calculated using GC-RMA normalization. Genes were identified as differentially expressed if they showed a fold-change (FC) of at least 1.5 and a p-value <0.05. A false-discovery rate of 5% was applied. Ten genes with potential biologic relevance were examined further with semiquantitative real-time RT–PCR. Results Comparing mRNA expression levels between wild-type and homozygous Egr-1 knockout mice, we found 73 differentially expressed genes at the age of 30 days and 135 genes at the age of 42 days. Testing for differences in gene expression between the two ages (30 versus 42 days), 54 genes were differently expressed in wild-type mice and 215 genes in homozygous animals. Based on three networks proposed by Ingenuity pathway analysis software, nine differently expressed genes in the homozygous Egr-1 knockout mice were chosen for further validation by real-time RT–PCR, three genes in each network. In addition, the gene that was most prominently regulated in the knockout mice, compared to wild-type, at both 30 days and 42 days of age (protocadherin beta-9 [Pcdhb9]), was tested with real-time RT–PCR. Changes in four of the ten genes could be confirmed by real-time RT–PCR: nuclear prelamin A recognition factor (Narf), oxoglutarate dehydrogenase (Ogdh), selenium binding protein 1 (Selenbp1), and Pcdhb9. Except for Pcdhb9, the genes whose mRNA expression levels were validated were listed in one of the networks proposed by Ingenuity pathway analysis software. In addition to these genes, the software proposed several key-regulators which did not change in our study: retinoic acid, vascular endothelial growth factor A (VEGF-A), FBJ murine osteosarcoma viral oncogene homolog (cFos), and others. Conclusions Identification of genes that are differentially regulated during the development period between postnatal day 30 (when both homozygous and wild-type mice still have the same axial length) and day 42 (where the difference in eye length is apparent) could improve the understanding of mechanisms for the control of axial eye growth and may lead to potential targets for pharmacological intervention. With the aid of pathway-analysis software, a coarse picture of possible biochemical pathways could be generated. Although the mRNA expression levels of proteins proposed by the software, like VEGF, FOS, retinoic acid (RA) receptors, or cellular RA binding protein, did not show any changes in our experiment, these molecules have previously been implicated in the signaling cascades controlling axial eye growth. According to the pathway-analysis software, they represent links between several proteins whose mRNA expression was changed in our study. PMID:20019881

Microarray analysis of retinal gene expression in Egr-1 knockout mice.

PubMed

Schippert, Ruth; Schaeffel, Frank; Feldkaemper, Marita Pauline

2009-12-10

We found earlier that 42 day-old Egr-1 knockout mice had longer eyes and a more myopic refractive error compared to their wild-types. To identify genes that could be responsible for the temporarily enhanced axial eye growth, a microarray analysis was performed in knockout and wild-type mice at the postnatal ages of 30 and 42 days. The retinas of homozygous and wild-type Egr-1 knockout mice (Taconic, Ry, Denmark) were prepared for RNA isolation (RNeasy Mini Kit, Qiagen) at the age of 30 or 42 days, respectively (n=12 each). Three retinas were pooled and labeled cRNA was made. The samples were hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Hybridization signals were calculated using GC-RMA normalization. Genes were identified as differentially expressed if they showed a fold-change (FC) of at least 1.5 and a p-value <0.05. A false-discovery rate of 5% was applied. Ten genes with potential biologic relevance were examined further with semiquantitative real-time RT-PCR. Comparing mRNA expression levels between wild-type and homozygous Egr-1 knockout mice, we found 73 differentially expressed genes at the age of 30 days and 135 genes at the age of 42 days. Testing for differences in gene expression between the two ages (30 versus 42 days), 54 genes were differently expressed in wild-type mice and 215 genes in homozygous animals. Based on three networks proposed by Ingenuity pathway analysis software, nine differently expressed genes in the homozygous Egr-1 knockout mice were chosen for further validation by real-time RT-PCR, three genes in each network. In addition, the gene that was most prominently regulated in the knockout mice, compared to wild-type, at both 30 days and 42 days of age (protocadherin beta-9 [Pcdhb9]), was tested with real-time RT-PCR. Changes in four of the ten genes could be confirmed by real-time RT-PCR: nuclear prelamin A recognition factor (Narf), oxoglutarate dehydrogenase (Ogdh), selenium binding protein 1 (Selenbp1), and Pcdhb9. Except for Pcdhb9, the genes whose mRNA expression levels were validated were listed in one of the networks proposed by Ingenuity pathway analysis software. In addition to these genes, the software proposed several key-regulators which did not change in our study: retinoic acid, vascular endothelial growth factor A (VEGF-A), FBJ murine osteosarcoma viral oncogene homolog (cFos), and others. Identification of genes that are differentially regulated during the development period between postnatal day 30 (when both homozygous and wild-type mice still have the same axial length) and day 42 (where the difference in eye length is apparent) could improve the understanding of mechanisms for the control of axial eye growth and may lead to potential targets for pharmacological intervention. With the aid of pathway-analysis software, a coarse picture of possible biochemical pathways could be generated. Although the mRNA expression levels of proteins proposed by the software, like VEGF, FOS, retinoic acid (RA) receptors, or cellular RA binding protein, did not show any changes in our experiment, these molecules have previously been implicated in the signaling cascades controlling axial eye growth. According to the pathway-analysis software, they represent links between several proteins whose mRNA expression was changed in our study.

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

Epidermal growth factor impairs palatal shelf adhesion and fusion in the Tgf-β 3 null mutant.

PubMed

Barrio, M Carmen; Del Río, Aurora; Murillo, Jorge; Maldonado, Estela; López-Gordillo, Yamila; Paradas-Lara, Irene; Hernandes, Luzmarina; Catón, Javier; Martínez-Álvarez, Concepción

2014-01-01

The cleft palate presented by transforming growth factor-β3 (Tgf-β3) null mutant mice is caused by altered palatal shelf adhesion, cell proliferation, epithelial-to-mesenchymal transformation and cell death. The expression of epidermal growth factor (EGF), transforming growth factor-β1 (Tgf-β1) and muscle segment homeobox-1 (Msx-1) is modified in the palates of these knockout mice, and the cell proliferation defect is caused by the change in EGF expression. In this study, we aimed to determine whether this change in EGF expression has any effect on the other mechanisms altered in Tgf-β3 knockout mouse palates. We tested the effect of inhibiting EGF activity in vitro in the knockout palates via the addition of Tyrphostin AG 1478. We also investigated possible interactions between EGF, Tgf-β1 and Msx-1 in Tgf-β3 null mouse palate cultures. The results show that the inhibition of EGF activity in Tgf-β3 null mouse palate cultures improves palatal shelf adhesion and fusion, with a particular effect on cell death, and restores the normal distribution pattern of Msx-1 in the palatal mesenchyme. Inhibition of TGF-β1 does not affect either EGF or Msx-1 expression. © 2014 S. Karger AG, Basel.

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.

Impaired eye-blink conditioning in waggler, a mutant mouse with cerebellar BDNF deficiency.

PubMed

Bao, S; Chen, L; Qiao, X; Knusel, B; Thompson, R F

1998-01-01

In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expression of brain-derived neurotrophic factor (BDNF) was selectively absent in the cerebellar granule cells. The cytoarchitecture of the waggler cerebellum appeared to be normal at the light microscope level. The mutant mice exhibited no sensory deficits to auditory stimuli or heat-induced pain. However, they were massively impaired in classic eye-blink conditioning. These results suggest that BDNF may have a role in normal cerebellar neuronal function, which, in turn, is essential for classic eye-blink conditioning.

Diabetes-causing gene, kruppel-like factor 11, modulates the antinociceptive response of chronic ethanol intake.

PubMed

Ou, Xiao-Ming; Udemgba, Chinelo; Wang, Niping; Dai, Xiaoli; Lomberk, Gwen; Seo, Seungmae; Urrutia, Raul; Wang, Junming; Duncan, Jeremy; Harris, Sharonda; Fairbanks, Carolyn A; Zhang, Xiao

2014-02-01

Alcohol (EtOH [ethanol]) is an antinociceptive agent, working in part, by reducing sensitivity to painful stimuli. The transcription factor Kruppel-like factor 11 (KLF11), a human diabetes-causing gene that also regulates the neurotransmitter metabolic enzymes monoamine oxidase (MAO), has recently been identified as an EtOH-inducible gene. However, its role in antinociception remains unknown. Consequently, we investigated the function of KLF11 in chronic EtOH-induced antinociception using a genetically engineered knockout mouse model. Wild-type (Klf11(+/+) ) and KLF11 knockout (Klf11(-/-) ) mice were fed a liquid diet containing EtOH for 28 days with increasing amounts of EtOH from 0% up to a final concentration of 6.4%, representing a final diet containing 36% of calories primarily from EtOH. Control mice from both genotypes were fed liquid diet without EtOH for 28 days. The EtOH-induced antinociceptive effect was determined using the tail-flick test before and after EtOH exposure (on day 29). In addition, the enzyme activity and mRNA levels of MAO A and MAO B were measured by real-time RT-PCR and enzyme assays, respectively. EtOH produced an antinociceptive response to thermal pain in Klf11(+/+) mice, as expected. In contrast, deletion of KLF11 in the Klf11(-/-) mice abolished the EtOH-induced antinociceptive effect. The mRNA and protein levels of KLF11 were significantly increased in the brain prefrontal cortex of Klf11(+/+) mice exposed to EtOH compared with control Klf11(+/+) mice. Furthermore, MAO enzyme activities were affected differently in Klf11 wild-type versus Klf11 knockout mice exposed to chronic EtOH. Chronic EtOH intake significantly increased MAO B activity in Klf11(+/+) mice. The data show KLF11 modulation of EtOH-induced antinociception. The KLF11-targeted MAO B enzyme may contribute more significantly to EtOH-induced antinociception. Thus, this study revealed a new role for the KLF11 gene in the mechanisms underlying the antinociceptive effects of chronic EtOH exposure. Copyright © 2013 by the Research Society on Alcoholism.

Diabetes Causing Gene, Kruppel-Like Factor 11, Modulates the Antinociceptive Response of Chronic Ethanol Intake

PubMed Central

Ou, Xiao-Ming; Udemgba, Chinelo; Wang, Niping; Dai, Xiaoli; Lomberk, Gwen; Seo, Seungmae; Urrutia, Raul; Wang, Junming; Duncan, Jeremy; Harris, Sharonda; Fairbanks, Carolyn A.; Zhang, Xiao

2017-01-01

Background Alcohol (ethanol) is an antinociceptive agent, working in part, by reducing sensitivity to painful stimuli. The transcription factor, Kruppel-like factor 11 (KLF11), a human diabetes-causing gene that also regulates the neurotransmitter-metabolic enzymes monoamine oxidase (MAOs), has recently been identified as an ethanol-inducible gene. However, its role in antinociception remains unknown. Consequently, we investigated the function of KLF11 in chronic ethanol-induced antinociception using a genetically engineered knockout mouse model. Methods Wild-type (Klf11+/+) and KLF11 knockout (Klf11−/−) mice were fed a liquid diet containing ethanol for 28 days with increasing amounts of ethanol from 0% up to a final concentration of 6.4%, representing a final diet containing 36% of calories primarily from ethanol. Control mice from both genotypes were fed liquid diet without ethanol for 28 days. The ethanol-induced antinociceptive effect was determined using the tail-flick test before and after ethanol exposure (on day 29). In addition, the enzyme activity and mRNA levels of MAO A and MAO B were measured by Real-time RT-PCR and enzyme assays, respectively. Results Ethanol produced an antinociceptive response to thermal pain in Klf11+/+ mice, as expected. In contrast, deletion of KLF11 in the Klf11−/− mice abolished the ethanol-induced antinociceptive effect. The mRNA and protein levels of KLF11were significantly increased in the brain prefrontal cortex of Klf11+/+ mice exposed to ethanol compared to control Klf11+/+ mice. Furthermore, MAO enzyme activities were affected differently in Klf11 wild-type versus Klf11 knockout mice exposed to chronic ethanol. Chronic ethanol intake significantly increased MAO-B activity in Klf1+/+ mice. Conclusions The data show KLF11 modulation of ethanol-induced antinociception. The KLF11-targeted MAO B enzyme, may contribute more significantly to ethanol-induced antinociception. Thus, this study revealed a new role for the KLF11 gene in the mechanisms underlying the antinociceptive effects of chronic ethanol exposure. PMID:24428663

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

Phenotypic screening of hepatocyte nuclear factor (HNF) 4-{gamma} receptor knockout mice

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

Gerdin, Anna Karin; Surve, Vikas V.; Joensson, Marie

2006-10-20

Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-{gamma}. HNF4-{gamma} is expressed in the kidneys,more » gut, pancreas, and testis. First level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-{gamma}{sup +/+}), the HNF4-{gamma} knockout (HNF4-{gamma}{sup -/-}) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-{gamma}{sup -/-} mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.« less

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.

Phosphoproteomic analysis of the striatum from pleiotrophin knockout and midkine knockout mice treated with cocaine reveals regulation of oxidative stress-related proteins potentially underlying cocaine-induced neurotoxicity and neurodegeneration.

PubMed

Vicente-Rodríguez, Marta; Gramage, Esther; Herradón, Gonzalo; Pérez-García, Carmen

2013-12-06

The neurotrophic factors pleiotrophin (PTN) and midkine (MK) are highly upregulated in different brain areas relevant to drug addiction after administrations of different drugs of abuse, including psychostimulants. We have previously demonstrated that PTN and MK modulate amphetamine-induced neurotoxicity and that PTN prevents cocaine-induced cytotoxicity in NG108-15 and PC12 cells. In an effort to dissect the different mechanisms of action triggered by PTN and MK to exert their protective roles against psychostimulant neurotoxicity, we have now used a proteomic approach to study protein phosphorylation, in which we combined phosphoprotein enrichment, by immobilized metal affinity chromatography (IMAC), with two-dimensional gel electrophoresis and mass spectrometry, in order to identify the phosphoproteins regulated in the striatum of PTN knockout, MK knockout and wild type mice treated with a single dose of cocaine (15mg/kg, i.p.). We identified 7 differentially expressed phosphoproteins: 5'(3')-deoxyribonucleotidase, endoplasmic reticulum resident protein 60 (ERP60), peroxiredoxin-6 (PRDX6), glutamate dehydrogenase 1 (GLUD1), aconitase and two subunits of hemoglobin. Most of these proteins are related to neurodegeneration processes and oxidative stress and their variations specially affect the PTN knockout mice, suggesting a protective role of endogenous PTN against cocaine-induced neural alterations. Further studies are needed to validate these proteins as possible targets against neural alterations induced by cocaine. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Elevated body temperature during sleep in orexin knockout mice

PubMed Central

Mochizuki, Takatoshi; Klerman, Elizabeth B.; Sakurai, Takeshi; Scammell, Thomas E.

2008-01-01

Core body temperature (Tb) is influenced by many physiological factors, including behavioral state, locomotor activity, and biological rhythms. To determine the relative roles of these factors, we examined Tb in orexin knockout (KO) mice, which have a narcolepsy-like phenotype with severe sleep-wake fragmentation. Because orexin is thought to promote heat production during wakefulness, we hypothesized that orexin KO mice would have lower Tb while awake. Surprisingly, the Tb of orexin KO mice was 0.4°C higher than wild-type (WT) littermates during the dark period. Orexin KO mice had normal diurnal variations in Tb, but the ultradian rhythms of Tb, locomotor activity, and wakefulness were markedly reduced. During sustained wakefulness, Tb was the same in both groups. During the first 15 min of spontaneous sleep, the Tb of WT mice decreased by 1.0°C, but Tb in orexin KO mice decreased only 0.4°C. Even during intense recovery sleep after 8 hr of sleep deprivation, the Tb of orexin KO mice remained 0.7°C higher than in WT mice. This blunted fall in Tb during sleep may be due to inadequate activation of heat loss mechanisms or sustained activity in heat-generating systems. These observations reveal an unexpected role for orexin in thermoregulation. In addition, because heat loss is an essential aspect of sleep, the blunted fall in Tb of orexin KO mice may provide an explanation for the fragmented sleep of narcolepsy. PMID:16556901

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.

Identifying Key Networks Linked to Light-Independent Photoreceptor Degeneration in Visual Arrestin 1 Knockout Mice.

PubMed

Kim, Hwa Sun; Huang, Shun-Ping; Lee, Eun-Jin; Craft, Cheryl Mae

2018-01-01

When visual arrestin 1 (ARR1, S-antigen, 48 KDa protein) was genetically knocked out in mice (original Arr1 -/- , designated Arr1 -/-A ), rod photoreceptors degenerated in a light-dependent manner. Subsequently, a light-independent cone dystrophy was identified with minimal rod death in ARR1 knockout mice (Arr1 -/-A Arr4 +/+ , designated Arr1 -/-B ), which were F2 littermates from breeding the original Arr1 -/-A and cone arrestin knockout 4 (Arr4 -/- ) mice. To resolve the genetic and phenotypic differences between the two ARR1 knockouts, we performed Affymetrix™ exon array analysis to focus on the potential differential gene expression profile and to explore the molecular and cellular pathways leading to this observed susceptibility to cone dystrophy in Arr1 -/-B compared to Arr1 -/-A or control Arr1 +/+ Arr4 +/+ (wild type [WT]). Only in the Arr1 -/-B retina did we observe an up-regulation of retinal transcripts involved in the immune response, inflammatory response and JAK-STAT signaling molecules, OSMRβ and phosphorylation of STAT3. Of these responses, the complement system was significantly higher, and a variety of inflammatory responses by complement regulation and anti-inflammatory cytokine or factors were identified in Arr1 -/-B retinal transcripts. This discovery supports that Arr1 -/-B has a distinct genetic background from Arr1 -/-A that results in alterations in its retinal phenotype leading to susceptibility to cone degeneration induced by inappropriate inflammatory and immune responses.

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 ...

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

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.

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...

Ethanol Induction of CYP2A5: Role of CYP2E1-ROS-Nrf2 Pathway

PubMed Central

Lu, Yongke; Zhang, Xu Hannah

2012-01-01

Chronic ethanol consumption was previously shown to induce CYP2A5 in mice, and this induction of CYP2A5 by ethanol was CYP2E1 dependent. In this study, the mechanisms of CYP2E1-dependent ethanol induction of CYP2A5 were investigated. CYP2E1 was induced by chronic ethanol consumption to the same degree in wild-type (WT) mice and CYP2A5 knockout (Cyp2a5 –/–) mice, suggesting that unlike the CYP2E1-dependent ethanol induction of CYP2A5, ethanol induction of CYP2E1 is not CYP2A5 dependent. Microsomal ethanol oxidation was about 25% lower in Cyp2a5 –/– mice compared with that in WT mice, suggesting that CYP2A5 can oxidize ethanol although to a lesser extent than CYP2E1 does. CYP2A5 was induced by short-term ethanol consumption in human CYP2E1 transgenic knockin (Cyp2e1 –/– KI) mice but not in CYP2E1 knockout (Cyp2e1 –/–) mice. The redox-sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) was also induced by acute ethanol in Cyp2e1 –/– KI mice but not in Cyp2e1 –/– mice. Ethanol induction of CYP2A5 in Nrf2 knockout (Nrf2 –/–) mice was lower compared with that in WT mice, whereas CYP2E1 induction by ethanol was comparable in WT and Nrf2 –/– mice. Antioxidants (N-acetyl-cysteine and vitamin C), which blocked oxidative stress induced by chronic ethanol in WT mice and acute ethanol in Cyp2e1 –/– KI mice, also blunted the induction of CYP2A5 and Nrf2 by ethanol but not the induction of CYP2E1 by ethanol. These results suggest that oxidative stress induced by ethanol via induction of CYP2E1 upregulates Nrf2 activity, which in turn regulates ethanol induction of CYP2A5. Results obtained from primary hepatocytes, mice gavaged with binge ethanol or fed chronic ethanol, show that Nrf2-regulated ethanol induction of CYP2A5 protects against ethanol-induced steatosis. PMID:22552773

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.

Failure of Pelvic Organ Support in Mice Deficient In Fibulin-3

PubMed Central

Rahn, David D.; Acevedo, Jesús F.; Roshanravan, Shayzreen; Keller, Patrick W.; Davis, Elaine C.; Marmorstein, Lihua Y.; Word, R. Ann

2009-01-01

Fibulin-5 is crucial for normal elastic fiber synthesis in the vaginal wall; more than 90% of fibulin-5-knockout mice develop pelvic organ prolapse by 20 weeks of age. In contrast, fibulin-1 and -2 deficiencies do not result in similar pathologies, and fibulin-4-knockout mice die shortly after birth. EFEMP1 encodes fibulin-3, an extracellular matrix protein important in the maintenance of abdominal fascia. Herein, we evaluated the role of fibulin-3 in pelvic organ support. Pelvic organ support was impaired significantly in female Efemp1 knockout mice (Fbln3−[supi]/−), and overt vaginal, perineal, and rectal prolapse occurred in 26.9% of animals. Prolapse severity increased with age but not parity. Fibulin-5 was up-regulated in vaginal tissues from Fbln3−[supi]/− mice regardless of prolapse. Despite increased expression of fibulin-5 in the vaginal wall, pelvic organ support failure occurred in Fbln3−[supi]/− animals, suggesting that factors related to aging led to prolapse. Elastic fiber abnormalities in vaginal tissues from young Fbln3−[supi]/− mice progressed to severe elastic fiber disruption with age, and vaginal matrix metalloprotease activity was increased significantly in Fbln3−[supi]/− animals with prolapse compared with Fbln3−[supi]/− mice without prolapse. Overall, these results indicate that both fibulin-3 and -5 are important in maintaining pelvic organ support in mice. We suggest that increased vaginal protease activity and abnormal elastic fibers in the vaginal wall are important components in the pathogenesis of pelvic organ prolapse. PMID:19095964

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

Promyelocytic Leukemia Protein Is a Cell-Intrinsic Factor Inhibiting Parvovirus DNA Replication

PubMed Central

Mitchell, Angela M.; Hirsch, Matthew L.; Li, Chengwen

2014-01-01

Tripartite motif proteins are important viral restriction factors and affect processes ranging from uncoating to transcription to immune signaling. Specifically, the promyelocytic leukemia protein (TRIM19; also called PML) is a viral restriction factor inhibiting processes from uncoating to transcription to cell survival. Here we investigated PML's effect on adeno-associated virus (AAV), a parvovirus used for gene delivery. Although dependovirus (AAV) and autonomous parvovirus (minute virus of mice) replication centers can colocalize with PML, PML's functional effect on parvoviruses is unknown. Using PML knockout mice, we determined that PML knockout enhances recombinant AAV2 (rAAV2) transduction at a range of vector doses in both male and female mice. In fact, male and female PML knockout mice exhibited up to 56-fold and 28-fold increases in transduction, respectively. PML inhibited several rAAV serotypes, suggesting a conserved mechanism, and organ specificity correlated with PML expression. Mechanistically, PML inhibited rAAV second-strand DNA synthesis, precluding inhibition of self-complementary rAAV, and did not affect the prior steps in transduction. Furthermore, we confirmed the effect of human PML on rAAV transduction through small interfering RNA (siRNA)-mediated knockdown in HuH7 cells and determined that the highest level of inhibition was due to effects of PML isoform II (PMLII). Overexpression of PMLII resulted in inhibition of second-strand synthesis, vector production, and genome replication. Moreover, wild-type AAV2 production and infectivity were also inhibited by PMLII, demonstrating a PML interaction with wild-type AAV. These data have important implications for AAV-mediated gene therapy. Additionally, PMLII inhibition of AAV second-strand synthesis and replication, which are processes necessary for all parvoviruses, suggests implications for replication of other parvoviruses. PMID:24198403

Promyelocytic leukemia protein is a cell-intrinsic factor inhibiting parvovirus DNA replication.

PubMed

Mitchell, Angela M; Hirsch, Matthew L; Li, Chengwen; Samulski, R Jude

2014-01-01

Tripartite motif proteins are important viral restriction factors and affect processes ranging from uncoating to transcription to immune signaling. Specifically, the promyelocytic leukemia protein (TRIM19; also called PML) is a viral restriction factor inhibiting processes from uncoating to transcription to cell survival. Here we investigated PML's effect on adeno-associated virus (AAV), a parvovirus used for gene delivery. Although dependovirus (AAV) and autonomous parvovirus (minute virus of mice) replication centers can colocalize with PML, PML's functional effect on parvoviruses is unknown. Using PML knockout mice, we determined that PML knockout enhances recombinant AAV2 (rAAV2) transduction at a range of vector doses in both male and female mice. In fact, male and female PML knockout mice exhibited up to 56-fold and 28-fold increases in transduction, respectively. PML inhibited several rAAV serotypes, suggesting a conserved mechanism, and organ specificity correlated with PML expression. Mechanistically, PML inhibited rAAV second-strand DNA synthesis, precluding inhibition of self-complementary rAAV, and did not affect the prior steps in transduction. Furthermore, we confirmed the effect of human PML on rAAV transduction through small interfering RNA (siRNA)-mediated knockdown in HuH7 cells and determined that the highest level of inhibition was due to effects of PML isoform II (PMLII). Overexpression of PMLII resulted in inhibition of second-strand synthesis, vector production, and genome replication. Moreover, wild-type AAV2 production and infectivity were also inhibited by PMLII, demonstrating a PML interaction with wild-type AAV. These data have important implications for AAV-mediated gene therapy. Additionally, PMLII inhibition of AAV second-strand synthesis and replication, which are processes necessary for all parvoviruses, suggests implications for replication of other parvoviruses.

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

Glucagon-receptor Signaling Regulates Energy Metabolism Via Hepatic Farnesoid X Receptor and Fibroblast Growth Factor 21.

PubMed

Kim, Teayoun; Nason, Shelly; Holleman, Cassie; Pepin, Mark; Wilson, Landon; Berryhill, Taylor F; Wende, Adam R; Steele, Chad; Young, Martin E; Barnes, Stephen; Drucker, Daniel J; Finan, Brian; DiMarchi, Richard; Perez-Tilve, Diego; Tschoep, Matthias; Habegger, Kirk M

2018-06-20

Glucagon, an essential regulator of glucose and lipid metabolism, also promotes weight loss, in part through potentiation of fibroblast-growth factor 21 (FGF21) secretion. However, FGF21 is only a partial mediator of metabolic actions ensuing from GcgR-activation, prompting us to search for additional pathways. Intriguingly, chronic GcgR agonism increases plasma bile acid levels. We hypothesized that GcgR agonism regulates energy metabolism, at least in part, through farnesoid X receptor (FXR). To test this hypothesis, we studied whole body and liver-specific FXR knockout ( Fxr ∆liver ) mice. Chronic GcgR agonist (IUB288) administration in diet-induced obese (DIO) Gcgr , Fgf21 and Fxr whole body or liver-specific knockout ( ∆liver ) mice failed to reduce body weight (BW) when compared to wildtype (WT) mice. IUB288 increased energy expenditure and respiration in DIO WT mice, but not FXR ∆liver mice. GcgR agonism increased [ 14 C]-palmitate oxidation in hepatocytes isolated from WT mice in a dose-dependent manner, an effect blunted in hepatocytes from Fxr ∆liver mice. Our data clearly demonstrate that control of whole body energy expenditure by GcgR agonism requires intact FXR signaling in the liver. This heretofore-unappreciated aspect of glucagon biology has implications for the use of GcgR agonism in the therapy of metabolic disorders. © 2018 by the American Diabetes Association.

Protein arginine methyltransferase 1 modulates innate immune responses through regulation of peroxisome proliferator-activated receptor γ-dependent macrophage differentiation.

PubMed

Tikhanovich, Irina; Zhao, Jie; Olson, Jody; Adams, Abby; Taylor, Ryan; Bridges, Brian; Marshall, Laurie; Roberts, Benjamin; Weinman, Steven A

2017-04-28

Arginine methylation is a common posttranslational modification that has been shown to regulate both gene expression and extranuclear signaling events. We recently reported defects in protein arginine methyltransferase 1 (PRMT1) activity and arginine methylation in the livers of cirrhosis patients with a history of recurrent infections. To examine the role of PRMT1 in innate immune responses in vivo , we created a cell type-specific knock-out mouse model. We showed that myeloid-specific PRMT1 knock-out mice demonstrate higher proinflammatory cytokine production and a lower survival rate after cecal ligation and puncture. We found that this defect is because of defective peroxisome proliferator-activated receptor γ (PPARγ)-dependent M2 macrophage differentiation. PPARγ is one of the key transcription factors regulating macrophage polarization toward a more anti-inflammatory and pro-resolving phenotype. We found that PRMT1 knock-out macrophages failed to up-regulate PPARγ expression in response to IL4 treatment resulting in 4-fold lower PPARγ expression in knock-out cells than in wild-type cells. Detailed study of the mechanism revealed that PRMT1 regulates PPARγ gene expression through histone H4R3me2a methylation at the PPARγ promoter. Supplementing with PPARγ agonists rosiglitazone and GW1929 was sufficient to restore M2 differentiation in vivo and in vitro and abrogated the difference in survival between wild-type and PRMT1 knock-out mice. Taken together these data suggest that PRMT1-dependent regulation of macrophage PPARγ expression contributes to the infection susceptibility in PRMT1 knock-out mice. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

PubMed

Wu, Melissa P; Doyle, Jamie R; Barry, Brenda; Beauvais, Ariane; Rozkalne, Anete; Piao, Xianhua; Lawlor, Michael W; Kopin, Alan S; Walsh, Christopher A; Gussoni, Emanuela

2013-12-01

Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities. © 2013 FEBS.

Inhibitory Effects of North American Wild Rice on Monocyte Adhesion and Inflammatory Modulators in Low-Density Lipoprotein Receptor-Knockout Mice.

PubMed

Moghadasian, Mohammed H; Zhao, Ruozhi; Ghazawwi, Nora; Le, Khuong; Apea-Bah, Franklin B; Beta, Trust; Shen, Garry X

2017-10-18

The present study examined the effects of wild rice on monocyte adhesion, inflammatory and fibrinolytic mediators in low-density lipoprotein receptor-knockout (LDLr-KO) mice. Male LDLr-KO mice received a cholesterol (0.06%, w/w)-supplemented diet with or without white or wild rice (60%, w/w) for 20 weeks. White rice significantly increased monocyte adhesion and abundances of monocyte chemoattractant protein-1, tissue necrosis factor-α, intracellular cell adhesion molecule-1, plasminogen activator inhibitor-1, urokinase plasminogen activator (uPA), and uPA receptor in aortae and hearts of LDLr-KO mice compared to the control diet. Wild rice inhibited monocyte adhesion to the aorta, atherosclerosis, and abundances of the inflammatory and fibrinolytic regulators in the cardiovascular tissue of LDLr-KO mice compared to white rice. White or wild rice did not significantly alter the levels of cholesterol, triglycerides, or antioxidant enzymes in plasma. The anti-atherosclerotic effect of wild rice may result from its inhibition on monocyte adhesion and inflammatory modulators in LDLr-KO mice.

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.

Independent effects of apolipoprotein AV and apolipoprotein CIII on plasma triglyceride concentrations

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

Baroukh, Nadine N.; Bauge, Eric; Akiyama, Jennifer

2003-08-15

Both the apolipoprotein A5 and C3 genes have repeatedly been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are negatively and positively correlated with APOA5 and APOC3 expression, respectively. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. The evolutionary relationship among these two apolipoprotein genes and their close proximity on human chromosome 11q23 have largely precluded the determination of their relative contribution to altered Both the apolipoprotein A5 and C3 genes have repeatedly been shownmore » to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are negatively and positively correlated with APOA5 and APOC3 expression, respectively. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. The evolutionary relationship among these two apolipoprotein genes and their close proximity on human chromosome 11q23 have largely precluded the determination of their relative contribution to altered triglycerides. To overcome these confounding factors and address their relationship, we generated independent lines of mice that either over-expressed (''double transgenic'') or completely lacked (''double knockout'') both apolipoprotein genes. We report that both ''double transgenic'' and ''double knockout'' mice display intermedia tetriglyceride concentrations compared to over-expression or deletion of either gene alone. Furthermore, we find that human ApoAV plasma protein levels in the ''double transgenic'' mice are approximately 500-fold lower than human ApoCIII levels, supporting ApoAV is a potent triglyceride modulator despite its low concentration. Together, these data indicate that APOA5 and APOC3 independently influence plasma triglyceride concentrations but in an opposing manner.« less

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.

Hypoxia-Induced Mitogenic Factor Promotes Cardiac Hypertrophy via Calcium-Dependent and Hypoxia-Inducible Factor-1α Mechanisms.

PubMed

Kumar, Santosh; Wang, Gang; Liu, Wenjuan; Ding, Wenwen; Dong, Ming; Zheng, Na; Ye, Hongyu; Liu, Jie

2018-06-11

HIMF (hypoxia-induced mitogenic factor/found in inflammatory zone 1/resistin like α) is a secretory and cytokine-like protein and serves as a critical stimulator of hypoxia-induced pulmonary hypertension. With a role for HIMF in heart disease unknown, we explored the possible roles for HIMF in cardiac hypertrophy by overexpressing and knocking down HIMF in cardiomyocytes and characterizing HIMF gene ( himf ) knockout mice. We found that HIMF mRNA and protein levels were upregulated in phenylephrine-stimulated cardiomyocyte hypertrophy and our mouse model of transverse aortic constriction-induced cardiac hypertrophy, as well as in human hearts with dilated cardiomyopathy. Furthermore, HIMF overexpression could induce cardiomyocyte hypertrophy, as characterized by elevated protein expression of hypertrophic biomarkers (ANP [atrial natriuretic peptide] and β-MHC [myosin heavy chain-β]) and increased cell-surface area compared with controls. Conversely, HIMF knockdown prevented phenylephrine-induced cardiomyocyte hypertrophy and himf ablation in knockout mice significantly attenuated transverse aortic constriction-induced hypertrophic remodeling and cardiac dysfunction. HIMF overexpression increased the cytosolic Ca 2+ concentration and activated the CaN-NFAT (calcineurin-nuclear factor of activated T cell) and MAPK (mitogen-activated protein kinase) pathways; this effect could be prevented by reducing cytosolic Ca 2+ concentration with L-type Ca 2+ channel blocker nifedipine or inhibiting the CaSR (Ca 2+ sensing receptor) with Calhex 231. Furthermore, HIMF overexpression increased HIF-1α (hypoxia-inducible factor) expression in neonatal rat ventricular myocytes, and HIMF knockout inhibited HIF-1α upregulation in transverse aortic constriction mice. Knockdown of HIF-1α attenuated HIMF-induced cardiomyocyte hypertrophy. In conclusion, HIMF has a critical role in the development of cardiac hypertrophy, and targeting HIMF may represent a potential therapeutic strategy. © 2018 American Heart Association, Inc.

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

Transferrin Receptor 1 in Chronic Hypoxia-Induced Pulmonary Vascular Remodeling.

PubMed

Naito, Yoshiro; Hosokawa, Manami; Sawada, Hisashi; Oboshi, Makiko; Hirotani, Shinichi; Iwasaku, Toshihiro; Okuhara, Yoshitaka; Morisawa, Daisuke; Eguchi, Akiyo; Nishimura, Koichi; Soyama, Yuko; Fujii, Kenichi; Mano, Toshiaki; Ishihara, Masaharu; Tsujino, Takeshi; Masuyama, Tohru

2016-06-01

Iron is associated with the pathophysiology of several cardiovascular diseases, including pulmonary hypertension (PH). In addition, disrupted pulmonary iron homeostasis has been reported in several chronic lung diseases. Transferrin receptor 1 (TfR1) plays a key role in cellular iron transport. However, the role of TfR1 in the pathophysiology of PH has not been well characterized. In this study, we investigate the role of TfR1 in the development of hypoxia-induced pulmonary vascular remodeling. PH was induced by exposing wild-type (WT) mice and TfR1 hetero knockout mice to hypoxia for 4 weeks and evaluated via assessment of pulmonary vascular remodeling, right ventricular (RV) systolic pressure, and RV hypertrophy. In addition, we assessed the functional role of TfR1 in pulmonary artery smooth muscle cells in vitro. The morphology of pulmonary arteries did not differ between WT mice and TfR1 hetero knockout mice under normoxic conditions. In contrast, TfR1 hetero knockout mice exposed to 4 weeks hypoxia showed attenuated pulmonary vascular remodeling, RV systolic pressure, and RV hypertrophy compared with WT mice. In addition, the depletion of TfR1 by RNA interference attenuated human pulmonary artery smooth muscle cells proliferation induced by platelet-derived growth factor-BB (PDGF-BB) in vitro. These results suggest that TfR1 plays an important role in the development of hypoxia-induced pulmonary vascular remodeling. © American Journal of Hypertension, Ltd 2015. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

Genomic Profiling of Tumor Necrosis Factor Alpha (TNF-α) Receptor and Interleukin-1 Receptor Knockout Mice Reveals a Link between TNF-α Signaling and Increased Severity of 1918 Pandemic Influenza Virus Infection▿ †

PubMed Central

Belisle, Sarah E.; Tisoncik, Jennifer R.; Korth, Marcus J.; Carter, Victoria S.; Proll, Sean C.; Swayne, David E.; Pantin-Jackwood, Mary; Tumpey, Terrence M.; Katze, Michael G.

2010-01-01

The influenza pandemic of 1918 to 1919 was one of the worst global pandemics in recent history. The highly pathogenic nature of the 1918 virus is thought to be mediated in part by a dysregulation of the host response, including an exacerbated proinflammatory cytokine response. In the present study, we compared the host transcriptional response to infection with the reconstructed 1918 virus in wild-type, tumor necrosis factor (TNF) receptor-1 knockout (TNFRKO), and interleukin-1 (IL-1) receptor-1 knockout (IL1RKO) mice as a means of further understanding the role of proinflammatory cytokine signaling during the acute response to infection. Despite reported redundancy in the functions of IL-1β and TNF-α, we observed that reducing the signaling capacity of each of these molecules by genetic disruption of their key receptor genes had very different effects on the host response to infection. In TNFRKO mice, we found delayed or decreased expression of genes associated with antiviral and innate immune signaling, complement, coagulation, and negative acute-phase response. In contrast, in IL1RKO mice numerous genes were differentially expressed at 1 day postinoculation, including an increase in the expression of genes that contribute to dendritic and natural killer cell processes and cellular movement, and gene expression profiles remained relatively constant at later time points. We also observed a compensatory increase in TNF-α expression in virus-infected IL1RKO mice. Our data suggest that signaling through the IL-1 receptor is protective, whereas signaling through the TNF-α receptor increases the severity of 1918 virus infection. These findings suggest that manipulation of these pathways may have therapeutic benefit. PMID:20926563

Altered prostate epithelial development in mice lacking the androgen receptor in stromal fibroblasts.

PubMed

Yu, Shengqiang; Yeh, Chiuan-Ren; Niu, Yuanjie; Chang, Hong-Chiang; Tsai, Yu-Chieh; Moses, Harold L; Shyr, Chih-Rong; Chang, Chawnshang; Yeh, Shuyuan

2012-03-01

Androgens and the androgen receptor (AR) play important roles in the development of male urogenital organs. We previously found that mice with total AR knockout (ARKO) and epithelial ARKO failed to develop normal prostate with loss of differentiation. We have recently knocked out AR gene in smooth muscle cells and found the reduced luminal infolding and IGF-1 production in the mouse prostate. However, AR roles of stromal fibroblasts in prostate development remain unclear. To further probe the stromal fibroblast AR roles in prostate development, we generated tissue-selective knockout mice with the AR gene deleted in stromal fibroblasts (FSP-ARKO). We also used primary culture stromal cells to confirm the in vivo data and investigate mechanisms related to prostate development. The results showed cellular alterations in the FSP-ARKO mouse prostate with decreased epithelial proliferation, increased apoptosis, and decreased collagen composition. Further mechanistic studies demonstrated that FSP-ARKO mice have defects in the expression of prostate stromal growth factors. To further confirm these in vivo findings, we prepared primary cultured mouse prostate stromal cells and found knocking down the stromal AR could result in growth retardation of prostate stromal cells and co-cultured prostate epithelial cells, as well as decrease of some stromal growth factors. Our FSP-ARKO mice not only provide the first in vivo evidence in Cre-loxP knockout system for the requirement of stromal fibroblast AR to maintain the normal development of the prostate, but may also suggest the selective knockdown of stromal AR might become a potential therapeutic approach to battle prostate hyperplasia and cancer. Copyright © 2011 Wiley Periodicals, Inc.

Serotonin abnormalities in Engrailed-2 knockout mice: New insight relevant for a model of Autism Spectrum Disorder.

PubMed

Viaggi, Cristina; Gerace, Claudio; Pardini, Carla; Corsini, Giovanni U; Vaglini, Francesca

2015-08-01

Autism spectrum disorder (ASD) is a congenital neurodevelopmental behavioral disorder that appears in early childhood. Recent human genetic studies identified the homeobox transcription factor, Engrailed 2 (EN2), as a possible ASD susceptibility gene. En2 knockout mice (En2-/-) display subtle cerebellar neuropathological changes and reduced levels of tyrosine hydroxylase, noradrenaline and serotonin in the hippocampus and cerebral cortex similar to those ones which have been observed in the ASD brain. Furthermore other similarities link En2 knockout mice to ASD patients. Several lines of evidence suggest that serotonin may play an important role in the pathophysiology of the disease. In the present study we measured, by using an HPLC, the 5-HT levels in different brain areas and at different ages in En2-/- mice. In the frontal and occipital cortex, the content of 5HT was reduced in En2-/- 1 and 3 months old mice; in 6 month old mice, the difference was still present, but it was not statistically significant. The 5-HT content of cerebellar cortex was significantly reduced at 1 month old but significantly high when the KO mice reached 3 months of age. The increase was present even at 6 months of age. A similar trend was highlighted by SERT immunolabeling in En2-/- mice compared to control in the same areas and age analyzed. Our findings, in agreement with the current knowledge on the 5-HT system alterations in ASD, confirm the early neurotransmitter deficit with a late compensatory recovery in En2 KO-mice further suggesting that this experimental animal may be considered a good predictive model for the human disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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.

Trpc2 Depletion Protects RBC from Oxidative Stress-Induced Hemolysis

PubMed Central

Hirschler-Laszkiewicz, Iwona; Zhang, Wenyi; Keefer, Kerry; Conrad, Kathleen; Tong, Qin; Chen, Shu-jen; Bronson, Sarah; Cheung, Joseph Y.; Miller, Barbara A.

2011-01-01

Transient receptor potential channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca2+]i) through TRPC2 and TRPC3. Because modulation of [Ca2+]i is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild type littermate controls. Although the erythropoietin induced increase in [Ca2+]i was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenyhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. While erythropoietin induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion. PMID:21924222

Insulin and insulin-like growth factor-I (IGF-I) receptor phosphorylation in µ-calpain knockout mice

USDA-ARS?s Scientific Manuscript database

Numerous cellular processes are controlled by insulin and IGF-I signaling pathways. Due to previous work in our laboratories, we hypothesized that insulin (IR) and type 1 IGF-I (IGF-IR) receptor signaling is decreased due to increased protein tyrosine phosphatase 1B (PTP1B) activity. C57BL/6J mice...

Exacerbation of spontaneous autoimmune nephritis following regulatory T cell depletion in B cell lymphoma 2-interacting mediator knock-out mice.

PubMed

Wang, Y M; Zhang, G Y; Wang, Y; Hu, M; Zhou, J J; Sawyer, A; Cao, Q; Wang, Y; Zheng, G; Lee, V W S; Harris, D C H; Alexander, S I

2017-05-01

Regulatory T cells (T regs ) have been recognized as central mediators for maintaining peripheral tolerance and limiting autoimmune diseases. The loss of T regs or their function has been associated with exacerbation of autoimmune disease. However, the temporary loss of T regs in the chronic spontaneous disease model has not been investigated. In this study, we evaluated the role of T regs in a novel chronic spontaneous glomerulonephritis model of B cell lymphoma 2-interacting mediator (Bim) knock-out mice by transient depleting T regs . Bim is a pro-apoptotic member of the B cell lymphoma 2 (Bcl-2) family. Bim knock-out (Bim -/- ) mice fail to delete autoreactive T cells in thymus, leading to chronic spontaneous autoimmune kidney disease. We found that T reg depletion in Bim -/- mice exacerbated the kidney injury with increased proteinuria, impaired kidney function, weight loss and greater histological injury compared with wild-type mice. There was a significant increase in interstitial infiltrate of inflammatory cells, antibody deposition and tubular damage. Furthermore, the serum levels of cytokines interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17α, interferon (IFN)-γ and tumour necrosis factor (TNF)-α were increased significantly after T reg depletion in Bim -/- mice. This study demonstrates that transient depletion of T regs leads to enhanced self-reactive T effector cell function followed by exacerbation of kidney disease in the chronic spontaneous kidney disease model of Bim-deficient mice. © 2017 British Society for Immunology.

Effects on enantiomeric drug disposition and open-field behavior after chronic treatment with venlafaxine in the P-glycoprotein knockout mice model.

PubMed

Karlsson, Louise; Hiemke, Christoph; Carlsson, Björn; Josefsson, Martin; Ahlner, Johan; Bengtsson, Finn; Schmitt, Ulrich; Kugelberg, Fredrik C

2011-05-01

P-glycoprotein (P-gp) plays an important role in the efflux of drugs from the brain back into the bloodstream and can influence the pharmacokinetics and pharmacodynamics of drug molecules. To our knowledge, no studies have reported pharmacodynamic effects of any antidepressant drug in the P-gp knockout mice model. The aim of this study was to investigate the enantiomeric venlafaxine and metabolite concentrations in serum and brain of abcb1ab⁻/⁻ mice compared to wild-type mice upon chronic dosing, and to assess the effect of venlafaxine treatment on open-field behavior. P-gp knockout and wild-type mice received two daily intraperitoneal injections of venlafaxine (10 mg/kg) over ten consecutive days. Locomotor and rearing activities were assessed on days 7 and 9. After 10 days, drug and metabolite concentrations in brain and serum were determined using an enantioselective LC/MS/MS method. The brain concentrations of venlafaxine and its three demethylated metabolites were two to four times higher in abcb1ab⁻/⁻ mice compared to abcb1ab+/+ mice. The behavioral results indicated an impact on exploration-related behaviors in the open-field as center activity was increased, and rears were decreased by venlafaxine treatment. Our results show that P-gp at the blood-brain barrier plays an important role in limiting brain entry of the enantiomers of venlafaxine and its metabolites after chronic dosing. Taken together, the present pharmacokinetic and pharmacodynamic findings offer the possibility that the expression of P-gp in patients may be a contributing factor for limited treatment response.

Knockout of toll-like receptor-2 attenuates both the proinflammatory state of diabetes and incipient diabetic nephropathy.

PubMed

Devaraj, Sridevi; Tobias, Peter; Kasinath, Balakuntalam S; Ramsamooj, Rajendra; Afify, Alaa; Jialal, Ishwarlal

2011-08-01

Type 1 diabetes (T1DM) is a proinflammatory state and confers an increased risk for vascular complications. Toll-like receptors (TLR) could participate in diabetic vasculopathies. Whether TLR activation contributes to the proinflammatory state of T1DM and the pathogenesis of diabetic nephropathy remains unknown. We induced T1DM in TLR2 knockout mice (TLR2-/-) and wild-type littermates (C57BL/6J-WT) using streptozotocin (STZ). Fasting blood, peritoneal macrophages, and kidneys were obtained for flow cytometry, Western blot, microscopy, and cytokine assays at 6 and 14 weeks after induction of diabetes. Macrophage TLR2 expression and MyD88-dependent signaling were increased in diabetic mice (WT+STZ) compared with nondiabetic WT mice. These biomarkers were attenuated in diabetic TLR2-/- macrophages. WT+STZ mice showed increased kidney:body weight ratio due to cell hypertrophy, increased albuminuria, decreased kidney nephrin, podocin, and podocyte number and increased transforming growth factor-β and laminin compared with WT mice. Nephrin, podocin, and podocyte number and effacement were restored, and transforming growth factor-β and laminin levels were decreased in TLR2-/-+ STZ mice kidneys versus WT+STZ. Peritoneal and kidney macrophages were predominantly M1 phenotype in WT+STZ mice; this was attenuated in TLR2-/-+STZ mice. These data support a role for TLR2 in promoting inflammation and early changes of incipient diabetic nephropathy, in addition to albuminuria and podocyte loss.

Adipose-specific ablation of Nrf2 transiently delayed high-fat diet-induced obesity by altering glucose, lipid and energy metabolism of male mice.

PubMed

Zhang, Le; Dasuri, Kalavathi; Fernandez-Kim, Sun-Ok; Bruce-Keller, Annadora J; Keller, Jeffrey N

2016-01-01

Nuclear factor E2-related factor 2 (NRF2) is a well-known master controller of the cellular adaptive antioxidant and detoxification response. Recent studies demonstrated altered glucose, lipid and energy metabolism in mice with a global Nrf2 knockout. In the present study, we aim to determine the effects of an adipose-specific ablation of Nrf2 (ASAN) on diet-induced obesity (DIO) in male mice. The 6-week-old adipose-specific Nrf2 knockout (NK) and its Nrf2 control (NC) mice were fed with either control diet (CD) or high-fat diet (HFD) for 14 weeks. NK mice exhibited transiently delayed body weight (BW) growth from week 5 to week 11 of HFD feeding, higher daily physical activity levels and preferential use of fat over carbohydrates as a source of energy at week 8 of the CD-feeding period. After 14 weeks of feeding, NK mice showed comparable results with NC mice with respect to the overall BW and body fat content, but exhibited reduced blood glucose, reduced number but increased size of adipocytes, accompanied with elevated expression of many genes and proteins in the visceral fat related to glucose, lipid and energy metabolism (e.g. Fgf21 , Pgc1a ). These results indicated that NRF2 is an important mediator for glucose, lipid and energy metabolism in adipose tissue, and ASAN could have beneficial effect for prevention of DIO during the early development of mice.

Three gene-targeted mouse models of RNA splicing factor RP show late-onset RPE and retinal degeneration.

PubMed

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

2011-01-01

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

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.

Arap1 Deficiency Causes Photoreceptor Degeneration in Mice.

PubMed

Moshiri, Ala; Humpal, Devin; Leonard, Brian C; Imai, Denise M; Tham, Addy; Bower, Lynette; Clary, Dave; Glaser, Thomas M; Lloyd, K C Kent; Murphy, Christopher J

2017-03-01

Small guanosine triphosphatase (GTPase) ADP-ribosylation factors (Arfs) regulate membrane traffic and actin reorganization under the control of GTPase-activating proteins (GAPs). Arap1 is an Arf-directed GAP that inhibits the trafficking of epidermal growth factor receptor (EGFR) to the early endosome, but the diversity of its functions is incompletely understood. The aim of this study was to determine the role of Arap1 in the mammalian retina. Genetically engineered Arap1 knockout mice were screened for ocular abnormalities in the National Institutes of Health Knockout Mouse Production and Phenotyping (KOMP2) Project. Arap1 knockout and wild-type eyes were imaged using optical coherence tomography and fundus photography, and analyzed by immunohistochemistry. Arap1-/- mice develop a normal appearing retina, but undergo photoreceptor degeneration starting at 4 weeks postnatal age. The fundus appearance of mutants is notable for pigmentary changes, optic nerve pallor, vascular attenuation, and outer retinal thinning, reminiscent of retinitis pigmentosa in humans. Immunohistochemical studies suggest the cell death is predominantly in the outer nuclear layer. Functional evaluation of the retina by electroretinography reveals amplitudes are reduced. Arap1 is detected most notably in Müller glia, and not in photoreceptors, implicating a role for Müller glia in photoreceptor survival. Arap1 is necessary for normal photoreceptor survival in mice, and may be a novel gene relevant to human retinal degenerative processes, although its mechanism is unknown. Further studies in this mouse model of retinal degeneration will give insights into the cellular functions and signaling pathways in which Arap1 participates.

Haloperidol inhibits the development of atherosclerotic lesions in LDL receptor knockout mice.

PubMed

van der Sluis, Ronald J; Nahon, Joya E; Reuwer, Anne Q; Van Eck, Miranda; Hoekstra, Menno

2015-05-01

Antipsychotic drugs have been shown to modulate the expression of ATP-binding cassette transporter A1 (ABCA1), a key factor in the anti-atherogenic reverse cholesterol transport process, in vitro. Here we evaluated the potential of the typical antipsychotic drug haloperidol to modulate the cholesterol efflux function of macrophages in vitro and their susceptibility to atherosclerosis in vivo. Thioglycollate-elicited peritoneal macrophages were used for in vitro studies. Hyperlipidaemic low-density lipoprotein (LDL) receptor knockout mice were implanted with a haloperidol-containing pellet and subsequently fed a Western-type diet for 5 weeks to induce the development of atherosclerotic lesions in vivo. Haloperidol induced a 54% decrease in the mRNA expression of ABCA1 in peritoneal macrophages. This coincided with a 30% decrease in the capacity of macrophages to efflux cholesterol to apolipoprotein A1. Haloperidol treatment stimulated the expression of ABCA1 (+51%) and other genes involved in reverse cholesterol transport, that is, CYP7A1 (+98%) in livers of LDL receptor knockout mice. No change in splenic ABCA1 expression was noted. However, the average size of the atherosclerotic size was significantly smaller (-31%) in the context of a mildly more atherogenic metabolic phenotype upon haloperidol treatment. More importantly, haloperidol markedly lowered MCP-1 expression (-70%) and secretion (-28%) by peritoneal macrophages. Haloperidol treatment lowered the susceptibility of hyperlipidaemic LDL receptor knockout mice to develop atherosclerotic lesions. Our findings suggest that the beneficial effect of haloperidol on atherosclerosis susceptibility can be attributed to its ability to inhibit macrophage chemotaxis. © 2015 The British Pharmacological Society.

FSP1-specific SMAD2 knockout in renal tubular, endothelial, and interstitial cells reduces fibrosis and epithelial-to-mesenchymal transition in murine STZ-induced diabetic nephropathy.

PubMed

Loeffler, Ivonne; Liebisch, Marita; Allert, Stefanie; Kunisch, Elke; Kinne, Raimund W; Wolf, Gunter

2018-04-01

Extracellular matrix deposition during tubulointerstitial fibrosis (TIF), a central pathological process in patients with diabetic nephropathy (DN), is driven by locally activated, disease-relevant myofibroblasts. Myofibroblasts can arise from various cellular sources, e.g., tubular epithelial cells via a process named epithelial-to-mesenchymal transition (EMT). Transforming growth factor beta 1 (TGF-β1) and its downstream Smad signaling play a critical role in both TIF and EMT. Whereas Smad3 is one central mediator, the role of the other prominently expressed variant, Smad2, is not completely understood. In this study, we sought to analyze the role of renal Smad2 in the development of TIF and EMT during streptozotocin-induced DN by using a fibroblast-specific protein 1 (FSP1)-promotor-driven SMAD2 knockout mouse model with decreased tubular, endothelial, and interstitial Smad2 expression. In contrast to wild-type diabetic mice, diabetic SMAD2 knockout mice showed the following features: (1) significantly reduced DN and TIF (shown by KIM1 expression; periodic acid Schiff staining; collagen I and III, fibronectin, and connective tissue growth factor deposition); (2) significantly reduced tubular EMT-like changes (e.g., altered Snail1, E-cadherin, matrix metalloproteinase 2, and vimentin deposition); and (3) significantly decreased expression of myofibroblast markers (α-smooth muscle actin, FSP1). As one mechanism for the protection against diabetes-induced TIF and EMT, decreased Smad3 protein levels and, as a possible consequence, reduced TGF-β1 levels were observed in diabetic SMAD2 knockout mice. Our findings thus support the important role of Smad2 for pro-fibrotic TGF-β/Smad3 signaling in experimental DN.

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

Changes in Otx2 and parvalbumin immunoreactivity in the superior colliculus in the platelet-derived growth factor receptor-β knockout mice.

PubMed

Zhao, Juanjuan; Urakawa, Susumu; Matsumoto, Jumpei; Li, Ruixi; Ishii, Yoko; Sasahara, Masakiyo; Peng, Yuwen; Ono, Taketoshi; Nishijo, Hisao

2013-01-01

The superior colliculus (SC), a relay nucleus in the subcortical visual pathways, is implicated in socioemotional behaviors. Homeoprotein Otx2 and β subunit of receptors of platelet-derived growth factor (PDGFR- β ) have been suggested to play an important role in development of the visual system and development and maturation of GABAergic neurons. Although PDGFR- β -knockout (KO) mice displayed socio-emotional deficits associated with parvalbumin (PV-)immunoreactive (IR) neurons, their anatomical bases in the SC were unknown. In the present study, Otx2 and PV-immunolabeling in the adult mouse SC were investigated in the PDGFR- β KO mice. Although there were no differences in distribution patterns of Otx2 and PV-IR cells between the wild type and PDGFR- β KO mice, the mean numbers of both of the Otx2- and PV-IR cells were significantly reduced in the PDGFR- β KO mice. Furthermore, average diameters of Otx2- and PV-IR cells were significantly reduced in the PDGFR- β KO mice. These findings suggest that PDGFR- β plays a critical role in the functional development of the SC through its effects on Otx2- and PV-IR cells, provided specific roles of Otx2 protein and PV-IR cells in the development of SC neurons and visual information processing, respectively.

Olfactory behavior and physiology are disrupted in prion protein knockout mice.

PubMed

Le Pichon, Claire E; Valley, Matthew T; Polymenidou, Magdalini; Chesler, Alexander T; Sagdullaev, Botir T; Aguzzi, Adriano; Firestein, Stuart

2009-01-01

The prion protein PrP(C) is infamous for its role in disease, but its normal physiological function remains unknown. Here we found a previously unknown behavioral phenotype of Prnp(-/-) mice in an odor-guided task. This phenotype was manifest in three Prnp knockout lines on different genetic backgrounds, which provides strong evidence that the phenotype is caused by a lack of PrP(C) rather than by other genetic factors. Prnp(-/-) mice also showed altered behavior in a second olfactory task, suggesting that the phenotype is olfactory specific. Furthermore, PrP(C) deficiency affected oscillatory activity in the deep layers of the main olfactory bulb, as well as dendrodendritic synaptic transmission between olfactory bulb granule and mitral cells. Notably, both the behavioral and electrophysiological alterations found in Prnp(-/-) mice were rescued by transgenic neuronal-specific expression of PrP(C). These data suggest that PrP(C) is important in the normal processing of sensory information by the olfactory system.

Neuronal BDNF Signaling Is Necessary for the Effects of Treadmill Exercise on Synaptic Stripping of Axotomized Motoneurons

PubMed Central

Krakowiak, Joey; Liu, Caiyue; Papudesu, Chandana; Ward, P. Jillian; Wilhelm, Jennifer C.; English, Arthur W.

2015-01-01

The withdrawal of synaptic inputs from the somata and proximal dendrites of spinal motoneurons following peripheral nerve injury could contribute to poor functional recovery. Decreased availability of neurotrophins to afferent terminals on axotomized motoneurons has been implicated as one cause of the withdrawal. No reduction in contacts made by synaptic inputs immunoreactive to the vesicular glutamate transporter 1 and glutamic acid decarboxylase 67 is noted on axotomized motoneurons if modest treadmill exercise, which stimulates the production of neurotrophins by spinal motoneurons, is applied after nerve injury. In conditional, neuron-specific brain-derived neurotrophic factor (BDNF) knockout mice, a reduction in synaptic contacts onto motoneurons was noted in intact animals which was similar in magnitude to that observed after nerve transection in wild-type controls. No further reduction in coverage was found if nerves were cut in knockout mice. Two weeks of moderate daily treadmill exercise following nerve injury in these BDNF knockout mice did not affect synaptic inputs onto motoneurons. Treadmill exercise has a profound effect on synaptic inputs to motoneurons after peripheral nerve injury which requires BDNF production by those postsynaptic cells. PMID:25918648

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.

Genetic Rescue of Glycosylation-deficient Fgf23 in the Galnt3 Knockout Mouse

PubMed Central

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

2014-01-01

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

Genetic rescue of glycosylation-deficient Fgf23 in the Galnt3 knockout mouse.

PubMed

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

2014-10-01

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

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

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.

Comprehensive behavioral analysis of mice deficient in Rapgef2 and Rapgef6, a subfamily of guanine nucleotide exchange factors for Rap small GTPases possessing the Ras/Rap-associating domain.

PubMed

Maeta, Kazuhiro; Hattori, Satoko; Ikutomo, Junji; Edamatsu, Hironori; Bilasy, Shymaa E; Miyakawa, Tsuyoshi; Kataoka, Tohru

2018-05-10

Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap small GTPases, characterized by the possession of the Ras/Rap-associating domain. Previous genomic analyses suggested their possible involvement in the etiology of schizophrenia. We recently demonstrated the development of an ectopic cortical mass (ECM), which resembles the human subcortical band heterotopia, in the dorsal telencephalon-specific Rapgef2 conditional knockout (Rapgef2-cKO) brains. Additional knockout of Rapgef6 in Rapgef2-cKO mice resulted in gross enlargement of the ECM whereas knockout of Rapgef6 alone (Rapgef6-KO) had no discernible effect on the brain morphology. Here, we performed a battery of behavioral tests to examine the effects of Rapgef2 or Rapgef6 deficiency on higher brain functions. Rapgef2-cKO mice exhibited hyperlocomotion phenotypes. They showed decreased anxiety-like behavior in the elevated plus maze and the open-field tests as well as increased depression-like behavior in the Porsolt forced swim and tail suspension tests. They also exhibited increased sociability especially in novel environments. They showed defects in cognitive function as evidenced by reduced learning ability in the Barnes circular maze test and by impaired working memory in the T maze tests. In contrast, although Rapgef6 and Rapgef2 share similarities in biochemical roles, Rapgef6-KO mice exhibited mild behavioral abnormalities detected with a number of behavioral tests, such as hyperlocomotion phenotype in the open-field test and the social interaction test with a novel environment and working-memory defects in the T-maze test. In conclusion, although there were differences in their brain morphology and the magnitude of the behavioral abnormalities, Rapgef2-cKO mice and Rapgef6-KO mice exhibited hyperlocomotion phenotype and working-memory defect, both of which could be recognized as schizophrenia-like behavior.

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

Expression of Human Complement Factor H Prevents Age-Related Macular Degeneration–Like Retina Damage and Kidney Abnormalities in Aged Cfh Knockout Mice

PubMed Central

Ding, Jin-Dong; Kelly, Una; Landowski, Michael; Toomey, Christopher B.; Groelle, Marybeth; Miller, Chelsey; Smith, Stephanie G.; Klingeborn, Mikael; Singhapricha, Terry; Jiang, Haixiang; Frank, Michael M.; Bowes Rickman, Catherine

2016-01-01

Complement factor H (CFH) is an important regulatory protein in the alternative pathway of the complement system, and CFH polymorphisms increase the genetic risk of age-related macular degeneration dramatically. These same human CFH variants have also been associated with dense deposit disease. To mechanistically study the function of CFH in the pathogenesis of these diseases, we created transgenic mouse lines using human CFH bacterial artificial chromosomes expressing full-length human CFH variants and crossed these to Cfh knockout (Cfh−/−) mice. Human CFH protein inhibited cleavage of mouse complement component 3 and factor B in plasma and in retinal pigment epithelium/choroid/sclera, establishing that human CFH regulates activation of the mouse alternative pathway. One of the mouse lines, which express relatively higher levels of CFH, demonstrated functional and structural protection of the retina owing to the Cfh deletion. Impaired visual function, detected as a deficit in the scotopic electroretinographic response, was improved in this transgenic mouse line compared with Cfh−/− mice, and transgenics had a thicker outer nuclear layer and less sub–retinal pigment epithelium deposit accumulation. In addition, expression of human CFH also completely protected the mice from developing kidney abnormalities associated with loss of CFH. These humanized CFH mice present a valuable model for study of the molecular mechanisms of age-related macular degeneration and dense deposit disease and for testing therapeutic targets. PMID:25447048

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

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.

Farnesoid X receptor deficiency induces nonalcoholic steatohepatitis in low-density lipoprotein receptor-knockout mice fed a high-fat diet.

PubMed

Kong, Bo; Luyendyk, James P; Tawfik, Ossama; Guo, Grace L

2009-01-01

Nonalcoholic steatohepatitis (NASH) comprises dysregulation of lipid metabolism and inflammation. Identification of the various genetic and environmental susceptibility factors for NASH may provide novel treatments to limit inflammation and fibrosis in patients. This study utilized a mouse model of hypercholesterolemia, low-density lipoprotein receptor knockout (LDLr(-/-)) mice fed a high-fat diet for 5 months, to test the hypothesis that farnesoid X receptor (FXR) deficiency contributed to NASH development. Either the high-fat diet or FXR deficiency increased serum alanine aminotransferase activity, whereas only FXR deficiency increased bile acid and alkaline phosphatase levels. FXR deficiency and high-fat feeding increased serum cholesterol and triglycerides. Although high fat led to macrosteatosis and hepatocyte ballooning in livers of mice regardless of genotype, no inflammatory infiltrate was observed in the livers of LDLr(-/-) mice. In contrast, in the livers of LDLr(-/-)/FXR(-/-) mice, foci of inflammatory cells were observed occasionally when fed the control diet and were greatly increased when fed the high-fat diet. Consistent with enhanced inflammatory cells, hepatic levels of tumor necrosis factor alpha and intercellular adhesion molecule-1 mRNA were increased by the high-fat diet in LDLr(-/-)/FXR(-/-) mice. In agreement with elevated levels of procollagen 1 alpha 1 and TGF-beta mRNA, type 1 collagen protein levels were increased in livers of LDLr(-/-)/FXR(-/-) mice fed a high-fat diet. In conclusion, FXR deficiency induces pathologic manifestations required for NASH diagnosis in a mouse model of hypercholesterolemia, including macrosteatosis, hepatocyte ballooning, and inflammation, which suggest a combination of FXR deficiency and high-fat diet is a risk factor for NASH development, and activation of FXR may be a therapeutic intervention in the treatment of NASH.

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.

Myostatin-deficiency in mice increases global gene expression at the Dlk1-Dio3 locus in the skeletal muscle

PubMed Central

Hitachi, Keisuke; Tsuchida, Kunihiro

2017-01-01

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth and development. Myostatin inhibition leads to increased skeletal muscle mass in mammals; hence, myostatin is considered a potential therapeutic target for skeletal muscle wasting. However, downstream molecules of myostatin in the skeletal muscle have not been fully elucidated. Here, we identified the Dlk1-Dio3 locus at the mouse chromosome 12qF1, also called as the callipyge locus in sheep, as a novel downstream target of myostatin. In skeletal muscle of myostatin knockout mice, the expression of mature miRNAs at the Dlk1-Dio3 locus was significantly increased. The increased miRNA levels are caused by the transcriptional activation of the Dlk1-Dio3 locus, because a significant increase in the primary miRNA transcript was observed in myostatin knockout mice. In addition, we found increased expression of coding and non-coding genes (Dlk1, Gtl2, Rtl1/Rtl1as, and Rian) at the Dlk1-Dio3 locus in myostatin-deficient skeletal muscle. Moreover, epigenetic changes, associated with the regulation of the Dlk1-Dio3 locus, were observed in myostatin knockout mice. Taken together, this is the first report demonstrating the role of myostatin in regulating the Dlk1-Dio3 (the callipyge) locus in the skeletal muscle. PMID:27992376

Myostatin-deficiency in mice increases global gene expression at the Dlk1-Dio3 locus in the skeletal muscle.

PubMed

Hitachi, Keisuke; Tsuchida, Kunihiro

2017-01-24

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth and development. Myostatin inhibition leads to increased skeletal muscle mass in mammals; hence, myostatin is considered a potential therapeutic target for skeletal muscle wasting. However, downstream molecules of myostatin in the skeletal muscle have not been fully elucidated. Here, we identified the Dlk1-Dio3 locus at the mouse chromosome 12qF1, also called as the callipyge locus in sheep, as a novel downstream target of myostatin. In skeletal muscle of myostatin knockout mice, the expression of mature miRNAs at the Dlk1-Dio3 locus was significantly increased. The increased miRNA levels are caused by the transcriptional activation of the Dlk1-Dio3 locus, because a significant increase in the primary miRNA transcript was observed in myostatin knockout mice. In addition, we found increased expression of coding and non-coding genes (Dlk1, Gtl2, Rtl1/Rtl1as, and Rian) at the Dlk1-Dio3 locus in myostatin-deficient skeletal muscle. Moreover, epigenetic changes, associated with the regulation of the Dlk1-Dio3 locus, were observed in myostatin knockout mice. Taken together, this is the first report demonstrating the role of myostatin in regulating the Dlk1-Dio3 (the callipyge) locus in the skeletal muscle.

Absence of Wip1 partially rescues Atm deficiency phenotypes in mice

PubMed Central

Darlington, Yolanda; Nguyen, Thuy-Ai; Moon, Sung-Hwan; Herron, Alan; Rao, Pulivarthi; Zhu, Chengming; Lu, Xiongbin; Donehower, Lawrence A.

2011-01-01

Wildtype p53-Induced Phosphatase 1 (WIP1) is a serine/threonine phosphatase that dephosphorylates proteins in the ataxia telangiectasia mutated (ATM)-initiated DNA damage response pathway. WIP1 may play a homeostatic role in ATM signaling by returning the cell to a normal pre-stress state following completion of DNA repair. To better understand the effects of WIP1 on ATM signaling, we crossed Atm-deficient mice to Wip1-deficient mice and characterized phenotypes of the double knockout progeny. We hypothesized that the absence of Wip1 might rescue Atm deficiency phenotypes. Atm null mice, like ATM-deficient humans with the inherited syndrome ataxia telangiectasia, exhibit radiation sensitivity, fertility defects, and are T-cell lymphoma prone. Most double knockout mice were largely protected from lymphoma development and had a greatly extended lifespan compared to Atm null mice. Double knockout mice had increased p53 and H2AX phosphorylation and p21 expression compared to their Atm null counterparts, indicating enhanced p53 and DNA damage responses. Additionally, double knockout splenocytes displayed reduced chromosomal instability compared to Atm null mice. Finally, doubly null mice were partially rescued from infertility defects observed in Atm null mice. These results indicate that inhibition of WIP1 may represent a useful strategy for cancer treatment in general and A-T patients in particular. PMID:21765465

Ascl1 (Mash1) Knockout Perturbs Differentiation of Nonneuronal Cells in Olfactory Epithelium

PubMed Central

Jang, Woochan; Wildner, Hendrik; Schwob, James E.

2012-01-01

The embryonic olfactory epithelium (OE) generates only a very few olfactory sensory neurons when the basic helix-loop-helix transcription factor, ASCL1 (previously known as MASH1) is eliminated by gene mutation. We have closely examined the structure and composition of the OE of knockout mice and found that the absence of neurons dramatically affects the differentiation of multiple other epithelial cell types as well. The most prominent effect is observed within the two known populations of stem and progenitor cells of the epithelium. The emergence of horizontal basal cells, a multipotent progenitor population in the adult epithelium, is anomalous in the Ascl1 knockout mice. The differentiation of globose basal cells, another multipotent progenitor population in the adult OE, is also aberrant. All of the persisting globose basal cells are marked by SOX2 expression, suggesting a prominent role for SOX2 in progenitors upstream of Ascl1. However, NOTCH1-expressing basal cells are absent from the knockout; since NOTCH1 signaling normally acts to suppress Ascl1 via HES1 and drives sustentacular (Sus) cell differentiation during adult epithelial regeneration, its absence suggests reciprocity between neurogenesis and the differentiation of Sus cells. Indeed, the Sus cells of the mutant mice express a markedly lower level of HES1, strengthening that notion of reciprocity. Duct/gland development appears normal. Finally, the expression of cKIT by basal cells is also undetectable, except in those small patches where neurogenesis escapes the effects of Ascl1 knockout and neurons are born. Thus, persistent neurogenic failure distorts the differentiation of multiple other cell types in the olfactory epithelium. PMID:23284756

PKCδ knockout mice are protected from para-methoxymethamphetamine-induced mitochondrial stress and associated neurotoxicity in the striatum of mice.

PubMed

Shin, Eun-Joo; Dang, Duy-Khanh; Tran, Hai-Quyen; Nam, Yunsung; Jeong, Ji Hoon; Lee, Young Hun; Park, Kyung Tae; Lee, Yong Sup; Jang, Choon-Gon; Hong, Jau-Shyong; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2016-11-01

Para-methoxymethamphetamine (PMMA) is a para-ring-substituted amphetamine derivative sold worldwide as an illegal psychotropic drug. Although PMMA use has been reported to lead to severe intoxication and even death, little is known about the mechanism(s) by which PMMA exerts its neurotoxic effects. Here we found that PMMA treatment resulted in phosphorylation of protein kinase Cδ (PKCδ) and subsequent mitochondrial translocation of cleaved PKCδ. PMMA-induced oxidative stress was more pronounced in mitochondria than in the cytosol. Moreover, treatment with PMMA consistently resulted in significant reductions in mitochondrial membrane potential, mitochondrial complex I activity, and mitochondrial Mn superoxide dismutase-immunoreactivity. In contrast, PMMA treatment led to a significant increase in intramitochondrial Ca 2+ level. Treatment with PMMA also significantly increased ionized calcium binding adaptor molecule 1 (Iba-1)-labeled microglial activation and upregulated tumor necrosis factor alpha (TNF-α) gene expression. PKCδ knockout attenuated these mitochondrial effects and dampened the neurotoxic effects of PMMA. Importantly, TNF-α knockout mice were significantly protected from PMMA-induced increases in phospho-PKCδ expression, mitochondrial translocation of cleaved PKCδ, and Iba-1-labeled microgliosis. Both rottlerin, a pharmacological inhibitor of PKCδ, and etanercept, a pharmacological inhibitor of TNF-α, significantly protected against PMMA-mediated induction of apoptosis, as assessed by terminal deoxynucleotidyl transferase dUDP nick end labeling (TUNEL) assays. In addition, PKCδ knockout and TNF-α knockout both resulted in decreased PMMA-mediated induction of dopaminergic loss. Therefore, our results suggest that PKCδ mediates PMMA-induced neurotoxicity by facilitating oxidative stress (mitochondria > cytosol), mitochondrial dysfunction, microglial activation, and pro-apoptotic signaling. Our results also indicate that PMMA-induced PKCδ activation requires the proinflammatory cytokine TNF-α. Copyright © 2016. Published by Elsevier Ltd.

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.

Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver*

PubMed Central

Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S.; Sun, Baodong

2016-01-01

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. PMID:27358407

Effects of sex and COMT genotype on environmentally modulated cognitive control in mice

PubMed Central

Papaleo, Francesco; Erickson, Lucy; Liu, Guangping; Chen, Jingshan; Weinberger, Daniel R.

2012-01-01

Cognitive functioning differs between males and females, likely in part related to genetic dimorphisms. An example of a common genetic variation reported to have sexually dimorphic effects on cognition and temperament in humans is the Val/Met polymorphism in catechol-O-methyltransferase (COMT). We tested male and female wild-type mice (+/+) and their COMT knockout littermates (+/− and −/−) in the five-choice serial reaction time task (5CSRTT) to investigate the effects of sex, COMT genotype, and their interactions with environmental manipulations of cognitive functions such as attention, impulsivity, compulsivity, motivation, and rule-reversal learning. No sex- or COMT-dependent differences were present in the basic acquisition of the five-choice serial reaction time task. In contrast, specific environmental manipulations revealed a variety of sex- and COMT-dependent effects. Following an experimental change to trigger impulsive responding, the sexes showed similar increases in impulsiveness, but males eventually habituated whereas females did not. Moreover, COMT knockout mice were more impulsive compared with wild-type littermates. Manipulations involving mild stress adversely affected cognitive performance in males, and particularly COMT knockout males, but not in females. In contrast, following amphetamine treatment, subtle sex by genotype and sex by treatment interactions emerged primarily limited to compulsive behavior. After repeated testing, female mice showed improved performance, working harder and eventually outperforming males. Finally, removing the food-restriction condition enhanced sex and COMT differences, revealing that overall, females outperform males and COMT knockout males outperform their wild-type littermates. These findings illuminate complex sex- and COMT-related effects and their interactions with environmental factors to influence specific executive cognitive domains. PMID:23169629

Haloperidol inhibits the development of atherosclerotic lesions in LDL receptor knockout mice

PubMed Central

van der Sluis, Ronald J; Nahon, Joya E; Reuwer, Anne Q; Van Eck, Miranda; Hoekstra, Menno

2015-01-01

Background and Purpose Antipsychotic drugs have been shown to modulate the expression of ATP-binding cassette transporter A1 (ABCA1), a key factor in the anti-atherogenic reverse cholesterol transport process, in vitro. Here we evaluated the potential of the typical antipsychotic drug haloperidol to modulate the cholesterol efflux function of macrophages in vitro and their susceptibility to atherosclerosis in vivo. Experimental Approach Thioglycollate-elicited peritoneal macrophages were used for in vitro studies. Hyperlipidaemic low-density lipoprotein (LDL) receptor knockout mice were implanted with a haloperidol-containing pellet and subsequently fed a Western-type diet for 5 weeks to induce the development of atherosclerotic lesions in vivo. Key Results Haloperidol induced a 54% decrease in the mRNA expression of ABCA1 in peritoneal macrophages. This coincided with a 30% decrease in the capacity of macrophages to efflux cholesterol to apolipoprotein A1. Haloperidol treatment stimulated the expression of ABCA1 (+51%) and other genes involved in reverse cholesterol transport, that is, CYP7A1 (+98%) in livers of LDL receptor knockout mice. No change in splenic ABCA1 expression was noted. However, the average size of the atherosclerotic size was significantly smaller (−31%) in the context of a mildly more atherogenic metabolic phenotype upon haloperidol treatment. More importantly, haloperidol markedly lowered MCP-1 expression (−70%) and secretion (−28%) by peritoneal macrophages. Conclusions and Implications Haloperidol treatment lowered the susceptibility of hyperlipidaemic LDL receptor knockout mice to develop atherosclerotic lesions. Our findings suggest that the beneficial effect of haloperidol on atherosclerosis susceptibility can be attributed to its ability to inhibit macrophage chemotaxis. PMID:25572138

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

Conditional knockout of N-WASP in mouse fibroblast caused keratinocyte hyper proliferation and enhanced wound closure

PubMed Central

Jain, Neeraj; Kalailingam, Pazhanichamy; Tan, Kai Wei; Tan, Hui Bing; Sng, Ming Keat; Chan, Jeremy Soon Kiat; Tan, Nguan Soon; Thanabalu, Thirumaran

2016-01-01

Neural-Wiskott Aldrich Syndrome Protein (N-WASP) is expressed ubiquitously, regulates actin polymerization and is essential during mouse development. We have previously shown that N-WASP is critical for cell-ECM adhesion in fibroblasts. To characterize the role of N-WASP in fibroblast for skin development, we generated a conditional knockout mouse model in which fibroblast N-WASP was ablated using the Cre recombinase driven by Fibroblast Specific Protein promoter (Fsp-Cre). N-WASPFKO (N-WASPfl/fl; Fsp-cre) were born following Mendelian genetics, survived without any visible abnormalities for more than 1 year and were sexually reproductive, suggesting that expression of N-WASP in fibroblast is not critical for survival under laboratory conditions. Histological sections of N-WASPFKO mice skin (13 weeks old) showed thicker epidermis with higher percentage of cells staining for proliferation marker (PCNA), suggesting that N-WASP deficient fibroblasts promote keratinocyte proliferation. N-WASPFKO mice skin had elevated collagen content, elevated expression of FGF7 (keratinocyte growth factor) and TGFβ signaling proteins. Wound healing was faster in N-WASPFKO mice compared to control mice and N-WASP deficient fibroblasts were found to have enhanced collagen gel contraction properties. These results suggest that N-WASP deficiency in fibroblasts improves wound healing by growth factor-mediated enhancement of keratinocyte proliferation and increased wound contraction in mice. PMID:27909303

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.

PKCδ Knockout Mice Are Protected from Dextromethorphan-Induced Serotonergic Behaviors in Mice: Involvements of Downregulation of 5-HT1A Receptor and Upregulation of Nrf2-Dependent GSH Synthesis.

PubMed

Tran, Hai-Quyen; Lee, Youngho; Shin, Eun-Joo; Jang, Choon-Gon; Jeong, Ji Hoon; Mouri, Akihiro; Saito, Kuniaki; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2018-02-22

We investigated whether a specific serotonin (5-HT) receptor-mediated mechanism was involved in dextromethorphan (DM)-induced serotonergic behaviors. We firstly observed that the activation of 5-HT 1A receptor, but not 5-HT 2A receptor, contributed to DM-induced serotonergic behaviors in mice. We aimed to determine whether the upregulation of 5-HT 1A receptor induced by DM facilitates the specific induction of certain PKC isoform, because previous reports suggested that 5-HT 1A receptor activates protein kinase C (PKC). A high dose of DM (80 mg/kg, i.p.) induced a selective induction of PKCδ out of PKCα, PKCβI, PKCβII, PKCξ, and PKCδ in the hypothalamus of wild-type (WT) mice. More importantly, 5-HT 1A receptor co-immunoprecipitated PKCδ in the presence of DM. Consistently, rottlerin, a pharmacological inhibitor of PKCδ, or PKCδ knockout significantly protected against increases in 5-HT 1A receptor gene expression, 5-HT turnover rate, and serotonergic behaviors induced by DM. Treatment with DM resulted in an initial increase in nuclear factor erythroid-2-related factor 2 (Nrf2) nuclear translocation and DNA-binding activity, γ-glutamylcysteine (GCL) mRNA expression, and glutathione (GSH) level. This compensative induction was further potentiated by rottlerin or PKCδ knockout. However, GCL mRNA and GSH/GSSG levels were decreased 6 and 12 h post-DM. These decreases were attenuated by PKCδ inhibition. Our results suggest that interaction between 5-HT 1A receptor and PKCδ is critical for inducing DM-induced serotonergic behaviors and that inhibition of PKCδ attenuates the serotonergic behaviors via downregulation of 5-HT 1A receptor and upregulation of Nrf2-dependent GSH synthesis.

The Role of Beta-Adrenergic Receptors in the Regulation of Circadian Intraocular Pressure Rhythm in Mice.

PubMed

Tsuchiya, Shunsuke; Higashide, Tomomi; Toida, Kazunori; Sugiyama, Kazuhisa

2017-07-01

To investigate whether the elimination of β1- and β2-adrenergic receptors alters the diurnal intraocular pressure (IOP) rhythm in mice. β1-/β2-adrenergic receptor double-knockout and C57BL/6J mice were anesthetized intraperitoneally, with their IOPs measured via microneedle method. After entrainment to a 12-h light-dark (LD) cycle (light phase 6:00-18:00), IOPs were measured every 3 h from 9:00 to 24:00 (group 1, β1-/β2-adrenergic receptor double-knockout mice, n = 11; C57BL/6J, n = 15). The IOP measurements at 15:00 and 24:00 under a 12-h LD cycle and in the constant darkness (1 day and 8 days after exposure to darkness, respectively) were performed in another group of β1-/β2-adrenergic receptor double-knockout mice (group 2, n = 12). IOP variance throughout the day and mean IOP differences among time points were evaluated using a linear mixed model. β1-/β2-adrenergic receptor double-knockout and C57BL/6J mice showed biphasic IOP curves, low during the light phase and high during the dark phase; the fluctuation was significant (P < 0.001). The peak IOP (18.7 ± 1.4 mmHg) occurred at 24:00 and the trough IOP (13.5 ± 1.5 mmHg) occurred at 15:00 in β1-/β2-adrenergic receptor double-knockout mice group. IOP curves of β1-/β2-adrenergic receptor double-knockout and C57BL/6J were nearly parallel, and the IOPs of β1-/β2-adrenergic receptor double-knockout mice were significantly higher than those of C57BL/6J mice (P < 0.001). Under constant dark (DD) conditions, IOP at 24:00 (18.1 ± 1.5 mmHg) was significantly higher than that at 15:00 (13.3 ± 1.2 mmHg) (P < 0.001). The transition from the LD cycle to DD environment produced no significant change in IOP (P = 0.728). Elimination of both β1- and β2-adrenergic receptors did not disturb the biphasic diurnal IOP rhythm in mice.

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...

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.

Histomorphometric Parameters of the Growth Plate and Trabecular Bone in Wild-Type and Trefoil Factor Family 3 (Tff3)-Deficient Mice Analyzed by Free and Open-Source Image Processing Software.

PubMed

Bijelić, Nikola; Belovari, Tatjana; Stolnik, Dunja; Lovrić, Ivana; Baus Lončar, Mirela

2017-08-01

Trefoil factor family 3 (Tff3) peptide is present during intrauterine endochondral ossification in mice, and its deficiency affects cancellous bone quality in secondary ossification centers of mouse tibiae. The aim of this study was to quantitatively analyze parameters describing the growth plate and primary ossification centers in tibiae of 1-month-old wild-type and Tff3 knock-out mice (n=5 per genotype) by using free and open-source software. Digital photographs of the growth plates and trabecular bone were processed by open-source computer programs GIMP and FIJI. Histomorphometric parameters were calculated using measurements made with FIJI. Tff3 knock-out mice had significantly smaller trabecular number and significantly larger trabecular separation. Trabecular bone volume, trabecular bone surface, and trabecular thickness showed no significant difference between the two groups. Although such histomorphological differences were found in the cancellous bone structure, no significant differences were found in the epiphyseal plate histomorphology. Tff3 peptide probably has an effect on the formation and quality of the cancellous bone in the primary ossification centers, but not through disrupting the epiphyseal plate morphology. This work emphasizes the benefits of using free and open-source programs for morphological studies in life sciences.

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.

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.

Use Of Transgenic Mice In UDP-Glucuronosyltransferase (UGT) Studies

PubMed Central

Ou, Zhimin; Huang, Min; Zhao, Lizi; Xie, Wen

2009-01-01

Transgenic mouse models are useful to understand the function and regulation of drug metabolizing enzymes in vivo. This article is intended to describe the general strategies and to discuss specific examples on how to use transgenic, gene knockout, and humanized mice to study the function as well as genetic and pharmacological regulation of UDP-glucuronosyltransferases (UGTs). The physiological and pharmacological implications of transcription factor-mediated UGT regulation will also be discussed. The UGT-regulating transcription factors to be discussed in this article include nuclear hormone receptors (NRs), aryl hydrocarbon receptor (AhR), and nuclear factor erythroid 2-related factor 2 (Nrf2). PMID:20070245

Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons.

PubMed

Georgiev, Danko; Yoshihara, Toru; Kawabata, Rika; Matsubara, Takurou; Tsubomoto, Makoto; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

2016-07-01

In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis. © The Author 2016. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

Structural and functional cardiac cholinergic deficits in adult neurturin knockout mice.

PubMed

Mabe, Abigail M; Hoover, Donald B

2009-04-01

Previous work provided indirect evidence that the neurotrophic factor neurturin (NRTN) is required for normal cholinergic innervation of the heart. This study used nrtn knockout (KO) and wild-type (WT) mice to determine the effect of nrtn deletion on cardiac cholinergic innervation and function in the adult heart. Immunohistochemistry, confocal microscopy, and quantitative image analysis were used to directly evaluate intrinsic cardiac neuronal development. Atrial acetylcholine (ACh) levels were determined as an indirect index of cholinergic innervation. Cholinergic function was evaluated by measuring negative chronotropic responses to right vagal nerve stimulation in anaesthetized mice and responses of isolated atria to muscarinic agonists. KO hearts contained only 35% the normal number of cholinergic neurons, and the residual cholinergic neurons were 15% smaller than in WT. Cholinergic nerve density at the sinoatrial node was reduced by 87% in KOs, but noradrenergic nerve density was unaffected. Atrial ACh levels were substantially lower in KO mice (0.013 +/- 0.004 vs. 0.050 +/- 0.011 pmol/microg protein; P < 0.02) as expected from cholinergic neuron and nerve fibre deficits. Maximum bradycardia evoked by vagal stimulation was reduced in KO mice (38 +/- 6% vs. 69 +/- 3% decrease at 20 Hz; P < 0.001), and chronotropic responses took longer to develop and fade. In contrast to these deficits, isolated atria from KO mice had normal post-junctional sensitivity to carbachol and bethanechol. These findings demonstrate that NRTN is essential for normal cardiac cholinergic innervation and cholinergic control of heart rate. The presence of residual cardiac cholinergic neurons and vagal bradycardia in KO mice suggests that additional neurotrophic factors may influence this system.

Rodent nutritional model of steatohepatitis: effects of endotoxin (lipopolysaccharide) and tumor necrosis factor alpha deficiency.

PubMed

Kirsch, Richard; Clarkson, Vivian; Verdonk, Robert C; Marais, Adrian D; Shephard, Enid G; Ryffel, Bernard; de la M Hall, Pauline

2006-01-01

Intestinal endotoxin (lipopolysaccharide) is thought to contribute to liver injury in both alcoholic and nonalcoholic steatohepatitis (NASH). Tumor necrosis factor alpha (TNFalpha) is an important mediator of this process and is considered central to the inflammatory response in NASH. This study aimed to investigate the effects of lipopolysaccharide on liver injury in the methionine choline deficient (MCD) nutritional model of NASH, and to determine if TNFalpha is required for the development of steatohepatitis in this model. Male C57/BL6 mice received a MCD diet for 4 weeks, whilst a control group received an identical diet supplemented with 0.2% choline bitartrate and 0.3% methionine. At 4 weeks, mice received either an intraperitoneal injection of lipopolysaccharide (0.5 microg/g body mass) or sterile saline, and were killed 24 h thereafter. In a separate study, TNFalpha knockout and wild type C57BL/6 mice received either MCD or control diets for 4 weeks. Serum transaminase levels, liver histology (steatosis, inflammation and apoptosis), hepatic triglyceride concentration and hepatic lipid peroxidation products (conjugated dienes, lipid hydroperoxides and thiobarbituric reactive substances, free and total) were evaluated. Intraperitoneal administration of lipopolysaccharide augmented serum alanine aminotransferase (ALT) levels (P<0.02), hepatic inflammation (P<0.025), apoptosis (P<0.01) and free thiobarbituric acid reactive substances (P<0.025) in MCD mice. TNFalpha knockout mice fed the MCD diet developed steatohepatitis with histological and biochemical changes similar to those seen in wild type counterparts. Lipopolysaccharide augments liver injury in MCD mice, and TNFalpha is not required for the development of steatohepatitis in MCD mice.

Learning and memory depend on fibroblast growth factor receptor 2 functioning in hippocampus.

PubMed

Stevens, Hanna E; Jiang, Ginger Y; Schwartz, Michael L; Vaccarino, Flora M

2012-06-15

Fibroblast growth factor (FGF) signaling controls self-renewal of neural stem cells during embryonic telencephalic development. FGF receptor 2 (FGFR2) has a significant role in the production of cortical neurons during embryogenesis, but its role in the hippocampus during development and in adulthood has not been described. Here we dissociate the role of FGFR2 in the hippocampus during development and during adulthood with the use of embryonic knockout and inducible knockout mice. Embryonic knockout of FGFR2 causes a reduction of hippocampal volume and impairment in adult spatial memory in mice. Spatial reference memory, as assessed by performance on the water maze probe trial, was correlated with reduced hippocampal parvalbumin+ cells, whereas short-term learning was correlated with reduction in immature neurons in the dentate gyrus. Furthermore, short-term learning and newly generated neurons in the dentate gyrus were deficient even when FGFR2 was lacking only in adulthood. Taken together, these findings support a dual role for FGFR2 in hippocampal short-term learning and long-term reference memory, which appear to depend on the abundance of two separate cellular components, parvalbumin interneurons and newly generated granule cells in the hippocampus. Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Altered feto-placental vascularization, feto-placental malperfusion and fetal growth restriction in mice with Egfl7 loss of function.

PubMed

Lacko, Lauretta A; Hurtado, Romulo; Hinds, Samantha; Poulos, Michael G; Butler, Jason M; Stuhlmann, Heidi

2017-07-01

EGFL7 is a secreted angiogenic factor produced by embryonic endothelial cells. To understand its role in placental development, we established a novel Egfl7 knockout mouse. The mutant mice have gross defects in chorioallantoic branching morphogenesis and placental vascular patterning. Microangiography and 3D imaging revealed patchy perfusion of Egfl7 -/- placentas marked by impeded blood conductance through sites of narrowed vessels. Consistent with poor feto-placental perfusion, Egfl7 knockout resulted in reduced placental weight and fetal growth restriction. The placentas also showed abnormal fetal vessel patterning and over 50% reduction in fetal blood space. In vitro , placental endothelial cells were deficient in migration, cord formation and sprouting. Expression of genes involved in branching morphogenesis, Gcm1 , Syna and Synb , and in patterning of the extracellular matrix, Mmrn1 , were temporally dysregulated in the placentas. Egfl7 knockout did not affect expression of the microRNA embedded within intron 7. Collectively, these data reveal that Egfl7 is crucial for placental vascularization and embryonic growth, and may provide insight into etiological factors underlying placental pathologies associated with intrauterine growth restriction, which is a significant cause of infant morbidity and mortality. © 2017. Published by The Company of Biologists Ltd.

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.

The Essential Role of Mbd5 in the Regulation of Somatic Growth and Glucose Homeostasis in Mice

PubMed Central

Du, Yarui; Liu, Bo; Guo, Fan; Xu, Guifang; Ding, Yuqiang; Liu, Yong; Sun, Xin; Xu, Guoliang

2012-01-01

Methyl-CpG binding domain protein 5 (MBD5) belongs to the MBD family proteins, which play central roles in transcriptional regulation and development. The significance of MBD5 function is highlighted by recent studies implicating it as a candidate gene involved in human 2q23.1 microdeletion syndrome. To investigate the physiological role of Mbd5, we generated knockout mice. The Mbd5-deficient mice showed growth retardation, wasting and pre-weaning lethality. The observed growth retardation was associated with the impairment of GH/IGF-1 axis in Mbd5-null pups. Conditional knockout of Mbd5 in the brain resulted in the similar phenotypes as whole body deletion, indicating that Mbd5 functions in the nervous system to regulate postnatal growth. Moreover, the mutant mice also displayed enhanced glucose tolerance and elevated insulin sensitivity as a result of increased insulin signaling, ultimately resulting in disturbed glucose homeostasis and hypoglycemia. These results indicate Mbd5 as an essential factor for mouse postnatal growth and maintenance of glucose homeostasis. PMID:23077600

The essential role of Mbd5 in the regulation of somatic growth and glucose homeostasis in mice.

PubMed

Du, Yarui; Liu, Bo; Guo, Fan; Xu, Guifang; Ding, Yuqiang; Liu, Yong; Sun, Xin; Xu, Guoliang

2012-01-01

Methyl-CpG binding domain protein 5 (MBD5) belongs to the MBD family proteins, which play central roles in transcriptional regulation and development. The significance of MBD5 function is highlighted by recent studies implicating it as a candidate gene involved in human 2q23.1 microdeletion syndrome. To investigate the physiological role of Mbd5, we generated knockout mice. The Mbd5-deficient mice showed growth retardation, wasting and pre-weaning lethality. The observed growth retardation was associated with the impairment of GH/IGF-1 axis in Mbd5-null pups. Conditional knockout of Mbd5 in the brain resulted in the similar phenotypes as whole body deletion, indicating that Mbd5 functions in the nervous system to regulate postnatal growth. Moreover, the mutant mice also displayed enhanced glucose tolerance and elevated insulin sensitivity as a result of increased insulin signaling, ultimately resulting in disturbed glucose homeostasis and hypoglycemia. These results indicate Mbd5 as an essential factor for mouse postnatal growth and maintenance of glucose homeostasis.

β2-Adrenergic Receptor Knockout Mice Exhibit A Diabetic Retinopathy Phenotype

PubMed Central

Jiang, Youde; Zhang, Qiuhua; Liu, Li; Tang, Jie; Kern, Timothy S.; Steinle, Jena J.

2013-01-01

There is considerable evidence from our lab and others for a functional link between β-adrenergic receptor and insulin receptor signaling pathways in retina. Furthermore, we hypothesize that this link may contribute to lesions similar to diabetic retinopathy in that the loss of adrenergic input observed in diabetic retinopathy may disrupt normal anti-apoptotic insulin signaling, leading to retinal cell death. Our studies included assessment of neural retina function (ERG), vascular degeneration, and Müller glial cells (which express only β1 and β2-adrenergic receptor subtypes). In the current study, we produced β2-adrenergic receptor knockout mice to examine this deletion on retinal neurons and vasculature, and to identify specific pathways through which β2-adrenergic receptor modulates insulin signaling. As predicted from our hypothesis, β2-adrenergic receptor knockout mice display certain features similar to diabetic retinopathy. In addition, loss of β2-adrenergic input resulted in an increase in TNFα, a key inhibitor of insulin receptor signaling. Increased TNFα may be associated with insulin-dependent production of the anti-apoptotic factor, Akt. Since the effects occurred in vivo under normal glucose conditions, we postulate that aspects of the diabetic retinopathy phenotype might be triggered by loss of β2-adrenergic receptor signaling. PMID:23894672

p27(kip1) Knockout enhances collateralization in response to hindlimb ischemia.

PubMed

Ankri-Eliahoo, Galit; Weitz, Kevin; Cox, Timothy C; Tang, Gale L

2016-05-01

The natural response to arterial occlusive disease is enlargement of collaterals; however, the molecular factors that control collateralization are not well understood. The gene p27(Kip1) (p27) affects human response to arterial injury. Previous studies have shown that overexpression of p27 inhibits vascular endothelial and vascular smooth muscle cell (VSMC) proliferation and angiogenesis. To test the hypothesis that knockout of p27 would improve collateralization in reaction to ischemia, we performed in vivo and in vitro experiments using p27 knockout (p27(-/-)) and wild-type (wt) mice. Hindlimb ischemia was induced by left femoral artery ligation in p27(-/-) and wt (C57BL/6) female mice. The mice underwent weekly laser Doppler perfusion imaging of the footpads until sacrifice on postoperative day 28 followed by microcomputed tomography scanning of both hindlimbs. VSMCs were isolated from p27(-/-) and wt mice and used in migration and gel contraction assays in the absence and presence of the nonspecific matrix metalloproteinase (MMP) inhibitor BB94. MMP-2 and MMP-9 messenger RNA (mRNA) expression was measured by quantitative reverse transcription-polymerase chain reaction in p27(-/-) and wt VSMCs. p27(-/-) mice reperfused more effectively than wt mice by laser Doppler starting from day 7 (ischemic/nonischemic ratio, 0.33 ± 0.02 vs 0.25 ± 0.02; P < .05) and continuing through day 28 (0.45 ± 0.04 vs 0.31 ± 0.04; P < .05). The gracilis collateral diameter was similar for the nonischemic hindlimbs of the p27(-/-) and wt mice, and this collateral pathway increased similarly after ischemia as assessed by microcomputed tomography. However, the p27(-/-) mice significantly enlarged a novel collateral pathway that bridged directly between the femoral artery proximal to the ligation site and the saphenous or popliteal artery distal to the ligation site more than wt mice (158 ± 18.3 vs 82 ± 22 μm; P < .001). p27(-/-) VSMCs migrated more (79% ± 5% vs 56% ± 6%; P < .05) and caused more gel contraction (18% ± 5% of the initial area vs 43% ± 4%; P < .05) than wt cells. Migration and collagen contraction were abolished in p27(-/-) and wt cells by MMP inhibition. p27(-/-) cells expressed significantly more MMP-2 mRNA than wt cells did. Knockout of p27 enhances arterial collateralization in response to hindlimb ischemia through enlargement of a new collateral pathway. In vitro, knockout of p27 increases collagen gel contraction in addition to stimulating VSMC migration. We speculate that p27 may affect collateralization through its role in regulating MMP-2 expression. Published by Elsevier Inc.

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.

Parkin Knockout Inhibits Neuronal Development via Regulation of Proteasomal Degradation of p21

PubMed Central

Park, Mi Hee; Lee, Hwa-Jeong; Lee, Hye Lim; Son, Dong Ju; Ju, Jung Hoon; Hyun, Byung Kook; Jung, Sung Hee; Song, Ju-Kyoung; Lee, Dong Hun; Hwang, Chul Ju; Han, Sang Bae; Kim, Sanghyeon; Hong, Jin Tae

2017-01-01

PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21. PMID:28656059

Parkin Knockout Inhibits Neuronal Development via Regulation of Proteasomal Degradation of p21.

PubMed

Park, Mi Hee; Lee, Hwa-Jeong; Lee, Hye Lim; Son, Dong Ju; Ju, Jung Hoon; Hyun, Byung Kook; Jung, Sung Hee; Song, Ju-Kyoung; Lee, Dong Hun; Hwang, Chul Ju; Han, Sang Bae; Kim, Sanghyeon; Hong, Jin Tae

2017-01-01

PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.

Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver.

PubMed

Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S; Sun, Baodong

2016-08-05

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Selective deletion of Smad4 in postnatal germ cells does not affect spermatogenesis or fertility in mice.

PubMed

Hao, Xiao-Xia; Chen, Su-Ren; Tang, Ji-Xin; Li, Jian; Cheng, Jin-Mei; Jin, Cheng; Wang, Xiu-Xia; Liu, Yi-Xun

2016-07-01

SMAD4 is the central component of canonical signaling in the transforming growth factor beta (TGFβ) superfamily. Loss of Smad4 in Sertoli cells affects the expansion of the fetal testis cords, whereas selective deletion of Smad4 in Leydig cells alone does not appreciably alter fetal or adult testis development. Loss of Smad4 in Sertoli and Leydig cells, on the other hand, leads to testicular dysgenesis, and tumor formation in mice. Within the murine testes, Smad4 is also expressed in germ cells of the seminiferous tubules. We therefore, crossed Ngn3-Cre or Stra8-Cre transgenic mice with Smad4-flox mice to generate conditional knockout animals in which Smad4 was specifically deleted in postnatal germ cells to further uncover cell type-specific requirement of Smad4. Unexpectedly, these germ-cell-knockout mice were fertile and did not exhibit any detectable abnormalities in spermatogenesis, indicating that Smad4 is not required for the production of sperm; instead, these data indicate a cell type-specific requirement of Smad4 primarily during testis development. Mol. Reprod. Dev. 83: 615-623, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Fasting modulates GH/IGF-I axis and its regulatory systems in the mammary gland of female mice: Influence of endogenous cortistatin.

PubMed

Villa-Osaba, Alicia; Gahete, Manuel D; Cordoba-Chacon, José; de Lecea, Luis; Castaño, Justo P; Luque, Raúl M

2016-10-15

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are essential factors in mammary-gland (MG) development and are altered during fasting. However, no studies have investigated the alterations in the expression of GH/IGF-I and its regulatory systems (somatostatin/cortistatin and ghrelin) in MG during fasting. Therefore, this study was aimed at characterizing the regulation of GH/IGF-I/somatostatin/cortistatin/ghrelin-systems expression in MG of fasted female-mice (compared to fed-controls) and the influence of endogenous-cortistatin (using cortistatin-knockouts). Fasting decreased IGF-I while increased IGF-I/Insulin-receptors expression in MGs. Fasting provoked an increase in GH expression that might be associated to enhanced ghrelin-variants/ghrelin-O-acyl-transferase enzyme expression, while an upregulation of somatostatin-receptors was observed. However, cortistatin-knockouts mice showed a decrease in GH and somatostatin receptor-subtypes expression. Altogether, we demonstrate that GH/IGF-I, somatostatin/cortistatin and ghrelin systems expression is altered in MG during fasting, suggesting a relevant role in coordinating its response to metabolic stress, wherein endogenous cortistatin might be essential for an appropriate response. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Curcumin Protects against Atherosclerosis in Apolipoprotein E-Knockout Mice by Inhibiting Toll-like Receptor 4 Expression.

PubMed

Zhang, Shanshan; Zou, Jun; Li, Peiyang; Zheng, Xiumei; Feng, Dan

2018-01-17

Toll-like receptor 4 (TLR4) has been reported to play a critical role in the pathogenesis of atherosclerosis, the current study aimed to investigate whether curcumin suppresses atherosclerosis development in ApoE-knockout (ApoE -/- ) mice by inhibiting TLR4 expression. ApoE -/- mice were fed a high-fat diet supplemented with or without curcumin (0.1% w/w) for 16 weeks. Curcumin supplementation significantly reduced TLR4 expression and macrophage infiltration in atherosclerotic plaques. Curcumin also reduced aortic interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression, nuclear factor-κB (NF-κB) activity, and plasma IL-1β, TNF-α, soluble VCAM-1 and ICAM-1 levels. In addition, aortic sinus sections revealed that curcumin treatment reduced the extent of atherosclerotic lesions and inhibited atherosclerosis development. In vitro, curcumin inhibited NF-κB activation in macrophages and reduced TLR4 expression induced by lipopolysaccharide. Our results indicate that curcumin protects against atherosclerosis at least partially by inhibiting TLR4 expression and its related inflammatory reaction.

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

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

Cellular and Molecular Mechanisms of Anterior Chamber-Associated Immune Deviation (ACAID): What We Have Learned from Knockout Mice

PubMed Central

Vendomèle, Julie; Khebizi, Quentin; Fisson, Sylvain

2017-01-01

Anterior chamber-associated immune deviation (ACAID) is a well-known phenomenon that can occur after an antigen is introduced without any danger signal into the anterior chamber of a murine eye. It is reported to lead to an antigen-specific immune deviation throughout the body. Despite the relatively little evidence of this phenomenon in humans, it has been suggested as a potential prophylactic strategy in allograft rejections and in several autoimmune diseases. Cellular and molecular mechanisms of ACAID have been explored in different murine models mainly as proofs of concept, first by direct analyses of immune components in normal immunocompetent settings and by cell transfer experiments. Later, use of knockout (KO) mice has helped considerably to decipher ACAID mechanisms. However, several factors raise questions about the reliability and validity of studies using KO murine models. This mini-review summarizes results obtained with KO mice and discusses their advantages, their potential weaknesses, and their potential methods for further progress. PMID:29250068

Deconstructing mammalian reproduction: using knockouts to define fertility pathways.

PubMed

Roy, Angshumoy; Matzuk, Martin M

2006-02-01

Reproduction is the sine qua non for the propagation of species and continuation of life. It is a complex biological process that is regulated by multiple factors during the reproductive life of an organism. Over the past decade, the molecular mechanisms regulating reproduction in mammals have been rapidly unraveled by the study of a vast number of mouse gene knockouts with impaired fertility. The use of reverse genetics to generate null mutants in mice through targeted disruption of specific genes has enabled researchers to identify essential regulators of spermatogenesis and oogenesis in vivo and model human disorders affecting reproduction. This review focuses on the merits, utility, and the variations of the knockout technology in studies of reproduction in mammals.

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.

Integrated transcriptomic and proteomic analyses uncover regulatory roles of Nrf2 in the kidney

PubMed Central

Walsh, Joanne; Jenkins, Rosalind E.; Wong, Michael H. L.; Rowe, Cliff; Ricci, Emanuele; Ressel, Lorenzo; Fang, Yongxiang; Demougin, Philippe; Vukojevic, Vanja; O’Neill, Paul M.; Goldring, Christopher E.; Kitteringham, Neil R.; Park, B. Kevin; Odermatt, Alex; Copple, Ian M.

2015-01-01

The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 hours, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared to wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared to vehicle control. In light of their sensitivity to genetic and pharmacological modulation of renal Nrf2 activity, genes/proteins that regulate xenobiotic disposition, redox balance, the intra/extracellular transport of small molecules, and the supply of NADPH and other cellular fuels were found to be positively regulated by Nrf2 in the kidney. This was verified by qPCR, immunoblotting, pathway analysis and immunohistochemistry. In addition, the levels of NADPH and glutathione were found to be significantly decreased in the kidneys of Nrf2 knockout mice. Thus, Nrf2 regulates genes that coordinate homeostatic processes in the kidney, highlighting its potential as a novel therapeutic target. PMID:26422507

Role of the Norrie disease pseudoglioma gene in sprouting angiogenesis during development of the retinal vasculature.

PubMed

Luhmann, Ulrich F O; Lin, Jihong; Acar, Niyazi; Lammel, Stefanie; Feil, Silke; Grimm, Christian; Seeliger, Mathias W; Hammes, Hans-Peter; Berger, Wolfgang

2005-09-01

To characterize developmental defects and the time course of Norrie disease in retinal and hyaloid vasculature during retinal development and to identify underlying molecular angiogenic pathways that may be affected in Norrie disease, exudative vitreoretinopathy, retinopathy of prematurity, and Coats' disease. Norrie disease pseudoglioma homologue (Ndph)-knockout mice were studied during retinal development at early postnatal (p) stages (p5, p10, p15, and p21). Histologic techniques, quantitative RT-PCR, ELISA, and Western blot analyses provided molecular data, and scanning laser ophthalmoscopy (SLO) angiography and electroretinography (ERG) were used to obtain in vivo data. The data showed that regression of the hyaloid vasculature of Ndph-knockout mice occurred but was drastically delayed. The development of the superficial retinal vasculature was strongly delayed, whereas the deep retinal vasculature did not form because of the blockage of vessel outgrowth into the deep retinal layers. Subsequently, microaneurysm-like lesions formed. Several angiogenic factors were differentially transcribed during retinal development. Increased levels of hypoxia inducible factor-1alpha (HIF1alpha) and VEGFA, as well as a characteristic ERG pattern, confirmed hypoxic conditions in the inner retina of the Ndph-knockout mouse. These data provide evidence for a crucial role of Norrin in hyaloid vessel regression and in sprouting angiogenesis during retinal vascular development, especially in the development of the deep retinal capillary networks. They also suggest an early and a late phase of Norrie disease and may provide an explanation for similar phenotypic features of allelic retinal diseases in mice and patients as secondary consequences of pathologic hypoxia.

Murine recombinant angiotensin-converting enzyme 2 attenuates kidney injury in experimental Alport syndrome.

PubMed

Bae, Eun Hui; Fang, Fei; Williams, Vanessa R; Konvalinka, Ana; Zhou, Xiaohua; Patel, Vaibhav B; Song, Xuewen; John, Rohan; Oudit, Gavin Y; Pei, York; Scholey, James W

2017-06-01

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase in the renin-angiotensin system that catalyzes the breakdown of angiotensin II to angiotensin 1-7. We have reported that ACE2 expression in the kidney is reduced in experimental Alport syndrome but the impact of this finding on disease progression has not been studied. Accordingly, we evaluated effects of murine recombinant ACE2 treatment in Col4a3 knockout mice, a model of Alport syndrome characterized by proteinuria and progressive renal injury. Murine recombinant ACE2 (0.5 mg/kg/day) was administered from four to seven weeks of age via osmotic mini-pump. Pathological changes were attenuated by murine recombinant ACE2 treatment which ameliorated kidney fibrosis as shown by decreased expression of COL1α1 mRNA, less accumulation of extracellular matrix proteins, and inhibition of transforming growth factor-β signaling. Further, increases in proinflammatory cytokine expression, macrophage infiltration, inflammatory signaling pathway activation, and heme oxygenase-1 levels in Col4a3 knockout mice were also reduced by murine recombinant ACE2 treatment. Lastly, murine recombinant ACE2 influenced the turnover of renal ACE2, as it suppressed the expression of tumor necrosis factor-α converting enzyme, a negative regulator of ACE2. Thus, treatment with exogenous ACE2 alters angiotensin peptide metabolism in the kidneys of Col4a3 knockout mice and attenuates the progression of Alport syndrome nephropathy. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Ultrasonic vocalizations in mouse models for speech and socio-cognitive disorders: insights into the evolution of vocal communication

PubMed Central

Fischer, J; Hammerschmidt, K

2011-01-01

Comparative analyses used to reconstruct the evolution of traits associated with the human language faculty, including its socio-cognitive underpinnings, highlight the importance of evolutionary constraints limiting vocal learning in non-human primates. After a brief overview of this field of research and the neural basis of primate vocalizations, we review studies that have addressed the genetic basis of usage and structure of ultrasonic communication in mice, with a focus on the gene FOXP2 involved in specific language impairments and neuroligin genes (NL-3 and NL-4) involved in autism spectrum disorders. Knockout of FoxP2 leads to reduced vocal behavior and eventually premature death. Introducing the human variant of FoxP2 protein into mice, in contrast, results in shifts in frequency and modulation of pup ultrasonic vocalizations. Knockout of NL-3 and NL-4 in mice diminishes social behavior and vocalizations. Although such studies may provide insights into the molecular and neural basis of social and communicative behavior, the structure of mouse vocalizations is largely innate, limiting the suitability of the mouse model to study human speech, a learned mode of production. Although knockout or replacement of single genes has perceptible effects on behavior, these genes are part of larger networks whose functions remain poorly understood. In humans, for instance, deficiencies in NL-4 can lead to a broad spectrum of disorders, suggesting that further factors (experiential and/or genetic) contribute to the variation in clinical symptoms. The precise nature as well as the interaction of these factors is yet to be determined. PMID:20579107

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.

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.

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

Elimination of Kalrn Expression in POMC Cells Reduces Anxiety-Like Behavior and Contextual Fear Learning

PubMed Central

Mandela, Prashant; Yan, Yan; LaRese, Taylor; Eipper, Betty A.; Mains, Richard E.

2014-01-01

Kalirin, a Rho GDP/GTP exchange factor for Rac1 and RhoG, is known to play an essential role in the formation and maintenance of excitatory synapses and in the secretion of neuropeptides. Mice unable to express any of the isoforms of Kalrn in cells that produce POMC at any time during development (POMC cells) exhibited reduced anxiety-like behavior and reduced acquisition of passive avoidance behavior, along with sex-specific alteration in the corticosterone response to restraint stress. Strikingly, lack of Kalrn expression in POMC cells closely mimicked the effects of global Kalrn knockout on anxiety-like behavior and passive avoidance conditioning without causing the other deficits noted in Kalrn knockout mice. Our data suggest that deficits in excitatory inputs onto POMC neurons are responsible for the behavioral phenotypes observed. PMID:25014196

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) ...

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

Lentivirus-ABCG1 instillation reduces lipid accumulation and improves lung compliance in GM-CSF knock-out mice

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

Malur, Anagha; Huizar, Isham; Wells, Greg

2011-11-18

Highlights: Black-Right-Pointing-Pointer Lentivirus-ABCG1 reduces lipid accumulation in lungs of GM-CSF knock-out mice. Black-Right-Pointing-Pointer Up-regulation of ABCG1 improves lung function. Black-Right-Pointing-Pointer Upregulation of ABCG1 improves surfactant metabolism. -- Abstract: We have shown decreased expression of the nuclear transcription factor, peroxisome proliferator-activated receptor-gamma (PPAR{gamma}) and the PPAR{gamma}-regulated ATP-binding cassette transporter G1 (ABCG1) in alveolar macrophages from patients with pulmonary alveolar proteinosis (PAP). PAP patients also exhibit neutralizing antibodies to granulocyte-macrophage colony stimulating factor (GM-CSF), an upregulator of PPAR{gamma}. In association with functional GM-CSF deficiency, PAP lung is characterized by surfactant-filled alveolar spaces and lipid-filled alveolar macrophages. Similar pathology characterizes GM-CSF knock-out (KO)more » mice. We reported previously that intratracheal instillation of a lentivirus (lenti)-PPAR{gamma} plasmid into GM-CSF KO animals elevated ABCG1 and reduced alveolar macrophage lipid accumulation. Here, we hypothesized that instillation of lenti-ABCG1 might be sufficient to decrease lipid accumulation and improve pulmonary function in GM-CSF KO mice. Animals received intratracheal instillation of lenti-ABCG1 or control lenti-enhanced Green Fluorescent Protein (eGFP) plasmids and alveolar macrophages were harvested 10 days later. Alveolar macrophage transduction efficiency was 79% as shown by lenti-eGFP fluorescence. Quantitative PCR analyses indicated a threefold (p = 0.0005) increase in ABCG1 expression with no change of PPAR{gamma} or ABCA1 in alveolar macrophages of lenti-ABCG1 treated mice. ABCG1 was unchanged in control lenti-eGFP and PBS-instilled groups. Oil Red O staining detected reduced intracellular neutral lipid in alveolar macrophages from lenti-ABCG1 treated mice. Extracellular cholesterol and phospholipids were also decreased as shown by analysis of bronchoalveolar lavage fluid. Lung compliance was diminished in untreated GMCSF KO mice but improved significantly after lenti-ABCG1 treatment. Data demonstrate that in vivo instillation of lenti-ABCG1 in GM-CSF KO mice is sufficient to restore pulmonary homeostasis by: (1) upregulating ABCG1; (2) reducing intra and extracellular lipids; and (3) improving lung function. Results suggest that the ABCG1 lipid transporter is the key downstream target of GM-CSF-induced PPAR{gamma} necessary for surfactant catabolism.« less

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.

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.

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.

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

In vivo regulation of the heme oxygenase-1 gene in humanized transgenic mice

PubMed Central

Kim, Junghyun; Zarjou, Abolfazl; Traylor, Amie M.; Bolisetty, Subhashini; Jaimes, Edgar A.; Hull, Travis D.; George, James F.; Mikhail, Fady M.; Agarwal, Anupam

2012-01-01

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation producing equimolar amounts of carbon monoxide, iron, and biliverdin. Induction of HO-1 is a beneficial response to tissue injury in diverse animal models of diseases including acute kidney injury. In vitro analysis has shown that the human HO-1 gene is transcriptionally regulated by changes in chromatin conformation but whether such control occurs in vivo is not known. To enable such analysis, we generated transgenic mice, harboring an 87-kb bacterial artificial chromosome expressing human HO-1 mRNA and protein and bred these mice with HO-1 knockout mice to generate humanized BAC transgenic mice. This successfully rescued the phenotype of the knockout mice including reduced birth rates, tissue iron overload, splenomegaly, anemia, leukocytosis, dendritic cell abnormalities and survival after acute kidney injury induced by rhabdomyolysis or cisplatin nephrotoxicity. Transcription factors such as USF1/2, JunB, Sp1, and CTCF were found to associate with regulatory regions of the human HO-1 gene in the kidney following rhabdomyolysis. Chromosome Conformation Capture and ChIP-loop assays confirmed this in the formation of chromatin looping in vivo. Thus, these bacterial artificial chromosome humanized HO-1 mice are a valuable model to study the human HO-1 gene providing insight to the in vivo architecture of the gene in acute kidney injury and other diseases. PMID:22495295

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) enhances vascular and renal damage induced by hyperlipidemic diet in ApoE-knockout mice.

PubMed

Muñoz-García, Begoña; Moreno, Juan Antonio; López-Franco, Oscar; Sanz, Ana Belén; Martín-Ventura, José Luis; Blanco, Julia; Jakubowski, Aniela; Burkly, Linda C; Ortiz, Alberto; Egido, Jesús; Blanco-Colio, Luis Miguel

2009-12-01

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily of cytokines. TWEAK binds and activates the Fn14 receptor, and may regulate apoptosis, inflammation, and angiogenesis, in different pathological conditions. We have evaluated the effect of exogenous TWEAK administration as well as the role of endogenous TWEAK on proinflammatory cytokine expression and vascular and renal injury severity in hyperlipidemic ApoE-knockout mice. ApoE(-/-) mice were fed with hyperlipidemic diet for 4 to 10 weeks, then randomized and treated with saline (controls), TWEAK (10 microg/kg/d), anti-TWEAK neutralizing mAb (1000 microg/kg/d), TWEAK plus anti-TWEAK antibody (10 microg TWEAK +1000 microg anti-TWEAK/kg/d), or nonspecific IgG (1000 microg/kg/d) daily for 9 days. In ApoE(-/-) mice, exogenous TWEAK administration in ApoE(-/-) mice induced activation of NF-kappaB, a key transcription factor implicated in the regulation of the inflammatory response, in vascular and renal lesions. Furthermore, TWEAK treatment increased chemokine expression (RANTES and MCP-1), as well as macrophage infiltration in atherosclerotic plaques and renal lesions. These effects were associated with exacerbation of vascular and renal damage. Conversely, treatment of ApoE(-/-) mice with an anti-TWEAK blocking mAb decreased NF-kappaB activation, proinflammatory cytokine expression, macrophage infiltration, and vascular and renal injury severity, indicating a pathological role for endogenous TWEAK. Finally, in murine vascular smooth muscle cells or tubular cells, either ox-LDL or TWEAK treatment increased expression and secretion of both RANTES and MCP-1. Furthermore, ox-LDL and TWEAK synergized for induction of MCP-1 and RANTES expression and secretion. Our results suggest that TWEAK exacerbates the inflammatory response associated with a high lipid-rich diet. TWEAK may be a novel therapeutic target to prevent vascular and renal damage associated with hyperlipidemia.

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.

A cardiomyocyte-specific Wdr1 knockout demonstrates essential functional roles for actin disassembly during myocardial growth and maintenance in mice.

PubMed

Yuan, Baiyin; Wan, Ping; Chu, Dandan; Nie, Junwei; Cao, Yunshan; Luo, Wen; Lu, Shuangshuang; Chen, Jiong; Yang, Zhongzhou

2014-07-01

Actin dynamics are critical for muscle development and function, and mutations leading to deregulation of actin dynamics cause various forms of heritable muscle diseases. AIP1 is a major cofactor of the actin depolymerizing factor/cofilin in eukaryotes, promoting actin depolymerizing factor/cofilin-mediated actin disassembly. Its function in vertebrate muscle has been unknown. To investigate functional roles of AIP1 in myocardium, we generated conditional knockout (cKO) mice with cardiomyocyte-specific deletion of Wdr1, the mammalian homolog of yeast AIP1. Wdr1 cKO mice began to die at postnatal day 13 (P13), and none survived past P24. At P12, cKO mice exhibited cardiac hypertrophy and impaired contraction of the left ventricle. Electrocardiography revealed reduced heart rate, abnormal P wave, and abnormal T wave at P10 and prolonged QT interval at P12. Actin filament (F-actin) accumulations began at P10 and became prominent at P12 in the myocardium of cKO mice. Within regions of F-actin accumulation in myofibrils, the sarcomeric components α-actinin and tropomodulin-1 exhibited disrupted patterns, indicating that F-actin accumulations caused by Wdr1 deletion result in disruption of sarcomeric structure. Ectopic cofilin colocalized with F-actin aggregates. In adult mice, Wdr1 deletion resulted in similar but much milder phenotypes of heart hypertrophy, F-actin accumulations within myofibrils, and lethality. Taken together, these results demonstrate that AIP1-regulated actin dynamics play essential roles in heart function in mice. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Regulation of extinction of cocaine-induced place preference by midkine is related to a differential phosphorylation of peroxiredoxin 6 in dorsal striatum.

PubMed

Gramage, Esther; Pérez-García, Carmen; Vicente-Rodríguez, Marta; Bollen, Silke; Rojo, Loreto; Herradón, Gonzalo

2013-09-15

The neurotrophic factors Midkine (MK) and Pleiotrophin (PTN) have been suggested to modulate drugs of abuse-induced effects. To test this hypothesis, cocaine (10 and 15mg/kg)-induced conditioned place preference (CPP) was rendered in PTN knockout (PTN-/-), MK knockout (MK-/-) and wild type (WT+/+) mice, and then extinguished after repeated saline injections (distributed in 4 extinction sessions). Cocaine induced a similar CPP in all the three genotypes. We found a significantly increased percentage of MK-/- mice that did not extinguish cocaine CPP at the end of the extinction sessions. Particularly, 40% of MK-/- mice did not extinguish cocaine (15mg/kg)-induced CPP compared to WT+/+ and PTN-/- mice (∼0-6%). Interestingly, we found that a greater magnitude of extinction of CPP after the first extinction session (5 days after last administration of cocaine) correlates with increased tyrosine phosphorylation of the enzyme peroxiredoxin 6 in the dorsal striatum of MK-/- mice. On the other hand, a greater magnitude of CPP extinction correlates with increased tyrosine phosphorylation of aconitase 2 in the prefrontal cortex of WT+/+ mice. In contrast, a lower magnitude of CPP extinction correlates with increased phosphorylation of aconitase 2 in the prefrontal cortex of PTN-/- mice, suggesting that the correlation between the tyrosine phosphorylation levels of aconitase 2 and magnitude of CPP extinction depends on the genotype considered. The data demonstrate that MK is a novel genetic factor that plays a role in the extinction of cocaine-induced CPP by mechanisms that may involve specific phosphorylation of striatal peroxiredoxin 6. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

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.

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.

MitoNEET in Perivascular Adipose Tissue Blunts Atherosclerosis under Mild Cold Condition in Mice

PubMed Central

Xiong, Wenhao; Zhao, Xiangjie; Garcia-Barrio, Minerva T.; Zhang, Jifeng; Lin, Jiandie; Chen, Y. Eugene; Jiang, Zhisheng; Chang, Lin

2017-01-01

Background: Perivascular adipose tissue (PVAT), which surrounds most vessels, is de facto a distinct functional vascular layer actively contributing to vascular function and dysfunction. PVAT contributes to aortic remodeling by producing and releasing a large number of undetermined or less characterized factors that could target endothelial cells and vascular smooth muscle cells, and herein contribute to the maintenance of vessel homeostasis. Loss of PVAT in mice enhances atherosclerosis, but a causal relationship between PVAT and atherosclerosis and the possible underlying mechanisms remain to be addressed. The CDGSH iron sulfur domain 1 protein (referred to as mitoNEET), a mitochondrial outer membrane protein, regulates oxidative capacity and adipose tissue browning. The roles of mitoNEET in PVAT, especially in the development of atherosclerosis, are unknown. Methods: The brown adipocyte-specific mitoNEET transgenic mice were subjected to cold environmental stimulus. The metabolic rates and PVAT-dependent thermogenesis were investigated. Additionally, the brown adipocyte-specific mitoNEET transgenic mice were cross-bred with ApoE knockout mice. The ensuing mice were subsequently subjected to cold environmental stimulus and high cholesterol diet challenge for 3 months. The development of atherosclerosis was investigated. Results: Our data show that mitoNEET mRNA was downregulated in PVAT of both peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α)- and beta (Pgc1β)-knockout mice which are sensitive to cold. MitoNEET expression was higher in PVAT of wild type mice and increased upon cold stimulus. Transgenic mice with overexpression of mitoNEET in PVAT were cold resistant, and showed increased expression of thermogenic genes. ApoE knockout mice with mitoNEET overexpression in PVAT showed significant downregulation of inflammatory genes and showed reduced atherosclerosis development upon high fat diet feeding when kept in a 16°C environment. Conclusion: mitoNEET in PVAT is associated with PVAT-dependent thermogenesis and prevents atherosclerosis development. The results of this study provide new insights on PVAT and mitoNEET biology and atherosclerosis in cardiovascular diseases. PMID:29311966

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.

Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice.

PubMed

Autio, Kaija J; Schmitz, Werner; Nair, Remya R; Selkälä, Eija M; Sormunen, Raija T; Miinalainen, Ilkka J; Crick, Peter J; Wang, Yuqin; Griffiths, William J; Reddy, Janardan K; Baes, Myriam; Hiltunen, J Kalervo

2014-07-01

Cholesterol is catabolized to bile acids by peroxisomal β-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this β-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

Altered lipid and salt taste responsivity in ghrelin and GOAT null mice.

PubMed

Cai, Huan; Cong, Wei-Na; Daimon, Caitlin M; Wang, Rui; Tschöp, Matthias H; Sévigny, Jean; Martin, Bronwen; Maudsley, Stuart

2013-01-01

Taste perception plays an important role in regulating food preference, eating behavior and energy homeostasis. Taste perception is modulated by a variety of factors, including gastric hormones such as ghrelin. Ghrelin can regulate growth hormone release, food intake, adiposity, and energy metabolism. Octanoylation of ghrelin by ghrelin O-acyltransferase (GOAT) is a specific post-translational modification which is essential for many biological activities of ghrelin. Ghrelin and GOAT are both widely expressed in many organs including the gustatory system. In the current study, overall metabolic profiles were assessed in wild-type (WT), ghrelin knockout (ghrelin(-/-)), and GOAT knockout (GOAT(-/-)) mice. Ghrelin(-/-) mice exhibited decreased food intake, increased plasma triglycerides and increased ketone bodies compared to WT mice while demonstrating WT-like body weight, fat composition and glucose control. In contrast GOAT(-/-) mice exhibited reduced body weight, adiposity, resting glucose and insulin levels compared to WT mice. Brief access taste behavioral tests were performed to determine taste responsivity in WT, ghrelin(-/-) and GOAT(-/-) mice. Ghrelin and GOAT null mice possessed reduced lipid taste responsivity. Furthermore, we found that salty taste responsivity was attenuated in ghrelin(-/-) mice, yet potentiated in GOAT(-/-) mice compared to WT mice. Expression of the potential lipid taste regulators Cd36 and Gpr120 were reduced in the taste buds of ghrelin and GOAT null mice, while the salt-sensitive ENaC subunit was increased in GOAT(-/-) mice compared with WT mice. The altered expression of Cd36, Gpr120 and ENaC may be responsible for the altered lipid and salt taste perception in ghrelin(-/-) and GOAT(-/-) mice. The data presented in the current study potentially implicates ghrelin signaling activity in the modulation of both lipid and salt taste modalities.

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

Altered morphology and function of the lacrimal functional unit in protein kinase C{alpha} knockout mice.

PubMed

Chen, Zhuo; Li, Zhijie; Basti, Surendra; Farley, William J; Pflugfelder, Stephen C

2010-11-01

Protein kinase C (PKC) α plays a major role in the parasympathetic neural stimulation of lacrimal gland (LG) secretion. It also has been reported to have antiapoptotic properties and to promote cell survival. Therefore, the hypothesis for the present study was that PKCα knockout ((-/-)) mice have impaired ocular surface-lacrimal gland signaling, rendering them susceptible to desiccating stress and impaired corneal epithelial wound healing. In this study, the lacrimal function unit (LFU) and the stressed wound-healing response were examined in PKCα(-/-) mice. In PKCα(+/+) control mice and PKCα(-/-) mice, tear production, osmolarity, and clearance rate were evaluated before and after experimental desiccating stress. Histology and immunofluorescent staining of PKC and epidermal growth factor were performed in tissues of the LFU. Cornified envelope (CE) precursor protein expression and cell proliferation were evaluated. The time course of healing and degree of neutrophil infiltration was evaluated after corneal epithelial wounding. Compared with the PKCα(+/+) mice, the PKCα(-/-) mice were noted to have significantly increased lacrimal gland weight, with enlarged, carbohydrate-rich, PAS-positive acinar cells; increased corneal epithelia permeability, with reduced CE expression; and larger conjunctival epithelial goblet cells. The PKCα(-/-) mice showed more rapid corneal epithelial healing, with less neutrophil infiltration and fewer proliferating cells than did the PKCα(+/+) mice. The PKCα(-/-) mice showed lower tear production, which appeared to be caused by impaired secretion by the LG and conjunctival goblet cells. Despite their altered tear dynamics, the PKCα(-/-) mice demonstrated more rapid corneal epithelial wound healing, perhaps due to decreased neutrophil infiltration.

Sirt2 suppresses inflammatory responses in collagen-induced arthritis

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

Lin, Jiangtao; Department of Orthopaedics, Yantaishan Hospital, 91 Jiefang Road, Yantai, Shandong 264001; Sun, Bing

Highlights: •Sirt2 expression decreases in collagen-induced arthritis (CIA). •Sirt2 knockout aggravates severity of arthritis in mice with CIA. •Sirt2 knockout increases levels of pro-inflammatory factors in the serum. •Sirt2 deacetylates p65 and inhibits pro-inflammatory factors expression. •Sirt2 rescue abates severity of arthritis in mice with CIA. -- Abstract: Arthritis is a common autoimmune disease that is associated with progressive disability, systemic complications and early death. However, the underling mechanisms of arthritis are still unclear. Sirtuins are a NAD{sup +}-dependent class III deacetylase family, and regulate cellular stress, inflammation, genomic stability, carcinogenesis, and energy metabolism. Among the sirtuin family members, Sirt1more » and Sirt6 are critically involved in the development of arthritis. It remains unknown whether other sirtuin family members participate in arthritis. Here in this study, we demonstrate that Sirt2 inhibits collagen-induced arthritis (CIA) using in vivo and in vitro evidence. The protein and mRNA levels of Sirt2 significantly decreased in joint tissues of mice with CIA. When immunized with collagen, Sirt2-KO mice showed aggravated severity of arthritis based on clinical scores, hind paw thickness, and radiological and molecular findings. Mechanically, Sirt2 deacetylated p65 subunit of nuclear factor-kappa B (NF-κB) at lysine 310, resulting in reduced expression of NF-κB-dependent genes, including interleukin 1β (IL-1β), IL-6, monocyte chemoattractant protein 1(MCP-1), RANTES, matrix metalloproteinase 9 (MMP-9) and MMP-13. Importantly, our rescue experiment showed that Sirt2 re-expression abated the severity of arthritis in Sirt2-KO mice. Those findings strongly indicate Sirt2 as a considerably inhibitor of the development of arthritis.« less

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.

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...

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.

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

Biochemistry and physiology of hexose-6-phosphate knockout mice.

PubMed

Zielinska, Agnieszka E; Walker, Elizabeth A; Stewart, Paul M; Lavery, Gareth G

2011-04-10

Hexose-6-phosphate dehydrogenase (H6PDH) has emerged as an important factor in setting the redox status of the endoplasmic reticulum (ER) lumen. An important role of H6PDH is to generate a high NADPH/NADP(+) ratio which permits 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to act as an oxo-reductase, catalyzing the activation of glucocorticoids (GCs). In H6PDH knockout mice 11β-HSD1 assumes dehydrogenase activity and inactivates GCs, rendering the target cell relatively GC insensitive. Consequently, H6PDHKO mice have a phenotype consistent with defects in the permissive and adaptive actions of GCs upon physiology. H6PDHKO mice have also offered an insight into muscle physiology as they also present with a severe vacuolating myopathy, abnormalities of glucose homeostasis and activation of the unfolded protein response due to ER stress, and a number of mechanisms driving this phenotype are thought to be involved. This article will review what we understand of the redox control of GC hormone metabolism regulated by H6PDH, and how H6PDHKO mice have allowed an in-depth understanding of its potentially novel, GC-independent roles in muscle physiology. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Ethanol self-administration in serotonin transporter knockout mice: unconstrained demand and elasticity.

PubMed

Lamb, R J; Daws, L C

2013-10-01

Low serotonin function is associated with alcoholism, leading to speculation that increasing serotonin function could decrease ethanol consumption. Mice with one or two deletions of the serotonin transporter (SERT) gene have increased extracellular serotonin. To examine the relationship between SERT genotype and motivation for alcohol, we compared ethanol self-administration in mice with zero (knockout, KO), one (HET) or two copies (WT) of the SERT gene. All three genotypes learned to self-administer ethanol. The SSRI, fluvoxamine, decreased responding for ethanol in the HET and WT, but not the KO mice. When tested under a progressive ratio schedule, KO mice had lower breakpoints than HET or WT. As work requirements were increased across sessions, behavioral economic analysis of ethanol self-administration indicated that the decreased breakpoint in KO as compared to HET or WT mice was a result of lower levels of unconstrained demand, rather than differences in elasticity, i.e. the proportional decreases in ethanol earned with increasing work requirements were similar across genotypes. The difference in unconstrained demand was unlikely to result from motor or general motivational factors, as both WT and KO mice responded at high levels for a 50% condensed milk solution. As elasticity is hypothesized to measure essential value, these results indicate that KO value ethanol similarly to WT or HET mice despite having lower break points for ethanol. © 2013 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

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

Prolyl hydroxylase domain 2 deficiency promotes skeletal muscle fiber-type transition via a calcineurin/NFATc1-dependent pathway.

PubMed

Shin, Junchul; Nunomiya, Aki; Kitajima, Yasuo; Dan, Takashi; Miyata, Toshio; Nagatomi, Ryoichi

2016-01-01

Hypoxia exposure is known to induce an alteration in skeletal muscle fiber-type distribution mediated by hypoxia-inducible factor (HIF)-α. The downstream pathway of HIF-α leading to fiber-type shift, however, has not been elucidated. The calcineurin pathway is one of the pathways responsible for slow muscle fiber transition. Because calcineurin pathway is activated by vascular endothelial growth factor (VEGF), one of the factors induced by HIF-1α, we hypothesized that the stabilization of HIF-1α may lead to slow muscle fiber transition via the activation of calcineurin pathway in skeletal muscles. To induce HIF-1α stabilization, we used a loss of function strategy to abrogate Prolyl hydroxylase domain protein (PHD) 2 responsible for HIF-1α hydroxylation making HIF-1α susceptible to ubiquitin dependent degradation by proteasome. The purpose of this study was therefore to examine the effect of HIF-1α stabilization in PHD2 conditional knockout mouse on skeletal muscle fiber-type transition and to elucidate the involvement of calcineurin pathway on muscle fiber-type transition. PHD2 deficiency resulted in an increased capillary density in skeletal muscles due to the induction of vascular endothelial growth factor. It also elicited an alteration of skeletal muscle phenotype toward the type I fibers in both of the soleus (35.8 % in the control mice vs. 46.7 % in the PHD2-deficient mice, p < 0.01) and the gastrocnemius muscle (0.94 vs. 1.89 %, p < 0.01), and the increased proportion of type I fibers appeared to correspond to the area of increased capillary density. In addition, calcineurin and nuclear factor of activated T cell (NFATc1) protein levels were increased in both the gastrocnemius and soleus muscles, suggesting that the calcineurin/NFATc1 pathway was responsible for the type I fiber transition regardless of PGC-1α, which responded minimally to PHD2 deficiency. Indeed, we found that tacrolimus (FK-506), a calcineurin inhibitor, successfully suppressed slow fiber-type formation in PHD2-deficient mice. Taken together, stabilized HIF-1α induced by PHD2 conditional knockout resulted in the transition of muscle fibers toward a slow fiber type via a calcineurin/NFATc1 signaling pathway. PHD2 conditional knockout mice may serve as a model for chronic HIF-1α stabilization as in mice exposed to low oxygen concentration.

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.

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.

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.

Elimination of Kalrn expression in POMC cells reduces anxiety-like behavior and contextual fear learning.

PubMed

Mandela, Prashant; Yan, Yan; LaRese, Taylor; Eipper, Betty A; Mains, Richard E

2014-07-01

Kalirin, a Rho GDP/GTP exchange factor for Rac1 and RhoG, is known to play an essential role in the formation and maintenance of excitatory synapses and in the secretion of neuropeptides. Mice unable to express any of the isoforms of Kalrn in cells that produce POMC at any time during development (POMC cells) exhibited reduced anxiety-like behavior and reduced acquisition of passive avoidance behavior, along with sex-specific alteration in the corticosterone response to restraint stress. Strikingly, lack of Kalrn expression in POMC cells closely mimicked the effects of global Kalrn knockout on anxiety-like behavior and passive avoidance conditioning without causing the other deficits noted in Kalrn knockout mice. Our data suggest that deficits in excitatory inputs onto POMC neurons are responsible for the behavioral phenotypes observed. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Brain and muscle Arnt-like protein-1 (BMAL1), a component of the molecular clock, regulates adipogenesis

PubMed Central

Shimba, Shigeki; Ishii, Norimasa; Ohta, Yuki; Ohno, Toshiharu; Watabe, Yuichi; Hayashi, Mitsuaki; Wada, Taira; Aoyagi, Toshinori; Tezuka, Masakatsu

2005-01-01

Brain and muscle Arnt-like protein-1 (BMAL1; also known as MOP3 or Arnt3) is a transcription factor known to regulate circadian rhythm. Here, we established its involvement in the control of adipogenesis and lipid metabolism activity in mature adipocytes. During adipose differentiation in 3T3-L1 cells, the level of BMAL1 mRNA began to increase 4 days after induction and was highly expressed in differentiated cells. In white adipose tissues isolated from C57BL/6J mice, BMAL1 was predominantly expressed in a fraction containing adipocytes, as compared with the stromal-vascular fraction. BMAL1 knockout mice embryonic fibroblast cells failed to be differentiated into adipocytes. Importantly, adding BMAL1 back by adenovirus gene transfer restored the ability of BMAL1 knockout mice embryonic fibroblast cells to differentiate. Knock-down of BMAL1 expression in 3T3-L1 cells by an RNA interference technique allowed the cells to accumulate only minimum amounts of lipid droplets in the cells. Adenovirus-mediated expression of BMAL1 in 3T3-L1 adipocytes resulted in induction of several factors involved in lipogenesis. The promoter activity of these genes was stimulated in a BMAL1-dependent manner. Interestingly, expression of these factors showed clear circadian rhythm in mice adipose tissue. Furthermore, overexpression of BMAL1 in adipocytes increased lipid synthesis activity. These results indicate that BMAL1, a master regulator of circadian rhythm, also plays important roles in the regulation of adipose differentiation and lipogenesis in mature adipocytes. PMID:16093318

Long-term deficiency of circulating and hippocampal insulin-like growth factor I induces depressive behavior in adult mice: A potential model of geriatric depression

PubMed Central

Mitschelen, Matthew; Yan, Han; Farley, Julie A.; Warrington, Junie P.; Han, Song; Hereñú, Claudia B.; Csiszar, Anna; Ungvari, Zoltan; Bailey-Downs, Lora C.; Bass, Caroline E.; Sonntag, William E.

2011-01-01

Numerous studies support the hypothesis that deficiency of insulin-like growth factor I (IGF-1) in adults contributes to depression, but direct evidence is limited. Many psychological and pro-cognitive effects have been attributed to IGF-1, but appropriate animal models of adult-onset IGF-1 deficiency are lacking. In this study, we use a viral-mediated Cre-loxP system to knockout the Igf1 gene in either the liver, neurons of the CA1 region of the hippocampus, or both. Knockout of liver Igf1 reduced serum IGF-1 levels by 40% and hippocampal IGF-1 levels by 26%. Knockout of Igf1 in CA1 reduced hippocampal IGF-1 levels by 13%. The most severe reduction in hippocampal IGF-1 occurred in the group with knockouts in both liver and CA1 (36% reduction), and was associated with a 3.5-fold increase in immobility in the forced swim test. Reduction of either circulating or hippocampal IGF-1 levels did not alter anxiety measured in an open field and elevated plus maze, nor locomotion in the open field. Furthermore, local compensation for deficiencies in circulating IGF-1 did not occur in the hippocampus, nor were serum levels of IGF-1 upregulated in response to the moderate decline of hippocampal IGF-1 caused by the knockouts in CA1. We conclude that adult-onset IGF-1 deficiency alone is sufficient to induce a depressive phenotype in mice. Furthermore, our results suggest that individuals with low brain levels of IGF-1 are at increased risk for depression and these behavioral effects are not ameliorated by increased local IGF-1 production or transport. Our study supports the hypothesis that the natural IGF-1 decline in aging humans may contribute to geriatric depression. PMID:21524689

Novel Insights into the Genetic Controls of Primitive and Definitive Hematopoiesis from Zebrafish Models

PubMed Central

Sood, Raman; Liu, Paul

2012-01-01

Hematopoiesis is a dynamic process where initiation and maintenance of hematopoietic stem cells, as well as their differentiation into erythroid, myeloid and lymphoid lineages, are tightly regulated by a network of transcription factors. Understanding the genetic controls of hematopoiesis is crucial as perturbations in hematopoiesis lead to diseases such as anemia, thrombocytopenia, or cancers, including leukemias and lymphomas. Animal models, particularly conventional and conditional knockout mice, have played major roles in our understanding of the genetic controls of hematopoiesis. However, knockout mice for most of the hematopoietic transcription factors are embryonic lethal, thus precluding the analysis of their roles during the transition from embryonic to adult hematopoiesis. Zebrafish are an ideal model organism to determine the function of a gene during embryonic-to-adult transition of hematopoiesis since bloodless zebrafish embryos can develop normally into early larval stage by obtaining oxygen through diffusion. In this review, we discuss the current status of the ontogeny and regulation of hematopoiesis in zebrafish. By providing specific examples of zebrafish morphants and mutants, we have highlighted the contributions of the zebrafish model to our overall understanding of the roles of transcription factors in regulation of primitive and definitive hematopoiesis. PMID:22888355

Single allele Lmbrd1 knockout results in cardiac hypertrophy.

PubMed

Tseng, Linda Tzu-Ling; Lin, Chieh-Liang; Pan, Kuei-Hsiang; Tzen, Kai-Yuan; Su, Ming-Jai; Tsai, Chia-Ti; Li, Yi-Han; Li, Pai-Chi; Chiang, Fu-Tien; Chang, Shin C; Chang, Ming-Fu

2018-06-01

LMBD1 protein, a type IV-B plasma membrane protein possessing nine putative trans-membrane domains, was previously demonstrated at cellular level to play a critical part in the signaling cascade of insulin receptor through its involvement in regulating clathrin-mediated endocytosis. However, at physiological level, the significance of LMBD1 protein in cardiac development remains unclear. To understand the role of Lmbrd1 gene involved in the cardiac function, heterozygous knockout mice were used as an animal model system. The pathological outcomes were analyzed by micro-positron emission tomography, ECG acquisition, cardiac ultrasound, and immunohistochemistry. By studying the heterozygous knockout of Lmbrd1 (Lmbrd1 +/- ), we discovered that lack of Lmbrd1 not only resulted in the increase of cardiac-glucose uptake, pathological consequences were also observed. Here, we have distinguished that Lmbrd1 +/- is sufficient in causing cardiac diseases through a pathway independent of the recessive vitamin B 12 cblF cobalamin transport defect. Lmbrd1 +/- mice exhibited an increase in myocardial glucose uptake and insulin receptor signaling that is insensitive to the administration of additional insulin. Pathological symptoms such as cardiac hypertrophy, ventricular tissue fibrosis, along with the increase of heart rate and cardiac muscle contractility were observed. As Lmbrd1 +/- mice aged, the decrease in ejection fraction and fraction shortening showed signs of ventricular function deterioration. The results suggested that Lmbrd1 gene not only plays a significant role in mediating the energy homeostasis in cardiac tissue, it may also be a key factor in the regulation of cardiac function in mice. Copyright © 2017. Published by Elsevier B.V.

Beijing ambient particle exposure accelerates atherosclerosis in ApoE knockout mice.

PubMed

Chen, Tian; Jia, Guang; Wei, Yongjie; Li, Jiucun

2013-11-25

Air pollution is associated with significant adverse health effects including increased cardiovascular morbidity and mortality. However research on the cardiovascular effect of "real-world" exposure to ambient particulate matter (PM) in susceptible animal model is very limited. In this study, we aimed to investigate the association between Beijing ambient particle exposure and the atherosclerosis development in the apolipoprotein E knockout mice (ApoE(-/-) mice). Two parallel exposure chambers were used for whole body exposure among ApoE knockout mice. One of the chambers was supplied with untreated ambient air (PM group) and the other chamber was treated with ambient air filtered by high-efficiency particulate air (HEPA) filter (FA group). Twenty mice were divided into two groups and exposed to ambient PM (n=10 for PM group) or filtered air (n=10 for FA group) for two months from January 18th to March 18th, 2010. During the exposure, the mass concentrations of PM2.5 and PM10 in the two chambers were continuously monitored. Additionally, a receptor source apportionment model of chemical mass balance using 19 organic tracers was applied to determine the contributions of sources on the PM2.5 in terms of natural gas, diesel vehicle, gasoline vehicle, coal burning, vegetable debris, biomass burning and cooking. At the end of the two-month exposure, biomarkers of oxidative stress, inflammation and lipid metabolism in bronchoalveolar lavage fluid (BAL) and blood samples were determined and the plaque area on the aortic endothelium was quantified. In the experiment, the concentrations of PM10 and PM2.5 in PM chamber were 99.45μg/m(3) and 61.0μg/m(3) respectively, while PM2.5 in FA chamber was 17.6μg/m(3). Source apportionment analysis by organic tracers showed that gasoline vehicle (39.9%) and coal burning (24.3%) emission were the two major sources contributing to the mass concentration of PM2.5 in Beijing. Among the ApoE knockout mice, the PM group were significantly higher than the FA group in terms of serum total cholesterol, low-density lipoprotein, tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein as well as TNF-alpha and interleukin-6 in BAL. Also the total antioxidant capacity and oxidized low-density lipoprotein were significantly different between the two groups. In addition, pathological analysis of aortic arch reveals that the plaques area in the PM group increased significantly compared to the FA group. Our results demonstrated that ambient PM exposure could induce considerable oxidative stress and systemic inflammation in ApoE knockout mice and contribute to the progression of atherosclerosis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Substance P increases liver fibrosis by differential changes in senescence of cholangiocytes and hepatic stellate cells.

PubMed

Wan, Ying; Meng, Fanyin; Wu, Nan; Zhou, Tianhao; Venter, Julie; Francis, Heather; Kennedy, Lindsey; Glaser, Trenton; Bernuzzi, Francesca; Invernizzi, Pietro; Glaser, Shannon; Huang, Qiaobing; Alpini, Gianfranco

2017-08-01

Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct-ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin-1 receptor (NK-1R). To identify whether SP regulates liver fibrosis during cholestasis, wild-type or NK-1R knockout (NK-1R -/- ) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2 -/- ) mice treated with either an NK-1R antagonist (L-733,060) or saline were used. Additionally, wild-type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK-1R in both BDL and Mdr2 -/- mice compared to wild-type mice. Expression of tachykinin precursor 1 and NK-1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK-1R decreased BDL-induced liver fibrosis, and treatment with L-733,060 resulted in decreased liver fibrosis in Mdr2 -/- mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor-β1 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK-1R -/- mice with BDL surgery or Mdr2 -/- mice treated with L-733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L-733,060 inhibited SP-induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L-733,060 partially reversed the SP-induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP-induced increase of senescence-related gene expression in cultured cholangiocytes. Collectively, our results demonstrate the regulatory effects of the SP/NK-1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury. (Hepatology 2017;66:528-541). © 2017 by the American Association for the Study of Liver Diseases. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Upregulation of Atrogin-1/FBXO32 is not necessary for cartilage destruction in mouse models of osteoarthritis.

PubMed

Kim, H-E; Rhee, J; Park, S; Yang, J; Chun, J-S

2017-03-01

In a preliminary study, we found that recently identified catabolic regulators of osteoarthritis (OA), including hypoxia-inducible factor (HIF)-2α and members of the zinc-ZIP8-MTF1 axis, upregulate the E3 ubiquitin ligase, Atrogin-1 (encoded by Fbxo32), in chondrocytes. As the ubiquitination/proteasomal degradation pathways are tightly regulated to modulate the expression of catabolic factors in chondrocytes, we examined the in vivo functions of Atrogin-1 in mouse models of OA. The mRNA and protein levels of Atrogin-1 and other regulators of OA were determined in primary cultured mouse chondrocytes, OA human cartilage, and OA cartilage from wild-type (WT) and Fbxo32-knockout (KO) mice subjected to destabilization of the medial meniscus or intra-articular (IA) injection of adenoviruses expressing HIF-2α (Ad-Epas1), ZIP8 (Ad-Zip8), or Atrogin-1 (Ad-Fbxo32). The effect of Atrogin-1 overexpression on the cartilage of WT mice was examined by IA injection of Ad-Fbxo32. Atrogin-1 mRNA levels in chondrocytes were markedly increased by treatment with interleukin-1β, HIF-2α, and members of the zinc-ZIP8-MTF1 axis. Atrogin-1 protein levels were also increased in OA cartilage from humans and various mouse OA models. However, the forced overexpression of Atrogin-1 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrix-degrading enzymes. Moreover, overexpression of Atrogin-1 in the mouse joint tissues failed to cause OA pathogenesis, and Fbxo32 knockout failed to affect post-traumatic OA cartilage destruction in mice. Although Atrogin-1 is upregulated in OA cartilage, overexpression of Atrogin-1 in the joint tissues or knockout of Fbxo32 does not affect OA cartilage destruction in mice. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Genetic Ablation of CCAAT/Enhancer Binding Protein α in Epidermis Reveals Its Role in Suppression of Epithelial Tumorigenesis

PubMed Central

Loomis, Kari D.; Zhu, Songyun; Yoon, Kyungsil; Johnson, Peter F.; Smart, Robert C.

2013-01-01

CCAAT/enhancer binding protein y (C/EBPα) is a basic leucine zipper transcription factor that inhibits cell cycle progression and regulates differentiation in various cell types. C/EBPα is inactivated by mutation in acute myeloid leukemia (AML) and is considered a human tumor suppressor in AML. Although C/EBPα mutations have not been observed in malignancies other than AML, greatly diminished expression of C/EBPα occurs in numerous human epithelial cancers including lung, liver, endometrial, skin, and breast, suggesting a possible tumor suppressor function. However, direct evidence for C/EBPα as an epithelial tumor suppressor is lacking due to the absence of C/EBPα mutations in epithelial tumors and the lethal effect of C/EBPα deletion in mouse model systems. To examine the function of C/EBPα in epithelial tumor development, an epidermal-specific C/EBPα knockout mouse was generated. The epidermal-specific C/EBPα knockout mice survived and displayed no detectable abnormalities in epidermal keratinocyte proliferation, differentiation, or apoptosis, showing that C/EBPα is dispensable for normal epidermal homeostasis. In spite of this, the epidermal-specific C/EBPα knockout mice were highly susceptible to skin tumor development involving oncogenic Ras. These mice displayed decreased tumor latency and striking increases in tumor incidence, multiplicity, growth rate, and the rate of malignant progression. Mice hemizygous for C/EBPα displayed an intermediate-enhanced tumor phenotype. Our results suggest that decreased expression of C/EBPα contributes to deregulation of tumor cell proliferation. C/EBPα had been proposed to block cell cycle progression through inhibition of E2F activity. We observed that C/EBPα blocked Ras-induced and epidermal growth factor-induced E2F activity in keratinocytes and also blocked Ras-induced cell transformation and cell cycle progression. Our study shows that C/EBPα is dispensable for epidermal homeostasis and provides genetic evidence that C/EBPα is a suppressor of epithelial tumorigenesis. PMID:17638888

Stress-induced neuroinflammation is mediated by GSK3-dependent TLR4 signaling that promotes susceptibility to depression-like behavior

PubMed Central

Cheng, Yuyan; Pardo, Marta; de Souza Armini, Rubia; Martinez, Ana; Mouhsine, Hadley; Zagury, Jean-Francois; Jope, Richard S.; Beurel, Eleonore

2016-01-01

Most psychiatric and neurological diseases are exacerbated by stress. Because this may partially result from stress-induced inflammation, we examined factors involved in this stress response. After a paradigm of inescapable foot shock stress that causes learned helplessness depression-like behavior, eighteen cytokines and chemokines increased in mouse hippocampus, peaking 6 to 12 hr after stress. A 24 hr prior pre-conditioning stress accelerated the rate of stress-induced hippocampal cytokine and chemokine increases, with most reaching peak levels after 1 to 3 hr, often without altering the maximal levels. Toll-like receptor 4 (TLR4) was involved in this response because most stress-induced hippocampal cytokines and chemokines were attenuated in TLR4 knockout mice. Stress activated glycogen synthase kinase-3 (GSK3) in wild-type mouse hippocampus, but not in TLR4 knockout mice. Administration of the antidepressant fluoxetine or the GSK3 inhibitor TDZD-8 reduced the stress-induced increases of most hippocampal cytokines and chemokines. Stress increased hippocampal levels of the danger-associated molecular pattern (DAMP) protein high mobility group box 1 (HMGB1), activated the inflammatory transcription factor NF-κB, and the NLRP3 inflammasome. Knockdown of HMGB1 blocked the acceleration of cytokine and chemokine increases in the hippocampus caused by two successive stresses. Fluoxetine treatment blocked stress-induced up-regulation of HMGB1 and subsequent NF-κB activation, whereas TDZD-8 administration attenuated NF-κB activation downstream of HMGB1. To test if stress-induced cytokines and chemokines contribute to depression-like behavior, the learned helplessness model was assessed. Antagonism of TNFα modestly reduced susceptibility to learned helplessness induction, whereas TLR4 knockout mice were resistant to learned helplessness. Thus, stress-induces a broad inflammatory response in mouse hippocampus that involves TLR4, GSK3, and downstream inflammatory signaling, and these stress responses contribute to susceptibility to depression-like behavior in mice. PMID:26772151

Stress-induced neuroinflammation is mediated by GSK3-dependent TLR4 signaling that promotes susceptibility to depression-like behavior.

PubMed

Cheng, Yuyan; Pardo, Marta; Armini, Rubia de Souza; Martinez, Ana; Mouhsine, Hadley; Zagury, Jean-Francois; Jope, Richard S; Beurel, Eleonore

2016-03-01

Most psychiatric and neurological diseases are exacerbated by stress. Because this may partially result from stress-induced inflammation, we examined factors involved in this stress response. After a paradigm of inescapable foot shock stress that causes learned helplessness depression-like behavior, eighteen cytokines and chemokines increased in mouse hippocampus, peaking 6-12h after stress. A 24h prior pre-conditioning stress accelerated the rate of stress-induced hippocampal cytokine and chemokine increases, with most reaching peak levels after 1-3h, often without altering the maximal levels. Toll-like receptor 4 (TLR4) was involved in this response because most stress-induced hippocampal cytokines and chemokines were attenuated in TLR4 knockout mice. Stress activated glycogen synthase kinase-3 (GSK3) in wild-type mouse hippocampus, but not in TLR4 knockout mice. Administration of the antidepressant fluoxetine or the GSK3 inhibitor TDZD-8 reduced the stress-induced increases of most hippocampal cytokines and chemokines. Stress increased hippocampal levels of the danger-associated molecular pattern (DAMP) protein high mobility group box 1 (HMGB1), activated the inflammatory transcription factor NF-κB, and the NLRP3 inflammasome. Knockdown of HMGB1 blocked the acceleration of cytokine and chemokine increases in the hippocampus caused by two successive stresses. Fluoxetine treatment blocked stress-induced up-regulation of HMGB1 and subsequent NF-κB activation, whereas TDZD-8 administration attenuated NF-κB activation downstream of HMGB1. To test if stress-induced cytokines and chemokines contribute to depression-like behavior, the learned helplessness model was assessed. Antagonism of TNFα modestly reduced susceptibility to learned helplessness induction, whereas TLR4 knockout mice were resistant to learned helplessness. Thus, stress-induces a broad inflammatory response in mouse hippocampus that involves TLR4, GSK3, and downstream inflammatory signaling, and these stress responses contribute to susceptibility to depression-like behavior in mice. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of Mom12 and Mom13, two novel modifier loci of Apc (Min) -mediated intestinal tumorigenesis.

PubMed

Crist, Richard C; Roth, Jacquelyn J; Lisanti, Michael P; Siracusa, Linda D; Buchberg, Arthur M

2011-04-01

Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) Apc (Min/+) mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. Apc (Min/+) mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The Apc (Min) model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter Apc (Min) -mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 Apc (Min/+) mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than Apc (Min/+) controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: 1) Mom12, a dominant modifier linked to the congenic region on chromosome 6, and 2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines.

Voluntary exercise prevents the obese and diabetic metabolic syndrome of the melanocortin-4 receptor knockout mouse.

PubMed

Haskell-Luevano, Carrie; Schaub, Jay W; Andreasen, Amy; Haskell, Kim R; Moore, Marcus C; Koerper, Lorraine M; Rouzaud, Francois; Baker, Henry V; Millard, William J; Walter, Glenn; Litherland, S A; Xiang, Zhimin

2009-02-01

Exercise is a mechanism for maintenance of body weight in humans. Morbidly obese human patients have been shown to possess single nucleotide polymorphisms in the melanocortin-4 receptor (MC4R). MC4R knockout mice have been well characterized as a genetic model that possesses phenotypic metabolic disorders, including obesity, hyperphagia, hyperinsulinemia, and hyperleptinemia, similar to those observed in humans possessing dysfunctional hMC4Rs. Using this model, we examined the effect of voluntary exercise of MC4R knockout mice that were allowed access to a running wheel for a duration of 8 wk. Physiological parameters that were measured included body weight, body composition of fat and lean mass, food consumption, body length, and blood levels of cholesterol and nonfasted glucose, insulin, and leptin. At the termination of the experiment, hypothalamic mRNA expression levels of neuropeptide Y (NPY), agouti-related protein (AGRP), proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), orexin, brain-derived neurotropic factor (BDNF), phosphatase with tensin homology (Pten), melanocortin-3 receptor (MC3R), and NPY-Y1R were determined. In addition, islet cell distribution and function in the pancreas were examined. In the exercising MC4R knockout mice, the pancreatic islet cell morphology and other physiological parameters resembled those observed in the wild-type littermate controls. Gene expression profiles identified exercise as having a significant effect on hypothalamic POMC, orexin, and MC3R levels. Genotype had a significant effect on AGRP, POMC, CART, and NPY-Y1R, with an exercise and genotype interaction effect on NPY gene expression. These data support the hypothesis that voluntary exercise can prevent the genetic predisposition of melanocortin-4 receptor-associated obesity and diabetes.

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.

Myostatin signaling regulates Akt activity via the regulation of miR-486 expression.

PubMed

Hitachi, Keisuke; Nakatani, Masashi; Tsuchida, Kunihiro

2014-02-01

Myostatin, also known as growth and differentiation factor-8, is a pivotal negative regulator of skeletal muscle mass and reduces muscle protein synthesis by inhibiting the insulin-like growth factor-1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. However, the precise mechanism by which myostatin inhibits the IGF-1/Akt/mTOR pathway remains unclear. In this study, we investigated the global microRNA expression profile in myostatin knockout mice and identified miR-486, a positive regulator of the IGF-1/Akt pathway, as a novel target of myostatin signaling. In myostatin knockout mice, the expression level of miR-486 in skeletal muscle was significantly increased. In addition, we observed increased expression of the primary transcript of miR-486 (pri-miR-486) and Ankyrin 1.5 (Ank1.5), the host gene of miR-486, in myostatin knockout mice. In C2C12 cells, myostatin negatively regulated the expression of Ank1.5. Moreover, canonical myostatin signaling repressed the skeletal muscle-specific promoter activity of miR-486/Ank1.5. This repression was partially mediated by the E-box elements in the proximal region of the promoter. We also show that overexpression of miR-486 induced myotube hypertrophy in vitro and that miR-486 was essential to maintain skeletal muscle size both in vitro and in vivo. In addition, inhibition of miR-486 led to a decrease in Akt activity in C2C12 myotubes. Our findings indicate that miR-486 is one of the intermediary molecules connecting myostatin signaling and the IGF-1/Akt/mTOR pathway in the regulation of skeletal muscle size. Copyright © 2013 Elsevier Ltd. 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.

Hypolocomotion, anxiety and serotonin syndrome-like behavior contribute to the complex phenotype of serotonin transporter knockout mice.

PubMed

Kalueff, A V; Fox, M A; Gallagher, P S; Murphy, D L

2007-06-01

Although mice with a targeted disruption of the serotonin transporter (SERT) have been studied extensively using various tests, their complex behavioral phenotype is not yet fully understood. Here we assess in detail the behavior of adult female SERT wild type (+/+), heterozygous (+/-) and knockout (-/-) mice on an isogenic C57BL/6J background subjected to a battery of behavioral paradigms. Overall, there were no differences in the ability to find food or a novel object, nest-building, self-grooming and its sequencing, and horizontal rod balancing, indicating unimpaired sensory functions, motor co-ordination and behavioral sequencing. In contrast, there were striking reductions in exploration and activity in novelty-based tests (novel object, sticky label and open field tests), accompanied by pronounced thigmotaxis, suggesting that combined hypolocomotion and anxiety (rather than purely anxiety) influence the SERT -/- behavioral phenotype. Social interaction behaviors were also markedly reduced. In addition, SERT -/- mice tended to move close to the ground, frequently displayed spontaneous Straub tail, tics, tremor and backward gait - a phenotype generally consistent with 'serotonin syndrome'-like behavior. In line with replicated evidence of much enhanced serotonin availability in SERT -/- mice, this serotonin syndrome-like state may represent a third factor contributing to their behavioral profile. An understanding of the emerging complexity of SERT -/- mouse behavior is crucial for a detailed dissection of their phenotype and for developing further neurobehavioral models using these mice.

Effect of Shenxinning decoction on ventricular remodeling in AT1 receptor-knockout mice with chronic renal insufficiency.

PubMed

Yang, Xuejun; Zhou, Hua; Qu, Huiyan; Liu, Weifang; Huang, Xiaojin; Shun, Yating; He, Liqun

2014-01-01

To observe the efficacy of Shenxinning Decoction (SXND) in ventricular remodeling in AT1 receptor-knockout (AT1-KO) mice with chronic renal insufficiency (CRI). AT1-KO mice modeled with subtotal (5/6) nephrectomy were intervened with SXND for 12 weeks. Subsequently, blood urea nitrogen (BUN), serum creatinine (SCr), brain natriuretic peptide (BNP), echocardiography (left ventricular end-diastolic diameter, LVDD; left ventricular end-systolic diameter, LVDS; fractional shortening, FS; and ejection fraction, EF), collagen types I and III in the heart and kidney, myocardial mitochondria, and cardiac transforming growth factor-β1 (TGF-β1) of the AT1-KO mice were compared with the same model with nephrectomy only and untreated with SXND. AT1-KO mice did not affect the process of CRI but it could significantly affect cardiac remodeling process. SXND decreased to some extent the AT1-KO mice's BUN, SCr, BNP, and cardiac LVDD, LVDS, and BNP, improved FS and EF, lowered the expression of collagen type I and III in heart and kidney, increased the quantity of mitochondria and ameliorated their structure, and down-regulated the expression of TGF-β1. SXND may antagonize the renin-angiotensin system (RAS) and decrease uremia toxins, thereby ameliorating ventricular remodeling in CRI. Furthermore, SXND has a mechanism correlated with the improvement of myocardial energy metabolism and the down-regulation of TGF-β1.

Hepcidin regulation in wild-type and Hfe knockout mice in response to alcohol consumption: evidence for an alcohol-induced hypoxic response.

PubMed

Heritage, Mandy L; Murphy, Therese L; Bridle, Kim R; Anderson, Gregory J; Crawford, Darrell H G; Fletcher, Linda M

2009-08-01

Expression of Hamp1, the gene encoding the iron regulatory peptide hepcidin, is inappropriately low in HFE-associated hereditary hemochromatosis and Hfe knockout mice (Hfe(-/-)). Since chronic alcohol consumption is also associated with disturbances in iron metabolism, we investigated the effects of alcohol consumption on hepcidin mRNA expression in Hfe(-/-) mice. Hfe(-/-) and C57BL/6 (wild-type) mice were pair-fed either an alcohol liquid diet or control diet for up to 8 weeks. The mRNA levels of hepcidin and ferroportin were measured at the mRNA level by RT-PCR and protein expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was measured by western blot. Hamp1 mRNA expression was significantly decreased and duodenal ferroportin expression was increased in alcohol-fed wild-type mice at 8 weeks. Time course experiments showed that the decrease in hepcidin mRNA was not immediate, but was significant by 4 weeks. Consistent with the genetic defect, Hamp1 mRNA was decreased and duodenal ferroportin mRNA expression was increased in Hfe(-/-) mice fed on the control diet compared with wild-type animals and alcohol further exacerbated these effects. HIF-1alpha protein levels were elevated in alcohol-fed wild-type animals compared with controls. Alcohol may decrease Hamp1 gene expression independently of the HFE pathway possibly via alcohol-induced hypoxia.

The Tumor Suppressor Gene, RASSF1A, Is Essential for Protection against Inflammation -Induced Injury

PubMed Central

Fiteih, Yahya; Law, Jennifer; Volodko, Natalia; Mohamed, Anwar; El-Kadi, Ayman O. S.; Liu, Lei; Odenbach, Jeff; Thiesen, Aducio; Onyskiw, Christina; Ghazaleh, Haya Abu; Park, Jikyoung; Lee, Sean Bong; Yu, Victor C.; Fernandez-Patron, Carlos; Alexander, R. Todd; Wine, Eytan; Baksh, Shairaz

2013-01-01

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a+/−, Rassf1a−/− and an intestinal epithelial cell specific knockout mouse (Rassf1a IEC-KO) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a−/− mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a−/− background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a−/− mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis. PMID:24146755

Age-Dependent Deficits in Fear Learning in Heterozygous BDNF Knock-Out Mice

ERIC Educational Resources Information Center

Endres, Thomas; Lessmann, Volkmar

2012-01-01

Beyond its trophic function, the neurotrophin BDNF (brain-derived neurotrophic factor) is well known to crucially mediate synaptic plasticity and memory formation. Whereas recent studies suggested that acute BDNF/TrkB signaling regulates amygdala-dependent fear learning, no impairments of cued fear learning were reported in heterozygous BDNF…

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

Internal Pudental Artery Dysfunction in Diabetes Mellitus Is Mediated by NOX1-Derived ROS-, Nrf2-, and Rho Kinase-Dependent Mechanisms.

PubMed

Alves-Lopes, Rhéure; Neves, Karla B; Montezano, Augusto C; Harvey, Adam; Carneiro, Fernando S; Touyz, Rhian M; Tostes, Rita C

2016-10-01

Oxidative stress plays an important role in diabetes mellitus (DM)-associated vascular injury. DM is an important risk factor for erectile dysfunction. Functional and structural changes in internal pudendal arteries (IPA) can lead to erectile dysfunction. We hypothesized that downregulation of nuclear factor E2-related factor 2 (Nrf2), consequent to increased nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1)-derived reactive oxygen species (ROS), impairs IPA function in DM. IPA and vascular smooth muscle cells from C57BL/6 (control) and NOX1 knockout mice were used. DM was induced by streptozotocin in C57BL/6 mice. Functional properties of IPA were assessed using a myograph, protein expression and peroxiredoxin oxidation by Western blot, RNA expression by polymerase chain reaction, carbonylation by oxyblot assay, ROS generation by lucigenin, nitrotyrosine, and amplex red, and Rho kinase activity and nuclear accumulation of Nrf2 by ELISA. IPA from diabetic mice displayed increased contractions to phenylephrine (control 138.5±9.5 versus DM 191.8±15.5). ROS scavenger, Nrf2 activator, NOX1 and Rho kinase inhibitors normalized vascular function. High glucose increased ROS generation in IPA vascular smooth muscle cell. This effect was abrogated by Nrf2 activation and not observed in NOX1 knockout vascular smooth muscle cell. High glucose also increased levels of nitrotyrosine, protein oxidation/carbonylation, and Rho kinase activity, but reduced Nrf2 activity and expression of Nrf2-regulated genes (catalase [25.6±0.05%], heme oxygenase-1 [21±0.1%], and quinone oxidoreductase 1 [22±0.1%]) and hydrogen peroxide levels. These effects were not observed in vascular smooth muscle cell from NOX1 knockout mice. In these cells, high glucose increased hydrogen peroxide levels. In conclusion, Rho kinase activation, via NOX1-derived ROS and downregulation of Nrf2 system, impairs IPA function in DM. These data suggest that Nrf2 is vasoprotective in DM-associated erectile dysfunction. © 2016 American Heart Association, Inc.

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.

Host resistance of CD18 knockout mice against systemic infection with Listeria monocytogenes

NASA Technical Reports Server (NTRS)

Wu, Huaizhu; Prince, Joseph E.; Brayton, Cory F.; Shah, Chirayu; Zeve, Daniel; Gregory, Stephen H.; Smith, C. Wayne; Ballantyne, Christie M.

2003-01-01

Mice with targeted mutations of CD18, the common beta2 subunit of CD11/CD18 integrins, have leukocytosis, impaired transendothelial neutrophil emigration, and reduced host defense to Streptococcus pneumoniae, a gram-positive extracellular bacterium. Previous studies using blocking monoclonal antibodies suggested roles for CD18 and CD11b in hepatic neutrophil recruitment and host innate response to Listeria monocytogenes, a gram-positive intracellular bacterium. We induced systemic listeriosis in CD18 knockout (CD18-ko) and wild-type (WT) mice by tail vein injection with Listeria. By 14 days postinjection (dpi), 8 of 10 WT mice died, compared with 2 of 10 CD18-ko mice (P < 0.01). Quantitative organ culture showed that numbers of Listeria organisms in livers and spleens were similar in both groups at 20 min postinfection. By 3, 5, and 7 dpi, however, numbers of Listeria organisms were significantly lower in livers and spleens of CD18-ko mice than in WT mice. Histopathology showed that following Listeria infection, CD18-ko mice had milder inflammatory and necrotizing lesions in both spleens and livers than did WT mice. Cytokine assays indicated that baseline interleukin-1beta and granulocyte colony-stimulating factor (G-CSF) levels were higher in CD18-ko mice than in WT mice and that CD18-ko splenocytes produced higher levels of interleukin-1beta and G-CSF than WT splenocytes under the same amount of Listeria stimulation. These findings show that CD18 is not an absolute requirement for antilisterial innate immunity or hepatic neutrophil recruitment. We propose that the absence of CD18 in the mice results in the priming of innate immunity, as evidenced by elevated cytokine expression, and neutrophilic leukocytosis, which augments antilisterial defense.

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

Mechanism of Hyperphagia Contributing to Obesity in Brain-Derived Neurotrophic Factor Knockout Mice

PubMed Central

Fox, Edward A.; Biddinger, Jessica E.; Jones, Kevin R.; McAdams, Jennifer; Worman, Amber

2012-01-01

Global-heterozygous and brain-specific homozygous knockouts (KO's) of brain-derived neurotrophic factor (BDNF) cause late- and early-onset obesity, respectively, both involving hyperphagia. Little is known about the mechanism underlying this hyperphagia or whether BDNF loss from peripheral tissues could contribute to overeating. Since global-homozygous BDNF-KO is perinatal lethal, a BDNF-KO that spared sufficient brainstem BDNF to support normal health was utilized to begin to address these issues. Meal pattern and microstructure analyses suggested overeating of BDNF-KO mice was mediated by deficits in both satiation and satiety that resulted in increased meal size and frequency and implicated a reduction of vagal signaling from gut-to-brain. Meal-induced c-Fos activation in the nucleus of the solitary tract, a more direct measure of vagal afferent signaling, however, was not decreased in BDNF-KO mice, and thus was not consistent with a vagal afferent role. Interestingly though, meal-induced c-Fos activation was increased in the dorsal vagal motor nucleus (DMV) of BDNF-KO mice. This could imply that augmentation of vago-vagal digestive reflexes occurred (e.g., accommodation), which would support increased meal size and possibly increased meal number by reducing the increase in intragastric pressure produced by a given amount of ingesta. Additionally, vagal sensory neuron number in BDNF-KO mice was altered in a manner consistent with the increased meal-induced activation of the DMV. These results suggest reduced BDNF causes satiety and satiation deficits that support hyperphagia, possibly involving augmentation of vago-vagal reflexes mediated by central pathways or vagal afferents regulated by BDNF levels. PMID:23069761

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.

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

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.

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.

Differential Requirements for c-Myc in Chronic Hematopoietic Hyperplasia and Acute Hematopoietic Malignancies in Pten-null Mice

PubMed Central

Zhang, Jun; Xiao, Yechen; Guo, Yinshi; Breslin, Peter; Zhang, Shubin; Wei, Wei; Zhang, Zhou; Zhang, Jiwang

2011-01-01

Myeloproliferative disorders (MPDs), lymphoproliferative disorders (LPDs), acute T-lymphocytic or myeloid leukemia and T-lymphocytic lymphoma were developed in inducible Pten-knockout (Pten−/−) mice. The appearance of these multiple diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously. To study whether Myc function is required for the development of these hematopoietic disorders in Pten−/− mice, we generated inducible Pten/Myc double-knockout mice (Pten−/−/Myc−/−). By comparing the hematopoietic phenotypes of these double-knockout mice with those of Pten−/− mice, we found that both sets of animals developed MPDs and LPDs. However, none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPDs which developed in Pten−/− mice which are dominated by granulocytes, megakaryocytes predominate in the MPDs of Pten−/−/Myc−/− mice. Our study suggests that the deregulation of PI3K/Akt signaling in Pten−/− hematopoietic cells protects these cells from apoptotic cell death, resulting in chronic proliferative disorders. But due to the differential requirement for Myc in granulocyte as compared to megakaryocyte proliferation, Myc deletion converts Pten−/− MPDs from granulocyte-dominated to megakaryocyte-dominated conditions. Myc is absolutely required for the development of acute hematopoietic malignancies. PMID:21926961

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

PubMed Central

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

2008-01-01

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

Germinated Brown Rice Attenuates Atherosclerosis and Vascular Inflammation in Low-Density Lipoprotein Receptor-Knockout Mice.

PubMed

Zhao, Ruozhi; Ghazzawi, Nora; Wu, Jiansu; Le, Khuong; Li, Chunyang; Moghadasian, Mohammed H; Siow, Yaw L; Apea-Bah, Franklin B; Beta, Trust; Yin, Zhengfeng; Shen, Garry X

2018-05-02

The present study investigates the impact of germinated brown rice (GBR) on atherosclerosis and the underlying mechanism in low-density lipoprotein receptor-knockout (LDLr-KO) mice. The intensity of atherosclerosis in aortas of LDLr-KO mice receiving diet supplemented with 60% GBR (weight/weight) was significantly less than that in mice fed with 60% white rice (WR) or control diet ( p < 0.05); all diets contained 0.06% cholesterol. WR or GBR diet did not significantly alter plasma total or LDL-cholesterol, fecal sterols, or glucose, or the activities of antioxidant enzymes, compared to the control diet. The adhesion of monocytes to aortas from LDLr-KO mice fed with WR diet was significantly more than that from mice receiving the control diet ( p < 0.01). GBR diet decreased monocyte adhesion to aortas compared to WR diet ( p < 0.01). GBR diet also reduced the levels of plasminogen activator inhibitor-1 (PAI-1), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) in plasma, and the abundances of MCP-1, PAI-1, TNF-α, intracellular cell adhesion molecule-1, toll-like receptor-4, PAI-1, LDLr-like protein, and urokinase plasminogen activator and its receptor in aortas or hearts from LDLr-KO mice in comparison to the WR diet ( p < 0.05, 0.01, respectively). The findings suggest that GBR administration attenuated atherosclerosis and vascular inflammation in LDLr-KO mice compared to WR. The anti-atherosclerotic effect of GBR in LDLr-KO mice at least in part results from its anti-inflammatory activity.

Constitutive Knockout of Kalirin-7 Leads to Increased Rates of Cocaine Self-Administration

PubMed Central

Kiraly, Drew D.; Nemirovsky, Natali E.; LaRese, Taylor P.; Tomek, Seven E.; Yahn, Stephanie L.; Olive, M. Foster; Eipper, Betty A.

2013-01-01

Kalirin-7 (Kal7) is a Rho-guanine nucleotide exchange factor that is localized in neuronal postsynaptic densities. Kal7 interacts with the NR2B subunit of the NMDA receptor and regulates aspects of dendritic spine dynamics both in vitro and in vivo. Chronic treatment with cocaine increases dendritic spine density in the nucleus accumbens (NAc) of rodents and primates. Kal7 mRNA and protein are upregulated in the NAc following cocaine treatment, and the presence of Kal7 is necessary for the normal proliferation of dendritic spines following cocaine use. Mice that constitutively lack Kal7 [Kalirin-7 knockout mice (Kal7KO)] demonstrate increased locomotor sensitization to cocaine and a decreased place preference for cocaine. Here, using an intravenous cocaine self-administration paradigm, Kal7KO mice exhibit increased administration of cocaine at lower doses as compared with wild-type (Wt) mice. Analyses of mRNA transcript levels from the NAc of mice that self-administered saline or cocaine reveal that larger splice variants of the Kalrn gene are increased by cocaine more dramatically in Kal7KO mice than in Wt mice. Additionally, transcripts encoding the NR2B subunit of the NMDA receptor increased in Wt mice that self-administered cocaine but were unchanged in similarly experienced Kal7KO mice. These findings suggest that Kal7 participates in the reinforcing effects of cocaine, and that Kal7 and cocaine interact to alter the expression of genes related to critical glutamatergic signaling pathways in the NAc. PMID:23894151

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

SARM1 deletion restrains NAFLD induced by high fat diet (HFD) through reducing inflammation, oxidative stress and lipid accumulation.

PubMed

Pan, Zhen-Guo; An, Xu-Sheng

2018-04-06

SARM1 (Sterile alpha and armadillo motif-containing protein 1) is the recently identified TIR domain-containing cytosolic protein, which is involved in toll-like receptors (TLRs) signaling transduction. In the present study, the role of SARM1 in high fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) progression was explored. We found that SARM1 was expressed highly in fatty liver. And SARM1-knockout (KO) reduced steatohepatitis and metabolic disorders induced by HFD. SARM1-deletion decreased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in HFD-fed mice. Additionally, inflammatory response caused by HFD was alleviated by SARM1-deletion through inactivating TLR4/7/9 and nuclear factor kappa B (NF-κB) pathways. Of note, SARM1-deletion also reduced the expressions of inflammation-associated molecules in hypothalamus of HFD-fed mice. Furthermore, HFD administration led to oxidative stress in liver of mice, while being decreased in SARM1-KO mice. Moreover, SARM1-ablation improved lipid dyslipidemia by suppressing the mRNA levels of genes, linked to glycolysis, lipogenesis and transcriptional regulation. Insulin resistance was also attenuated by SARM1-deficiency through enhancing the activation of liver Akt/glycogen synthase kinase-3β (GSK3β) and insulin receptor substrate-1 (IRS1)/FOXO1 pathways in HFD-fed mice. Also, SARM1-knockout improved neuropeptide Y (NPY), Pro-Opiomelanocortins (POMC), Agouti-related Protein (AGRP) and Cocaine-and-Amphetamine Responsive Transcript 1 (CART1) expressions in hypothalamus of mice after HFD administration. In vitro, we found that the reduction of inflammatory response, oxidative stress and dyslipidemia induced by SARM1-knockout in primary hepatocytes after fructose stimulation was largely attributed to its suppression to TLR4/7/9. Together, the findings demonstrated that SARM1 might be an effective target for developing effective therapeutic strategies against NAFLD. Copyright © 2018. Published by Elsevier Inc.

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

PubMed Central

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

2015-01-01

Abstract 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 Ca2+ 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. Key points Acute inhibition of purinergic receptors with a selective P2X3 antagonist prevents transmission of information from taste buds to sensory nerves. The P2X3 antagonist has no effect on taste-evoked release of ATP, confirming the effect is postsynaptic. The results confirm previous results with P2X2/3 double knockout mice that ATP is required for transmission of all taste qualities, including sour and salty. Previously, ATP was confirmed to be required for bitter, sweet and umami tastes, but was questioned for salty and sour tastes due to pleomorphic deficits in the double knockout mice. The geniculate ganglion in mouse contains two populations of ganglion cells with different subunit composition of P2X2 and P2X3 receptors making them differently susceptible to pharmacological block and, presumably, desensitization. PMID:25524179

Salmonella infections in the absence of the major histocompatibility complex II

NASA Technical Reports Server (NTRS)

Chapes, S. K.; Beharka, A. A.; Spooner, B. S. (Principal Investigator)

1998-01-01

We examined the pathogenesis of the facultative intracellular bacterium, Salmonella typhimurium in MHCII-/-, C2D knock-out mice, and wild-type C57BL/6J mice. The MHCII knock-out shortened the kinetics of animal death and reduced the dose of S. typhimurium needed to kill mice. We measured the physiological and cytokine responses of both mouse strains after S. typhimurium injection. Animal weight loss, spleen weights, liver weights, thymus weights, and serum corticosterone concentrations were comparable after injection with several doses of bacteria. The only physiological differences observed between the two strains were observed 3 days after injection of the highest dose of bacteria tested. Serum concentrations of tumor necrosis factor alpha, interleukin-2, and interleukin-6 increased in a dose-dependent fashion irrespective of mouse MHCII expression. Therefore, even in the absence of MHCII, mice are able to mount relatively normal physiological and immunological responses. Consistent with these normal responses, an increased percentage of MHCII-/- mice, primed with a low dose of bacteria 13 days earlier, were able to survive a lethal challenge of Salmonella compared with unprimed controls. Lastly, C2D mice had significantly higher serum interleukin-10 concentrations than C57BL/6J mice 48 h after infection with all doses of S. typhimurium. C2D macrophages also secreted significantly more IL-10 and less NO and O2- after lipopolysaccharide or phorbol ester stimulation in vitro than wild-type macrophages.

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.

Knockout of the norepinephrine transporter and pharmacologically diverse antidepressants prevent behavioral and brain neurotrophin alterations in two chronic stress models of depression.

PubMed

Haenisch, Britta; Bilkei-Gorzo, Andras; Caron, Marc G; Bönisch, Heinz

2009-10-01

Diverse factors such as changes in neurotrophins and brain plasticity have been proposed to be involved in the actions of antidepressant drugs (ADs). However, in mouse models of depression based on chronic stress, it is still unclear whether simultaneous changes in behavior and neurotrophin expression occur and whether these changes can be corrected or prevented comparably by chronic administration of ADs or genetic manipulations that produce antidepressant-like effects such as the knockout of the norepinephrine transporter (NET) gene. Here we show that chronic restraint or social defeat stress induce comparable effects on behavior and changes in the expression of neurotrophins in depression-related brain regions. Chronic stress caused down-regulation of BDNF, nerve growth factor, and neurotrophin-3 in hippocampus and cerebral cortex and up-regulation of these targets in striatal regions. In wild-type mice, these effects could be prevented by concomitant chronic administration of five pharmacologically diverse ADs. In contrast, NET knock out (NETKO) mice were resistant to stress-induced depressive-like changes in behavior and brain neurotrophin expression. Thus, the resistance of the NETKO mice to the stress-induced depression-associated behaviors and biochemical changes highlight the importance of noradrenergic pathways in the maintenance of mood. In addition, these mice represent a useful model to study depression-resistant behaviors, and they might help to provide deeper insights into the identification of downstream targets involved in the mechanisms of antidepressants.

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.

Altered Morphology and Function of the Lacrimal Functional Unit in Protein Kinase Cα Knockout Mice

PubMed Central

Chen, Zhuo; Li, Zhijie; Basti, Surendra; Farley, William J.

2010-01-01

Purpose. Protein kinase C (PKC) α plays a major role in the parasympathetic neural stimulation of lacrimal gland (LG) secretion. It also has been reported to have antiapoptotic properties and to promote cell survival. Therefore, the hypothesis for the present study was that PKCα knockout (−/−) mice have impaired ocular surface–lacrimal gland signaling, rendering them susceptible to desiccating stress and impaired corneal epithelial wound healing. In this study, the lacrimal function unit (LFU) and the stressed wound-healing response were examined in PKCα−/− mice. Methods. In PKCα+/+ control mice and PKCα−/− mice, tear production, osmolarity, and clearance rate were evaluated before and after experimental desiccating stress. Histology and immunofluorescent staining of PKC and epidermal growth factor were performed in tissues of the LFU. Cornified envelope (CE) precursor protein expression and cell proliferation were evaluated. The time course of healing and degree of neutrophil infiltration was evaluated after corneal epithelial wounding. Results. Compared with the PKCα+/+ mice, the PKCα−/− mice were noted to have significantly increased lacrimal gland weight, with enlarged, carbohydrate-rich, PAS-positive acinar cells; increased corneal epithelia permeability, with reduced CE expression; and larger conjunctival epithelial goblet cells. The PKCα−/− mice showed more rapid corneal epithelial healing, with less neutrophil infiltration and fewer proliferating cells than did the PKCα+/+ mice. Conclusions. The PKCα−/− mice showed lower tear production, which appeared to be caused by impaired secretion by the LG and conjunctival goblet cells. Despite their altered tear dynamics, the PKCα−/− mice demonstrated more rapid corneal epithelial wound healing, perhaps due to decreased neutrophil infiltration. PMID:20505191

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.

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.

Contribution of Egr1/zif268 to Activity-Dependent Arc/Arg3.1 Transcription in the Dentate Gyrus and Area CA1 of the Hippocampus

PubMed Central

Penke, Zsuzsa; Chagneau, Carine; Laroche, Serge

2011-01-01

Egr1, a member of the Egr family of transcription factors, and Arc are immediate early genes known to play major roles in synaptic plasticity and memory. Despite evidence that Egr family members can control Arc transcriptional regulation, demonstration of a selective role of Egr1 alone is lacking. We investigated the extent to which activity-dependent Arc expression is dependent on Egr1 by analyzing Arc mRNA expression using fluorescence in situ hybridization in the dorsal dentate gyrus and CA1 of wild-type (WT) and Egr1 knockout mice. Following electroconvulsive shock, we found biphasic expression of Arc in area CA1 in mice, consisting in a rapid (30 min) and transient wave followed by a second late-phase of expression (8 h), and a single but prolonged wave of expression in the dentate gyrus. Egr1 deficiency abolished the latest, but not the early wave of Arc expression in CA1, and curtailed that of the dentate gyrus. Since the early wave of Arc expression was not affected in Egr1 mutant mice, we next analyzed behaviorally induced Arc expression patterns as an index of neural ensemble activation in the dentate gyrus and area CA1 of WT and Egr1 mutant mice. Spatial exploration of novel or familiar environments induced in mice a single early and transient wave of Arc expression in the dentate gyrus and area CA1, which were not affected in Egr1 mutant mice. Analyses of Arc-expressing cells revealed that exploration recruits similar size dentate gyrus and CA1 neural ensembles in WT and Egr1 knockout mice. These findings suggest that hippocampal neural ensembles are normally activated immediately following spatial exploration in Egr1 knockout mice, indicating normal hippocampal encoding of information. They also provide evidence that in condition of strong activation Egr1 alone can control late-phases of activity-dependent Arc transcription in the dentate gyrus and area CA1 of the hippocampus. PMID:21887136

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

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.

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.

High-fat diet-induced obesity and insulin resistance were ameliorated via enhanced fecal bile acid excretion in tumor necrosis factor-alpha receptor knockout mice.

PubMed

Yamato, Mayumi; Shiba, Takeshi; Ide, Tomomi; Seri, Naoko; Kudo, Wataru; Ando, Makoto; Yamada, Ken-ichi; Kinugawa, Shintaro; Tsutsui, Hiroyuki

2012-01-01

Tumor necrosis factor-α (TNF-α) is one of the main mediators of inflammatory response activated by fatty acids in obesity, and this signaling through TNF-α receptor (TNFR) is responsible for obesity-associated insulin resistance. Recently, TNF-α has shown to affect lipid metabolism including the regulation of lipase activity and bile acid synthesis. However, there is scanty in vivo evidence for the involvement of TNF-α in this process, and the mechanistic role of TNFR remains unclear. In this study, TNFR2 knockout mice (R2KO) and wild-type (WT) mice were fed commercial normal diet (ND) or high-fat diet (HFD) for 8 weeks. In R2KO/HFD mice, the increase in body weight and the accumulation of fat were significantly ameliorated compared with WT/HFD mice in association with the decrease in plasma total cholesterol (137.7±3.1 vs. 98.6±3.1 mg/dL, P<0.005), glucose (221.9±14.7 vs. 167.3±8.1 mg/dL, P<0.01), and insulin (5.1±0.3 vs. 3.4±0.3 ng/mL, P<0.05). Fecal excretion of lipid contents was significantly increased in R2KO mice. In R2KO/HFD mice, the decrease in hepatic cholesterol-7a-hydroxylase activity, the rate-limiting enzyme in bile acid synthesis, was inhibited (1.7±0.2 vs. 8.1±1.0 pmol/min/mg protein, P<0.01). These results suggested that HFD-induced obesity with metabolic derangements could be ameliorated in mice lacking TNF-α receptor 2 via increasing fecal bile acid and lipid content excretion. Therefore, TNF-α signaling through TNFR2 is essentially involved in the bile acid synthesis and excretion of lipids, resulting in its beneficial effects.

[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.

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.

Msx homeobox gene family and craniofacial development.

PubMed

Alappat, Sylvia; Zhang, Zun Yi; Chen, Yi Ping

2003-12-01

Vertebrate Msx genes are unlinked, homeobox-containing genes that bear homology to the Drosophila muscle segment homeobox gene. These genes are expressed at multiple sites of tissue-tissue interactions during vertebrate embryonic development. Inductive interactions mediated by the Msx genes are essential for normal craniofacial, limb and ectodermal organ morphogenesis, and are also essential to survival in mice, as manifested by the phenotypic abnormalities shown in knockout mice and in humans. This review summarizes studies on the expression, regulation, and functional analysis of Msx genes that bear relevance to craniofacial development in humans and mice. Key words: Msx genes, craniofacial, tooth, cleft palate, suture, development, transcription factor, signaling molecule.

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.

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.

Albumin contributes to kidney disease progression in Alport syndrome

PubMed Central

Knutsen, Russell H.; Mecham, Robert P.

2016-01-01

Alport syndrome is a familial kidney disease caused by defects in the collagen type IV network of the glomerular basement membrane. Lack of collagen-α3α4α5(IV) changes the glomerular basement membrane morphologically and functionally, rendering it leaky to albumin and other plasma proteins. Filtered albumin has been suggested to be a cause of the glomerular and tubular injuries observed at advanced stages of Alport syndrome. To directly investigate the role that albumin plays in the progression of disease in Alport syndrome, we generated albumin knockout (Alb−/−) mice to use as a tool for removing albuminuria as a component of kidney disease. Mice lacking albumin were healthy and indistinguishable from control littermates, although they developed hypertriglyceridemia. Dyslipidemia was observed in Alb+/− mice, which displayed half the normal plasma albumin concentration. Alb mutant mice were bred to collagen-α3(IV) knockout (Col4a3−/−) mice, which are a model for human Alport syndrome. Lack of circulating and filtered albumin in Col4a3−/−;Alb−/− mice resulted in dramatically improved kidney disease outcomes, as these mice lived 64% longer than did Col4a3−/−;Alb+/+ and Col4a3−/−;Alb+/− mice, despite similar blood pressures and serum triglyceride levels. Further investigations showed that the absence of albumin correlated with reduced transforming growth factor-β1 signaling as well as reduced tubulointerstitial, glomerular, and podocyte pathology. We conclude that filtered albumin is injurious to kidney cells in Alport syndrome and perhaps in other proteinuric kidney diseases, including diabetic nephropathy. PMID:27147675

Albumin contributes to kidney disease progression in Alport syndrome.

PubMed

Jarad, George; Knutsen, Russell H; Mecham, Robert P; Miner, Jeffrey H

2016-07-01

Alport syndrome is a familial kidney disease caused by defects in the collagen type IV network of the glomerular basement membrane. Lack of collagen-α3α4α5(IV) changes the glomerular basement membrane morphologically and functionally, rendering it leaky to albumin and other plasma proteins. Filtered albumin has been suggested to be a cause of the glomerular and tubular injuries observed at advanced stages of Alport syndrome. To directly investigate the role that albumin plays in the progression of disease in Alport syndrome, we generated albumin knockout (Alb(-/-)) mice to use as a tool for removing albuminuria as a component of kidney disease. Mice lacking albumin were healthy and indistinguishable from control littermates, although they developed hypertriglyceridemia. Dyslipidemia was observed in Alb(+/-) mice, which displayed half the normal plasma albumin concentration. Alb mutant mice were bred to collagen-α3(IV) knockout (Col4a3(-/-)) mice, which are a model for human Alport syndrome. Lack of circulating and filtered albumin in Col4a3(-/-);Alb(-/-) mice resulted in dramatically improved kidney disease outcomes, as these mice lived 64% longer than did Col4a3(-/-);Alb(+/+) and Col4a3(-/-);Alb(+/-) mice, despite similar blood pressures and serum triglyceride levels. Further investigations showed that the absence of albumin correlated with reduced transforming growth factor-β1 signaling as well as reduced tubulointerstitial, glomerular, and podocyte pathology. We conclude that filtered albumin is injurious to kidney cells in Alport syndrome and perhaps in other proteinuric kidney diseases, including diabetic nephropathy. Copyright © 2016 the American Physiological Society.

Osteoblast Menin Regulates Bone Mass in Vivo*

PubMed Central

Kanazawa, Ippei; Canaff, Lucie; Abi Rafeh, Jad; Angrula, Aarti; Li, Jingjing; Riddle, Ryan C.; Boraschi-Diaz, Iris; Komarova, Svetlana V.; Clemens, Thomas L.; Murshed, Monzur; Hendy, Geoffrey N.

2015-01-01

Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-β (TGF-β) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-β, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1f/f) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1f/f mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance. PMID:25538250

Lean phenotype and resistance to diet-induced obesity in vitamin D receptor knockout mice correlates with induction of uncoupling protein-1 in white adipose tissue.

PubMed

Narvaez, Carmen J; Matthews, Donald; Broun, Emily; Chan, Michelle; Welsh, JoEllen

2009-02-01

Increased adiposity is a feature of aging in both mice and humans, but the molecular mechanisms underlying age-related changes in adipose tissue stores remain unclear. In previous studies, we noted that 18-month-old normocalcemic vitamin D receptor (VDR) knockout (VDRKO) mice exhibited atrophy of the mammary adipose compartment relative to wild-type (WT) littermates, suggesting a role for VDR in adiposity. Here we monitored body fat depots, food intake, metabolic factors, and gene expression in WT and VDRKO mice on the C57BL6 and CD1 genetic backgrounds. Regardless of genetic background, both sc and visceral white adipose tissue depots were smaller in VDRKO mice than WT mice. The lean phenotype of VDRKO mice was associated with reduced serum leptin and compensatory increased food intake. Similar effects on adipose tissue, leptin and food intake were observed in mice lacking Cyp27b1, the 1alpha-hydroxylase enzyme that generates 1,25-dihydroxyvitamin D(3), the VDR ligand. Although VDR ablation did not reduce expression of peroxisome proliferator-activated receptor-gamma or fatty acid synthase, PCR array screening identified several differentially expressed genes in white adipose tissue from WT and VDRKO mice. Uncoupling protein-1, which mediates dissociation of cellular respiration from energy production, was greater than 25-fold elevated in VDRKO white adipose tissue. Consistent with elevation in uncoupling protein-1, VDRKO mice were resistant to high-fat diet-induced weight gain. Collectively, these studies identify a novel role for 1,25-dihydroxyvitamin D(3) and the VDR in the control of adipocyte metabolism and lipid storage in vivo.

Characterization of the mucosal cell-mediated immune response in IL-2 knockout mice before and after the onset of colitis.

PubMed Central

McDonald, S A; Palmen, M J; Van Rees, E P; MacDonald, T T

1997-01-01

One of the major advances in the understanding of inflammatory bowel disease has been the observation that mice with immunoregulatory defects, such as interleukin-2 knockout (IL-2 -/-) mice, develop spontaneous gut inflammation. Here we have characterized the immune response in the ileum, caecum and colon of these mice before and after the onset of colitis by examining the cellular infiltrate, the cytokines produced by these cells and the mucosal vascular addressin MAdCAM-1. IL-2 -/- mice developed colitis after 35 days of age and before this the mice were apparently healthy. IL-2 -/- mice aged over 35 days with colitis had large numbers of CD4+, CD8+, alpha beta T-cell receptor (TCR)+ and gamma delta TCR+ T cells, macrophages, dendritic cells and MAdCAM-1+ endothelial cells in the caecum and colon. This was associated with an increase in the number of interferon-gamma (IFN-gamma), IL-1 and tumour necrosis factor-alpha (TNF-alpha) transcripts and a decrease in IL-4 and IL-10 transcripts. Treatment of IL-2 -/- mice with cyclosporin A significantly delayed mortality. Interestingly, IL-2 -/- mice under 35 days, although healthy, did show some subtle immunological signs of preclinical disease. There was a significant increase in the number of macrophages and dendritic cells in the colonic lamina propria and increased mRNA for IL-1 and TNF-alpha. There were also increased numbers of MAdCAM-1+ endothelial cells, but IFN-gamma transcripts were not elevated. These results suggest that T-cell-mediated colitis in IL-2 -/- mice may be secondary to an initial non-specific inflammation. Images Figure 2 Figure 5 PMID:9203968

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.

Essential role for Bim in mediating the apoptotic and antitumor activities of immunotoxins.

PubMed

Antignani, A; Segal, D; Simon, N; Kreitman, R J; Huang, D; FitzGerald, D J

2017-08-31

Protein synthesis is crucial for regulating cell homeostasis and, when unrestricted, it can lead to tumorigenesis. Immunotoxins derived from Pseudomonas exotoxin are antibody-toxin fusion proteins that inhibit protein synthesis of mammalian cells via ADP-ribosylation of the eukaryotic elongation factor-2. Here we investigate the role of the Bcl-2 family proteins in the response of cancer cells to immunotoxin challenge. Besides the well-known reduction of the prosurvival Bcl-2 family member, Mcl-1, following inhibition of protein synthesis, we show for the first time that immunotoxins also reduce the levels of selected proapoptotic BH-3-only proteins. Among these, only Bim protein levels correlated with the ability of immunotoxins to induce an apoptotic response. To support our findings, we verified that a Bim knockout completely abolished immunotoxin-mediated apoptosis. Further, mice bearing either wild-type or Bid knockout tumors responded to immunotoxin treatment with a decrease in growth kinetics, whereas mice engrafted with Bim knockout tumors showed no reduction in tumor size or prolongation of survival following immunotoxin treatment. From these results, we conclude that Bim expression is a major susceptibility factor for tumor cell death and, as such, constitutes a potential biomarker that could be evaluated before immunotoxin treatment. In support of this hypothesis, clinically, we analyzed patient cells before immunotoxin treatment and report that samples of hairy cell leukemia with high levels of Bim protein responded with a greater decrease in leukemic cell count compared with those samples expressing a low level of Bim.

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

Cystathionine γ-Lyase Deficiency Exacerbates CCl4-Induced Acute Hepatitis and Fibrosis in the Mouse Liver.

PubMed

Ci, Lei; Yang, Xingyu; Gu, Xiaowen; Li, Qing; Guo, Yang; Zhou, Ziping; Zhang, Mengjie; Shi, Jiahao; Yang, Hua; Wang, Zhugang; Fei, Jian

2017-07-20

The present study examined the role of cystathionine γ-lyase (CSE) in carbon tetrachloride (CCl 4 )-induced liver damage. A CSE gene knock-out and luciferase gene knock-in (KI) mouse model was constructed to study the function of CSE and to trace its expression in living status. CCl 4 or lipopolysaccharide markedly downregulated CSE expression in the liver of mice. CSE-deficient mice showed increased serum alanine aminotransferase and aspartate aminotransferase levels, and liver damage after CCl 4 challenge, whereas albumin and endogenous hydrogen sulfide (H 2 S) levels decreased significantly. CSE knockout mice showed increased serum homocysteine levels, upregulation of inflammatory cytokines, and increased autophagy and IκB-α degradation in the liver in response to CCl 4 treatment. The increase in pro-inflammatory cytokines, including tumor necrosis factor-alpha in CSE-deficient mice after CCl 4 challenge, was accompanied by a significant increase in liver tissue hydroxyproline and α-smooth muscle actin and histopathologic changes in the liver. However, H 2 S donor pretreatment effectively attenuated most of these imbalances. Here, a CSE knock-out and luciferase KI mouse model was established for the first time to study the transcriptional regulation of CSE expression in real time in a non-invasive manner, providing information on the effects and potential mechanisms of CSE on CCl 4 -induced liver injury. CSE deficiency increases pro-inflammatory cytokines in the liver and exacerbates acute hepatitis and liver fibrosis by reducing H 2 S production from L-cysteine in the liver. The present data suggest the potential of an H 2 S donor for the treatment of liver diseases such as toxic hepatitis and fibrosis. Antioxid. Redox Signal. 27, 133-149.

NLRP3 Inflammasome Mediates Aldosterone-Induced Vascular Damage.

PubMed

Bruder-Nascimento, Thiago; Ferreira, Nathanne S; Zanotto, Camila Z; Ramalho, Fernanda; Pequeno, Isabela O; Olivon, Vania C; Neves, Karla B; Alves-Lopes, Rheure; Campos, Eduardo; Silva, Carlos Alberto A; Fazan, Rubens; Carlos, Daniela; Mestriner, Fabiola L; Prado, Douglas; Pereira, Felipe V; Braga, Tarcio; Luiz, Joao Paulo M; Cau, Stefany B; Elias, Paula C; Moreira, Ayrton C; Câmara, Niels O; Zamboni, Dario S; Alves-Filho, Jose Carlos; Tostes, Rita C

2016-12-06

Inflammation is a key feature of aldosterone-induced vascular damage and dysfunction, but molecular mechanisms by which aldosterone triggers inflammation remain unclear. The NLRP3 inflammasome is a pivotal immune sensor that recognizes endogenous danger signals triggering sterile inflammation. We analyzed vascular function and inflammatory profile of wild-type (WT), NLRP3 knockout (NLRP3 -/- ), caspase-1 knockout (Casp-1 -/- ), and interleukin-1 receptor knockout (IL-1R -/- ) mice treated with vehicle or aldosterone (600 µg·kg -1 ·d -1 for 14 days through osmotic mini-pump) while receiving 1% saline to drink. Here, we show that NLRP3 inflammasome plays a central role in aldosterone-induced vascular dysfunction. Long-term infusion of aldosterone in mice resulted in elevation of plasma interleukin-1β levels and vascular abnormalities. Mice lacking the IL-1R or the inflammasome components NLRP3 and caspase-1 were protected from aldosterone-induced vascular damage. In vitro, aldosterone stimulated NLRP3-dependent interleukin-1β secretion by bone marrow-derived macrophages by activating nuclear factor-κB signaling and reactive oxygen species generation. Moreover, chimeric mice reconstituted with NLRP3-deficient hematopoietic cells showed that NLRP3 in immune cells mediates aldosterone-induced vascular damage. In addition, aldosterone increased the expression of NLRP3, active caspase-1, and mature interleukin-1β in human peripheral blood mononuclear cells. Hypertensive patients with hyperaldosteronism or normal levels of aldosterone exhibited increased activity of NLRP3 inflammasome, suggesting that the effect of hyperaldosteronism on the inflammasome may be mediated through high blood pressure. Together, these data demonstrate that NLRP3 inflammasome, through activation of IL-1R, is critically involved in the deleterious vascular effects of aldosterone, placing NLRP3 as a potential target for therapeutic interventions in conditions with high aldosterone levels. © 2016 American Heart Association, Inc.

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.

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.

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.

Anti-Atherosclerotic Action of Agmatine in ApoE-Knockout Mice

PubMed Central

Olszanecki, Rafał; Totoń-Żurańska, Justyna; Stachowicz, Aneta; Suski, Maciej; Gębska, Anna; Gajda, Mariusz; Jawień, Jacek; Korbut, Ryszard

2017-01-01

Atherosclerosis is an inflammatory disease in which dysfunction of mitochondria play an important role, and disorders of lipid management intensify this process. Agmatine, an endogenous polyamine formed by decarboxylation of arginine, exerts a protective effect on mitochondria and modulates fatty acid metabolism. We investigated the effect of exogenous agmatine on the development of atherosclerosis and changes in lipid profile in apolipoprotein E knockout (apoE-/-) mice. Agmatine caused an approximate 40% decrease of atherosclerotic lesions, as estimated by en face and cross-section methods with an influence on macrophage but not on smooth muscle content in the plaques. Agmatine treatment did not changed gelatinase activity within the plaque area. What is more, the action of agmatine was associated with an increase in the number of high density lipoproteins (HDL) in blood. Real-Time PCR analysis showed that agmatine modulates liver mRNA levels of many factors involved in oxidation of fatty acid and cholesterol biosynthesis. Two-dimensional electrophoresis coupled with mass spectrometry identified 27 differentially expressed mitochondrial proteins upon agmatine treatment in the liver of apoE-/- mice, mostly proteins related to metabolism and apoptosis. In conclusion, prolonged administration of agmatine inhibits atherosclerosis in apoE-/- mice; however, the exact mechanisms linking observed changes and elevations of HDL plasma require further investigation. PMID:28777310

Anti-Atherosclerotic Action of Agmatine in ApoE-Knockout Mice.

PubMed

Wiśniewska, Anna; Olszanecki, Rafał; Totoń-Żurańska, Justyna; Kuś, Katarzyna; Stachowicz, Aneta; Suski, Maciej; Gębska, Anna; Gajda, Mariusz; Jawień, Jacek; Korbut, Ryszard

2017-08-04

Atherosclerosis is an inflammatory disease in which dysfunction of mitochondria play an important role, and disorders of lipid management intensify this process. Agmatine, an endogenous polyamine formed by decarboxylation of arginine, exerts a protective effect on mitochondria and modulates fatty acid metabolism. We investigated the effect of exogenous agmatine on the development of atherosclerosis and changes in lipid profile in apolipoprotein E knockout (apoE-/-) mice. Agmatine caused an approximate 40% decrease of atherosclerotic lesions, as estimated by en face and cross-section methods with an influence on macrophage but not on smooth muscle content in the plaques. Agmatine treatment did not changed gelatinase activity within the plaque area. What is more, the action of agmatine was associated with an increase in the number of high density lipoproteins (HDL) in blood. Real-Time PCR analysis showed that agmatine modulates liver mRNA levels of many factors involved in oxidation of fatty acid and cholesterol biosynthesis. Two-dimensional electrophoresis coupled with mass spectrometry identified 27 differentially expressed mitochondrial proteins upon agmatine treatment in the liver of apoE-/- mice, mostly proteins related to metabolism and apoptosis. In conclusion, prolonged administration of agmatine inhibits atherosclerosis in apoE-/- mice; however, the exact mechanisms linking observed changes and elevations of HDL plasma require further investigation.

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.

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

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

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.

Dietary corn fractions reduce atherogenesis in low-density lipoprotein receptor knockout mice.

PubMed

Masisi, Kabo; Le, Khuong; Ghazzawi, Nora; Moghadasian, Mohammed H; Beta, Trust

2017-01-01

Accumulating evidence has suggested that intake of whole grains is a protective factor against pathogenesis of coronary artery disease. The exact mechanisms, however, are still not clearly understood. In this study, we hypothesized that adequate intake of corn fractions (aleurone, endosperm and germ) can modify lipid profiles in relation to atherosclerotic lesion development in low-density lipoprotein receptor knockout (LDLr-KO) mice. The purpose of the present study was to investigate the potential cardiovascular benefits of corn fractions in LDLr-KO mice through a number of biomarkers including lipid profile, and morphologic and morphometrical analysis of atherosclerotic lesions in aortic root. Four groups of male LDLr-KO mice were fed with the experimental diets supplemented with (3 treated) or without (control) 5% (wt/wt) of each of corn fractions for 10 weeks. All diets were supplemented with 0.06% (wt/wt) cholesterol. Compared with mice in the control group, atherosclerotic lesions in the aortic roots were significantly reduced (P=.003) in the mice that were fed diet supplemented with aleurone and germ fractions. This effect was associated with significant reductions in plasma total (P=.02) and LDL (P=.03) cholesterol levels, and an increase in fecal cholesterol excretion (P=.04). Furthermore, abdominal fat mass was significantly reduced by consumption of aleurone (P=.03). In summary, the consumption of aleurone and germ may help attenuate atherosclerosis by reducing plasma total and LDL cholesterol levels. Copyright © 2016 Elsevier Inc. All rights reserved.

Influence of metformin on mitochondrial subproteome in the brain of apoE knockout mice.

PubMed

Suski, Maciej; Olszanecki, Rafał; Chmura, Łukasz; Stachowicz, Aneta; Madej, Józef; Okoń, Krzysztof; Adamek, Dariusz; Korbut, Ryszard

2016-02-05

Neurodegenerative diseases are the set of progressive, age-related brain disorders, characterized by an excessive accumulation of mutant proteins in the certain regions of the brain. Such changes, collectively identified as causal factors of neurodegeneration, all impact mitochondria, imminently leading to their dysfunction. These observations predestine mitochondria as an attractive drug target for counteracting degenerative brain damage. The aim of this study was to use a differential proteomic approach to comprehensively assess the changes in mitochondrial protein expression in the brain of apoE-knockout mice (apoE(-/-)) and to investigate the influence of prolonged treatment with metformin - an indirect activator of AMP-activated protein kinase (AMPK) on the brain mitoproteome in apoE(-/-) mice. The quantitative assessment of the brain mitoproteome in apoE(-/-) revealed the changes in 10 proteins expression as compared to healthy C57BL/6J mice and 25 proteins expression in metformin-treated apoE(-/-) mice. Identified proteins mainly included apoptosis regulators, metabolic enzymes and structural proteins. In summary, our study provided proteomic characteristics suggesting the decrease of antioxidant defense and structural disturbances in the brain mitochondria of apoE(-/-) mice as compared to healthy controls. In this setting, the use of metformin changed the expression of several proteins primarily involved in metabolic processes, the regulation of apoptosis and the structural maintenance of mitochondria, what could potentially restore their native functionalities. Copyright © 2015 Elsevier B.V. All rights reserved.

Inducing a visceral organ to protect a peripheral capillary bed: stabilizing hepatic HIF-1α prevents oxygen-induced retinopathy.

PubMed

Hoppe, George; Lee, Tamara J; Yoon, Suzy; Yu, Minzhong; Peachey, Neal S; Rayborn, Mary; Zutel, M Julieta; Trichonas, George; Au, John; Sears, Jonathan E

2014-06-01

Activation of hypoxia-inducible factor (HIF) can prevent oxygen-induced retinopathy in rodents. Here we demonstrate that dimethyloxaloylglycine (DMOG)-induced retinovascular protection is dependent on hepatic HIF-1 because mice deficient in liver-specific HIF-1α experience hyperoxia-induced damage even with DMOG treatment, whereas DMOG-treated wild-type mice have 50% less avascular retina (P < 0.0001). Hepatic HIF stabilization protects retinal function because DMOG normalizes the b-wave on electroretinography in wild-type mice. The localization of DMOG action to the liver is further supported by evidence that i) mRNA and protein erythropoietin levels within liver and serum increased in DMOG-treated wild-type animals but are reduced by 60% in liver-specific HIF-1α knockout mice treated with DMOG, ii) triple-positive (Sca1/cKit/VEGFR2), bone-marrow-derived endothelial precursor cells increased twofold in DMOG-treated wild-type mice (P < 0.001) but are unchanged in hepatic HIF-1α knockout mice in response to DMOG, and iii) hepatic luminescence in the luciferase oxygen-dependent degradation domain mouse was induced by subcutaneous and intraperitoneal DMOG. These findings uncover a novel endocrine mechanism for retinovascular protection. Activating HIF in visceral organs such as the liver may be a simple strategy to protect capillary beds in the retina and in other peripheral tissues. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Effect of Shenxinning decoction on ventricular remodeling in AT1 receptor-knockout mice with chronic renal insufficiency

PubMed Central

Yang, Xuejun; Zhou, Hua; Qu, Huiyan; Liu, Weifang; Huang, Xiaojin; Shun, Yating; He, Liqun

2014-01-01

Objective: To observe the efficacy of Shenxinning Decoction (SXND) in ventricular remodeling in AT1 receptor-knockout (AT1-KO) mice with chronic renal insufficiency (CRI). Materials and Methods: AT1-KO mice modeled with subtotal (5/6) nephrectomy were intervened with SXND for 12 weeks. Subsequently, blood urea nitrogen (BUN), serum creatinine (SCr), brain natriuretic peptide (BNP), echocardiography (left ventricular end-diastolic diameter, LVDD; left ventricular end-systolic diameter, LVDS; fractional shortening, FS; and ejection fraction, EF), collagen types I and III in the heart and kidney, myocardial mitochondria, and cardiac transforming growth factor-β1 (TGF-β1) of the AT1-KO mice were compared with the same model with nephrectomy only and untreated with SXND. Results: AT1-KO mice did not affect the process of CRI but it could significantly affect cardiac remodeling process. SXND decreased to some extent the AT1-KO mice's BUN, SCr, BNP, and cardiac LVDD, LVDS, and BNP, improved FS and EF, lowered the expression of collagen type I and III in heart and kidney, increased the quantity of mitochondria and ameliorated their structure, and down-regulated the expression of TGF-β1. Conclusion: SXND may antagonize the renin–angiotensin system (RAS) and decrease uremia toxins, thereby ameliorating ventricular remodeling in CRI. Furthermore, SXND has a mechanism correlated with the improvement of myocardial energy metabolism and the down-regulation of TGF-β1. PMID:25097276

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.

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.

Mechanism of hyperphagia contributing to obesity in brain-derived neurotrophic factor knockout mice.

PubMed

Fox, E A; Biddinger, J E; Jones, K R; McAdams, J; Worman, A

2013-01-15

Global-heterozygous and brain-specific homozygous knockouts (KOs) of brain-derived neurotrophic factor (BDNF) cause late- and early-onset obesity, respectively, both involving hyperphagia. Little is known about the mechanism underlying this hyperphagia or whether BDNF loss from peripheral tissues could contribute to overeating. Since global-homozygous BDNF-KO is perinatal lethal, a BDNF-KO that spared sufficient brainstem BDNF to support normal health was utilized to begin to address these issues. Meal pattern and microstructure analyses suggested overeating of BDNF-KO mice was mediated by deficits in both satiation and satiety that resulted in increased meal size and frequency and implicated a reduction of vagal signaling from the gut to the brain. Meal-induced c-Fos activation in the nucleus of the solitary tract, a more direct measure of vagal afferent signaling, however, was not decreased in BDNF-KO mice, and thus was not consistent with a vagal afferent role. Interestingly though, meal-induced c-Fos activation was increased in the dorsal motor nucleus of the vagus nerve (DMV) of BDNF-KO mice. This could imply that augmentation of vago-vagal digestive reflexes occurred (e.g., accommodation), which would support increased meal size and possibly increased meal number by reducing the increase in intragastric pressure produced by a given amount of ingesta. Additionally, vagal sensory neuron number in BDNF-KO mice was altered in a manner consistent with the increased meal-induced activation of the DMV. These results suggest reduced BDNF causes satiety and satiation deficits that support hyperphagia, possibly involving augmentation of vago-vagal reflexes mediated by central pathways or vagal afferents regulated by BDNF levels. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

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

Contributions of β2-microglobulin–dependent molecules and lymphocytes to iron regulation: insights from HfeRag1−/− and β2mRag1−/− double knock-out mice

PubMed Central

Miranda, Carlos J.; Makui, Hortence; Andrews, Nancy C.; Santos, Manuela M.

2010-01-01

Genetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, coding for a β2-microglobulin (β2m)–associated major histocompatibility complex class I-like protein. However, iron accumulation in patients with HH can be highly variable. Previously, analysis of β2mRag1−/− double-deficient mice, lacking all β2m-dependent molecules and lymphocytes, demonstrated increased iron accumulation in the pancreas and heart compared with β2m single knock-out mice. To evaluate whether the observed phenotype in β2mRag1−/− mice was due solely to the absence of Hfe or to other β2m-dependent molecules, we generated HfeRag1−/− double-deficient mice. Our studies revealed that introduction of Rag1 deficiency in Hfe knock-out mice leads to heightened iron overload, mainly in the liver, whereas the heart and pancreas are relatively spared compared with β2mRag1−/− mice. These results suggest that other β2m-interacting protein(s) may be involved in iron regulation and that in the absence of functional Hfe molecules lymphocyte numbers may influence iron overload severity. PMID:14656877

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.

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

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.

Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1

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

Ilic, Zoran, E-mail: zxi01@health.state.ny.u; Crawford, Dana, E-mail: crawfod@mail.amc.ed; Egner, Patricia A., E-mail: pegner@jhsph.ed

Aflatoxin B1 (AFB1) is a major risk factor for hepatocellular carcinoma (HCC) in humans. However, mice, a major animal model for the study of AFB1 carcinogenesis, are resistant, due to high constitutive expression, in the mouse liver, of glutathione S-transferase A3 subunit (mGSTA3) that is lacking in humans. Our objective was to establish that a mouse model for AFB1 toxicity could be used to study mechanisms of toxicity that are relevant for human disease, i.e., an mGSTA3 knockout (KO) mouse that responds to toxicants such as AFB1 in a manner similar to humans. Exons 3-6 of the mGSTA3 were replacedmore » with a neomycin cassette by homologous recombination. Southern blotting, RT-PCR, Western blotting, and measurement of AFB1-N{sup 7}-DNA adduct formation were used to evaluate the mGSTA3 KO mice. The KO mice have deletion of exons 3-6 of the mGSTA3 gene, as expected, as well as a lack of mGSTA3 expression at the mRNA and protein levels. Three hours after injection of 5 mg/kg AFB1, mGSTA3 KO mice have more than 100-fold more AFB1-N{sup 7}-DNA adducts in their livers than do similarly treated wild-type (WT) mice. In addition, the mGSTA3 KO mice die of massive hepatic necrosis, at AFB1 doses that have minimal toxic effects in WT mice. We conclude that mGSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of AFB1, confirming the crucial role of GSTA3 subunit in protection of normal mice against AFB1 toxicity. We propose the mGSTA3 KO mouse as a useful model with which to study the interplay of risk factors leading to HCC development in humans, as well as for testing of additional possible functions of mGSTA3.« less

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.

Sex-dependent alterations in motor and anxiety-like behavior of aged bacterial peptidoglycan sensing molecule 2 knockout mice.

PubMed

Arentsen, Tim; Khalid, Roksana; Qian, Yu; Diaz Heijtz, Rochellys

2018-01-01

Peptidoglycan recognition proteins (PGRPs) are key sensing-molecules of the innate immune system that specifically detect bacterial peptidoglycan (PGN) and its derivates. PGRPs have recently emerged as potential key regulators of normal brain development and behavior. To test the hypothesis that PGRPs play a role in motor control and anxiety-like behavior in later life, we used 15-month old male and female peptidoglycan recognition protein 2 (Pglyrp2) knockout (KO) mice. Pglyrp2 is an N-acetylmuramyl-l-alanine amidase that hydrolyzes PGN between the sugar backbone and the peptide chain (which is unique among the mammalian PGRPs). Using a battery of behavioral tests, we demonstrate that Pglyrp2 KO male mice display decreased levels of anxiety-like behavior compared with wild type (WT) males. In contrast, Pglyrp2 KO female mice show reduced rearing activity and increased anxiety-like behavior compared to WT females. In the accelerated rotarod test, however, Pglyrp2 KO female mice performed better compared to WT females (i.e., they had longer latency to fall off the rotarod). Further, Pglyrp2 KO male mice exhibited decreased expression levels of synaptophysin, gephyrin, and brain-derived neurotrophic factor in the frontal cortex, but not in the amygdala. Pglyrp2 KO female mice exhibited increased expression levels of spinophilin and alpha-synuclein in the frontal cortex, while exhibiting decreased expression levels of synaptophysin, gephyrin and spinophilin in the amygdala. Our findings suggest a novel role for Pglyrp2asa key regulator of motor and anxiety-like behavior in late life. Copyright © 2017. Published by Elsevier Inc.

Analysis of Kalirin-7 Knockout Mice Reveals Different Effects in Female Mice

PubMed Central

Mazzone, Christopher M.; Larese, Taylor P.; Kiraly, Drew D.; Eipper, Betty A.

2012-01-01

Estradiol treatment of ovariectomized rodents is known to affect the morphology of dendritic spines and produce behavioral and cognitive effects. Kalirin-7 (Kal7), a postsynaptic density (PSD)-localized Rho-guanine nucleotide exchange factor, is important for dendritic spine formation and stability. Male Kal7 knockout [Kal7(KO)] mice exhibit a number of abnormal behavioral and biochemical phenotypes. Given that chronic 17β-estradiol (E2) replacement of ovariectomized rats enhanced Kal7 expression in the hippocampus and primary hippocampal cultures, we assessed the behavioral and biochemical effects of chronic E2 treatment of ovariectomized female wild-type and Kal7(KO) mice. Both intact and ovariectomized Kal7(KO) female mice exhibited decreased anxiety-like behavior compared with the corresponding wild type in the elevated zero maze and were unaffected by E2 treatment. Chronic E2 decreased locomotor activity in the open field and enhanced performance in a passive-avoidance fear conditioning task, which were both unaffected by genotype. Kal7(KO) female mice engaged in significantly more object exploration, both familiar and novel, than did wild-type females. E2 enhanced the acute locomotor response to cocaine, with no significant effect of genotype. Similar to Kal7(KO) males, Kal7(KO) females had decreased levels of N-methyl-d-aspartate receptor 2B in hippocampal PSD fractions with no effect of E2 treatment. The differing behavioral effects of Kal7 ablation in female and male mice may offer insight into the molecular underpinnings of these differences. PMID:22989522

DJ-1 activates autophagy in the repression of cardiac hypertrophy.

PubMed

Xue, Ruicong; Jiang, Jingzhou; Dong, Bin; Tan, Weiping; Sun, Yu; Zhao, Jingjing; Chen, Yili; Dong, Yugang; Liu, Chen

2017-11-01

Cardiac hypertrophy is the risk factor of heart failure when the heart is confronted with pressure overload or neurohumoral stimuli. Autophagy, a conserved degradative pathway, is one of the important mechanisms involved in the regulation of cardiac hypertrophy. DJ-1 is a traditional anti-oxidative protein and emerging evidence suggested that DJ-1 might modulate autophagy. However, the regulation of autophagy by DJ-1 in the process of cardiac hypertrophy remains unknown. In our study, we firstly discovered that the expression of DJ-1declined in the process of pressure overload cardiac hypertrophy, and its alteration was parallel with the impairment of autophagy. Furthermore, we proved that DJ-1 knockout mice exhibited a more hypertrophied phenotype than wildtype mice in cardiac hypertrophy which indicated that DJ-1 is responsible for the repression of cardiac hypertrophy. Furthermore, DJ-1 knockout significantly exacerbated pulmonary edema due to cardiac hypertrophy. In the process of cardiac hypertrophy, DJ-1 knockout significantly impaired autophagy activation and enhanced mTORC1 and mTORC2 phosphorylation were found. Similarly, our in vitro study proved that DJ-1 overexpression ameliorated phenylephrine (PE)-induced cardiac hypertrophy and promoted autophagy activation. Taken together, DJ-1 might repress both pressure overload and PE-induced cardiac hypertrophy via the activation of autophagy. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Selective deletion of apolipoprotein E in astrocytes ameliorates the spatial learning and memory deficits in Alzheimer's disease (APP/PS1) mice by inhibiting TGF-β/Smad2/STAT3 signaling.

PubMed

Zheng, Jin-Yu; Sun, Jian; Ji, Chun-Mei; Shen, Lin; Chen, Zhong-Jun; Xie, Peng; Sun, Yuan-Zhao; Yu, Ru-Tong

2017-06-01

Astrocytes and apolipoprotein E (apoE) play critical roles in cognitive function, not only under physiological conditions but also in some pathological situations, particularly in the pathological progression of Alzheimer's disease (AD). The regulatory mechanisms underlying the effect of apoE, derived from astrocytes, on cognitive deficits during AD pathology development are unclear. In this study, we generated amyloid precursor protein/apoE knockout (APP/apoE KO ) and APP/glial fibrillary acidic protein (GFAP)-apoE KO mice (the AD mice model used in this study was based on the APP-familial Alzheimer disease overexpression) to investigate the role of apoE, derived from astrocytes, in AD pathology and cognitive function. To explore the mechanism, we investigated the amyloidogenic process related transforming growth factor β/mothers against decapentaplegic homolog 2/signal transducer and activator of transcription 3 (TGF-β/Smad2/STAT3) signaling pathway and further confirmed by administering TGF-β-overexpression adeno-associated virus (specific to astrocytes) to APP/GFAP-apoE KO mice and TGF-β-inhibition adeno-associated virus (specific to astrocytes) to APP/WT mice. Whole body deletion of apoE significantly ameliorated the spatial learning and memory impairment, reduced amyloid β-protein production and inhibited astrogliosis in APP/apoE KO mice, as well as specific deletion apoE in astrocytes in APP/GFAP-apoE KO mice. Moreover, amyloid β-protein accumulation was increased due to promotion of amyloidogenesis of APP, and astrogliosis was upregulated by activation of TGF-β/Smad2/STAT3 signaling. Furthermore, the overexpression of TGF-β in astrocytes in APP/GFAP-apoE KO mice abrogated the effects of apoE knockout. In contrast, repression of TGF-β in astrocytes of APP/WT mice exerted a therapeutic effect similar to apoE knockout. These data suggested that apoE derived from astrocytes contributes to the risk of AD through TGF-β/Smad2/STAT3 signaling activation. These findings enhance our understanding of the role of apoE, derived from astrocytes, in AD and suggest it to be a potential biomarker and therapeutic target for AD. Copyright © 2017 Elsevier Inc. All rights reserved.

PTH/PTHrP Receptor Mediates Cachexia in Models of Kidney Failure and Cancer.

PubMed

Kir, Serkan; Komaba, Hirotaka; Garcia, Ana P; Economopoulos, Konstantinos P; Liu, Wei; Lanske, Beate; Hodin, Richard A; Spiegelman, Bruce M

2016-02-09

Cachexia is a wasting syndrome associated with elevated basal energy expenditure and loss of adipose and muscle tissues. It accompanies many chronic diseases including renal failure and cancer and is an important risk factor for mortality. Our recent work demonstrated that tumor-derived PTHrP drives adipose tissue browning and cachexia. Here, we show that PTH is involved in stimulating a thermogenic gene program in 5/6 nephrectomized mice that suffer from cachexia. Fat-specific knockout of PTHR blocked adipose browning and wasting. Surprisingly, loss of PTHR in fat tissue also preserved muscle mass and improved muscle strength. Similarly, PTHR knockout mice were resistant to cachexia driven by tumors. Our results demonstrate that PTHrP and PTH mediate wasting through a common mechanism involving PTHR, and there exists an unexpected crosstalk mechanism between wasting of fat tissue and skeletal muscle. Targeting the PTH/PTHrP pathway may have therapeutic uses in humans with cachexia. Copyright © 2016 Elsevier Inc. All rights reserved.

SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function

PubMed Central

Price, Nathan L.; Gomes, Ana P.; Ling, Alvin J.Y.; Duarte, Filipe V.; Martin-Montalvo, Alejandro; North, Brian J.; Agarwal, Beamon; Ye, Lan; Ramadori, Giorgio; Teodoro, Joao S.; Hubbard, Basil P.; Varela, Ana T.; Davis, James G.; Varamini, Behzad; Hafner, Angela; Moaddel, Ruin; Rolo, Anabela P.; Coppari, Roberto; Palmeira, Carlos M.; de Cabo, Rafael; Baur, Joseph A.; Sinclair, David A.

2012-01-01

SUMMARY Resveratrol induces mitochondrial biogenesis and protects against metabolic decline but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germ-line SIRT1 knockouts, we have developed the first inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation and increased NAD+ levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo. PMID:22560220

[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.

Relative axial myopia in Egr-1 (ZENK) knockout mice.

PubMed

Schippert, Ruth; Burkhardt, Eva; Feldkaemper, Marita; Schaeffel, Frank

2007-01-01

Experiments in chickens have implicated the transcription factor ZENK (also known as Egr-1, NGFI-A, zif268, tis8, cef5, and Krox24) in the feedback mechanisms for visual control of axial eye growth and myopia development. ZENK is upregulated in retinal glucagon amacrine cells when axial eye growth is inhibited by positive spectacle lens wear and is downregulated when it is enhanced by negative spectacle lens wear, suggesting that ZENK may be linked to an inhibitory signal for axial eye growth. This study was undertaken to determine whether a Egr-1(-/-) knockout mouse mutant, lacking ZENK completely, has longer eyes and more myopic refraction, than do Egr-1(+/)(-) heterozygous and Egr-1(+/+) wild-type mice with near-identical genetic backgrounds. Eye growth and refractive development were tracked from day P28 to P98. Corneal radius of curvature was measured with infrared photokeratometry, refractive state with infrared photoretinoscopy, and ocular dimensions with low-coherence interferometry. As a functional vision test, grating acuity was determined in an automated optomotor task. The abundance of ZENK protein in the retina was quantified by immunohistochemistry. Egr-1 knockout mice had longer eyes and a relative myopic shift in refraction, with additional minor effects on anterior chamber depth and corneal radius of curvature. Paraxial schematic eye modeling suggested changes in the optics of the crystalline lens as well. With increasing age, the differences between mutant and wild-type mice declined, although the differences in refraction persisted over the observation period. Grating acuity was not affected by the lack of the Egr-1 protein during development. Although it has been shown that different mouse strains may have differently large eyes, the present study shows that a specific gene knockout can produce relative myopia, compared with the wild-type with near-identical genetic background. Further experiments are needed to determine whether the observed effects of Egr-1 deletion are due to changes in function within the retina or other ocular tissues or to changes of function in other systems that may affect ocular growth from outside the eye.

Brain pyroglutamate amyloid-β is produced by cathepsin B and is reduced by the cysteine protease inhibitor E64d, representing a potential Alzheimer's disease therapeutic.

PubMed

Hook, Gregory; Yu, Jin; Toneff, Thomas; Kindy, Mark; Hook, Vivian

2014-01-01

Pyroglutamate amyloid-β peptides (pGlu-Aβ) are particularly pernicious forms of amyloid-β peptides (Aβ) present in Alzheimer's disease (AD) brains. pGlu-Aβ peptides are N-terminally truncated forms of full-length Aβ peptides (flAβ(1-40/42)) in which the N-terminal glutamate is cyclized to pyroglutamate to generate pGlu-Aβ(3-40/42). β-secretase cleavage of amyloid-β precursor protein (AβPP) produces flAβ(1-40/42), but it is not yet known whether the β-secretase BACE1 or the alternative β-secretase cathepsin B (CatB) participate in the production of pGlu-Aβ. Therefore, this study examined the effects of gene knockout of these proteases on brain pGlu-Aβ levels in transgenic AβPPLon mice, which express AβPP isoform 695 and have the wild-type (wt) β-secretase activity found in most AD patients. Knockout or overexpression of the CatB gene reduced or increased, respectively, pGlu-Aβ(3-40/42), flAβ(1-40/42), and pGlu-Aβ plaque load, but knockout of the BACE1 gene had no effect on those parameters in the transgenic mice. Treatment of AβPPLon mice with E64d, a cysteine protease inhibitor of CatB, also reduced brain pGlu-Aβ(3-42), flAβ(1-40/42), and pGlu-Aβ plaque load. Treatment of neuronal-like chromaffin cells with CA074Me, an inhibitor of CatB, resulted in reduced levels of pGlu-Aβ(3-40) released from the activity-dependent, regulated secretory pathway. Moreover, CatB knockout and E64d treatment has been previously shown to improve memory deficits in the AβPPLon mice. These data illustrate the role of CatB in producing pGlu-Aβ and flAβ that participate as key factors in the development of AD. The advantages of CatB inhibitors, especially E64d and its derivatives, as alternatives to BACE1 inhibitors in treating AD patients are discussed.

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

SF-1 a key player in the development and differentiation of steroidogenic tissues

PubMed Central

Val, Pierre; Lefrançois-Martinez, Anne-Marie; Veyssière, Georges; Martinez, Antoine

2003-01-01

Since its discovery in the early 1990s, the orphan nuclear receptor SF-1 has been attributed a central role in the development and differentiation of steroidogenic tissues. SF-1 controls the expression of all the steroidogenic enzymes and cholesterol transporters required for steroidogenesis as well as the expression of steroidogenesis-stimulating hormones and their cognate receptors. SF-1 is also an essential regulator of genes involved in the sex determination cascade. The study of SF-1 null mice and of human mutants has been of great value to demonstrate the essential role of this factor in vivo, although the complete adrenal and gonadal agenesis in knock-out animals has impeded studies of its function as a transcriptional regulator. In particular, the role of SF-1 in the hormonal responsiveness of steroidogenic genes promoters is still a subject of debate. This extensive review takes into account recent data obtained from SF-1 haploinsufficient mice, pituitary-specific knock-outs and from transgenic mice experiments carried out with SF-1 target gene promoters. It also summarizes the pros and cons regarding the presumed role of SF-1 in cAMP signalling. PMID:14594453

The transcription factor Prep1 controls hepatic insulin sensitivity and gluconeogenesis by targeting nuclear localization of FOXO1

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

Kulebyakin, Konstantin; Penkov, Dmitry; IFOM – the FIRC Institute of Molecular Oncology, Via Adamello 16, Milan, 20139

Liver plays a key role in controlling body carbohydrate homeostasis by switching between accumulation and production of glucose and this way maintaining constant level of glucose in blood. Increased blood glucose level triggers release of insulin from pancreatic β-cells. Insulin represses hepatic glucose production and increases glucose accumulation. Insulin resistance is the main cause of type 2 diabetes and hyperglycemia. Currently thiazolidinediones (TZDs) targeting transcriptional factor PPARγ are used as insulin sensitizers for treating patients with type 2 diabetes. However, TZDs are reported to be associated with cardiovascular and liver problems and stimulate obesity. Thus, it is necessary to searchmore » new approaches to improve insulin sensitivity. A promising candidate is transcriptional factor Prep1, as it was shown earlier it could affect insulin sensitivity in variety of insulin-sensitive tissues. The aim of the present study was to evaluate a possible involvement of transcriptional factor Prep1 in control of hepatic glucose accumulation and production. We created mice with liver-specific Prep1 knockout and discovered that hepatocytes derived from these mice are much more sensitive to insulin, comparing to their WT littermates. Incubation of these cells with 100 nM insulin results in almost complete inhibition of gluconeogenesis, while in WT cells this repression is only partial. However, Prep1 doesn't affect gluconeogenesis in the absence of insulin. Also, we observed that nuclear content of gluconeogenic transcription factor FOXO1 was greatly reduced in Prep1 knockout hepatocytes. These findings suggest that Prep1 may control hepatic insulin sensitivity by targeting FOXO1 nuclear stability. - Highlights: • A novel model of liver-specific Prep1 knockout is established. • Ablation of Prep1 in hepatocytes increases insulin sensitivity. • Prep1 controls hepatic insulin sensitivity by regulating localization of FOXO1. • Prep1 regulates localization of FOXO1 via Wnt/β-catenin signaling pathway.« less

Osteoblast-Specific γ-Glutamyl Carboxylase-Deficient Mice Display Enhanced Bone Formation With Aberrant Mineralization.

PubMed

Azuma, Kotaro; Shiba, Sachiko; Hasegawa, Tomoka; Ikeda, Kazuhiro; Urano, Tomohiko; Horie-Inoue, Kuniko; Ouchi, Yasuyoshi; Amizuka, Norio; Inoue, Satoshi

2015-07-01

Vitamin K is a fat-soluble vitamin that is necessary for blood coagulation. In addition, it has bone-protective effects. Vitamin K functions as a cofactor of γ-glutamyl carboxylase (GGCX), which activates its substrates by carboxylation. These substrates are found throughout the body and examples include hepatic blood coagulation factors. Furthermore, vitamin K functions as a ligand of the nuclear receptor known as steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR). We have previously reported on the bone-protective role of SXR/PXR signaling by demonstrating that systemic Pxr-knockout mice displayed osteopenia. Because systemic Ggcx-knockout mice die shortly after birth from severe hemorrhage, the GGCX-mediated effect of vitamin K on bone metabolism has been difficult to evaluate. In this work, we utilized Ggcx-floxed mice to generate osteoblast-specific GGCX-deficient (Ggcx(Δobl/Δobl)) mice by crossing them with Col1-Cre mice. The bone mineral density (BMD) of Ggcx(Δobl/Δobl) mice was significantly higher than that of control Col1-Cre (Ggcx(+/+)) mice. Histomorphometrical analysis of trabecular bones in the proximal tibia showed increased osteoid volume and a higher rate of bone formation in Ggcx(Δobl/Δobl) mice. Histomorphometrical analysis of cortical bones revealed a thicker cortical width and a higher rate of bone formation in Ggcx(Δobl/Δobl) mice. Electron microscopic examination revealed disassembly of mineralized nodules and aberrant calcification of collagen fibers in Ggcx(Δobl/Δobl) mice. The mechanical properties of bones from Ggcx(Δobl/Δobl) mice tended to be stronger than those from control Ggcx(+/+) mice. These results suggest that GGCX in osteoblasts functions to prevent abnormal mineralization in bone formation, although this function may not be a prerequisite for the bone-protective effect of vitamin K. © 2015 American Society for Bone and Mineral Research.

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.

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.

Characterization of liver injury, oval cell proliferation and cholangiocarcinogenesis in glutathione S-transferase A3 knockout mice.

PubMed

Crawford, Dana R; Ilic, Zoran; Guest, Ian; Milne, Ginger L; Hayes, John D; Sell, Stewart

2017-07-01

We recently generated glutathione S-transferase (GST) A3 knockout (KO) mice as a novel model to study the risk factors for liver cancer. GSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of aflatoxin B1 (AFB1), confirming the crucial role of GSTA3 in resistance to AFB1. We now report histopathological changes, tumor formation, biochemical changes and gender response following AFB1 treatment as well as the contribution of oxidative stress. Using a protocol of weekly 0.5 mg AFB1/kg administration, we observed extensive oval (liver stem) cell (OC) proliferation within 1-3 weeks followed by microvesicular lipidosis, megahepatocytes, nuclear inclusions, cholangiomas and small nodules. Male and female GSTA3 KO mice treated with 12 and 24 weekly AFB1 injections followed by a rest period of 12 and 6 months, respectively, all had grossly distorted livers with macro- and microscopic cysts, hepatocellular nodules, cholangiomas and cholangiocarcinomas and OC proliferation. We postulate that the prolonged AFB1 treatment leads to inhibition of hepatocyte proliferation, which is compensated by OC proliferation and eventually formation of cholangiocarcinoma (CCA). At low-dose AFB1, male KO mice showed less extensive acute liver injury, OC proliferation and AFB1-DNA adducts than female KO mice. There were no significant compensatory changes in KO mice GST subunits, GST enzymatic activity, epoxide hydrolase, or CYP1A2 and CYP3A11 levels. Finally, there was a modest increase in F2-isoprostane and isofuran in KO mice that confirmed putative GSTA3 hydroperoxidase activity in vivo for the first time. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effects of pyrophosphate delivery in a peritoneal dialysis solution on bone tissue of apolipoprotein-E knockout mice with chronic kidney disease.

PubMed

Barreto, Fellype C; de Oliveira, Rodrigo B; Benchitrit, Joyce; Louvet, Loïc; Rezg, Raja; Poirot, Sabrina; Jorgetti, Vanda; Drüeke, Tilman B; Riser, Bruce L; Massy, Ziad A

2014-11-01

Vascular calcification (VC) is a risk factor for cardiovascular mortality in the setting of chronic kidney disease (CKD). Pyrophosphate (PPi), an endogenous molecule that inhibits hydroxyapatite crystal formation, has been shown to prevent the development of VC in animal models of CKD. However, the possibility of harmful effects of exogenous administration of PPi on bone requires further investigation. To this end, we examined by histomorphometry the bone of CKD mice after intraperitoneal PPi administration. After CKD creation or sham surgery, 10-week-old female apolipoprotein-E knockout (apoE(-/-)) mice were randomized to one non-CKD group or 4 CKD groups (n = 10-35/group) treated with placebo or three distinct doses of PPi, and fed with standard diet. Eight weeks later, the animals were killed. Serum and femurs were sampled. Femurs were processed for bone histomorphometry. Placebo-treated CKD mice had significantly higher values of osteoid volume, osteoid surface and bone formation rate than sham-placebo mice with normal renal function. Slightly higher osteoid values were observed in CKD mice in response to very low PPi dose (OV/BV, O.Th and ObS/BS) and, for one parameter measured, to high PPi dose (O.Th), compared to placebo-treated CKD mice. Treatment with PPi did not modify any other structural parameters. Mineral apposition rates, and other parameters of bone formation and resorption were not significantly different among the treated animal groups or control CKD placebo group. In conclusion, PPi does not appear to be deleterious to bone tissue in apoE(-/-) mice with CKD, although a possible stimulatory PPi effect on osteoid formation may be worth further investigation.

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.

Divergent roles of growth factors in the GnRH regulation of puberty in mice

PubMed Central

DiVall, Sara A.; Williams, Tameeka R.; Carver, Sarah E.; Koch, Linda; Brüning, Jens C.; Kahn, C. Ronald; Wondisford, Fredric; Radovick, Sally; Wolfe, Andrew

2010-01-01

Pubertal onset, initiated by pulsatile gonadotropin-releasing hormone (GnRH), only occurs in a favorable, anabolic hormonal milieu. Anabolic factors that may signal nutritional status to the hypothalamus include the growth factors insulin and IGF-1. It is unclear which hypothalamic neuronal subpopulation these factors affect to ultimately regulate GnRH neuron function in puberty and reproduction. We examined the direct role of the GnRH neuron in growth factor regulation of reproduction using the Cre/lox system. Mice with the IR or IGF-1R deleted specifically in GnRH neurons were generated. Male and female mice with the IR deleted in GnRH neurons displayed normal pubertal timing and fertility, but male and female mice with the IGF-1R deleted in GnRH neurons experienced delayed pubertal development with normal fertility. With IGF-1 administration, puberty was advanced in control females, but not in females with the IGF-1R deleted in GnRH neurons, in control males, or in knockout males. These mice exhibited developmental differences in GnRH neuronal morphology but normal number and distribution of neurons. These studies define the role of IGF-1R signaling in the coordination of somatic development with reproductive maturation and provide insight into the mechanisms regulating pubertal timing in anabolic states. PMID:20628204

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

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.

Polarization Raman spectroscopy to explain rodent models of brittle bone

NASA Astrophysics Data System (ADS)

Makowski, Alexander J.; Nyman, Jeffry S.; Mahadevan-Jansen, Anita

2013-03-01

Activation Transcription Factor 4 (Atf-4) is essential for osteoblast maturation and proper collagen synthesis. We recently found that these bones demonstrate a rare brittleness phenotype, which is independent of bone strength. We utilized a confocal Renishaw Raman microscope (50x objective; NA=.75) to evaluate embedded, polished cross-sections of mouse tibia from both wild-type and knockout mice at 8 weeks of age (24 mice, n<=8 per group). Analysis of peak ratios indicated statistically significant changes in both mineral and collagen; however, compositional changes did not fully encompass biomechanical differences. To investigate the impact of material organization, we acquired colocalized spectra aligning the polarization angle parallel and perpendicular to the long bone axis from wet intact femurs. To validate our results, we used MMP9-/- mice, which have a brittleness phenotype that is not explained by compositional Raman measures. Polarization angle difference spectra show marked significant changes in orientation of these compositional differences when comparing wild type to knockout bones. Relative to wild-type, Atf4 -/- and MMP9 -/- bones show significant differences (t-test; p<0.05) in prominent collagen peaks. Further investigation of known peak ratios illustrates that this physical anisotropy of molecular organization is tightly clustered in brittle knockout bones. Such findings could have alternate interpretations about net collagen orientation or the angular distribution of collagen molecules. Use of polarization specific Raman measurements has implicated a structural profile that furthers our understanding of models of bone brittleness. Polarization content of Raman spectra may prove significant in future studies of brittle fracture and human fracture risk.

Loss of Notch3 Signaling in Vascular Smooth Muscle Cells Promotes Severe Heart Failure Upon Hypertension.

PubMed

Ragot, Hélène; Monfort, Astrid; Baudet, Mathilde; Azibani, Fériel; Fazal, Loubina; Merval, Régine; Polidano, Evelyne; Cohen-Solal, Alain; Delcayre, Claude; Vodovar, Nicolas; Chatziantoniou, Christos; Samuel, Jane-Lise

2016-08-01

Hypertension, which is a risk factor of heart failure, provokes adaptive changes at the vasculature and cardiac levels. Notch3 signaling plays an important role in resistance arteries by controlling the maturation of vascular smooth muscle cells. Notch3 deletion is protective in pulmonary hypertension while deleterious in arterial hypertension. Although this latter phenotype was attributed to renal and cardiac alterations, the underlying mechanisms remained unknown. To investigate the role of Notch3 signaling in the cardiac adaptation to hypertension, we used mice with either constitutive Notch3 or smooth muscle cell-specific conditional RBPJκ knockout. At baseline, both genotypes exhibited a cardiac arteriolar rarefaction associated with oxidative stress. In response to angiotensin II-induced hypertension, the heart of Notch3 knockout and SM-RBPJκ knockout mice did not adapt to pressure overload and developed heart failure, which could lead to an early and fatal acute decompensation of heart failure. This cardiac maladaptation was characterized by an absence of media hypertrophy of the media arteries, the transition of smooth muscle cells toward a synthetic phenotype, and an alteration of angiogenic pathways. A subset of mice exhibited an early fatal acute decompensated heart failure, in which the same alterations were observed, although in a more rapid timeframe. Altogether, these observations indicate that Notch3 plays a major role in coronary adaptation to pressure overload. These data also show that the hypertrophy of coronary arterial media on pressure overload is mandatory to initially maintain a normal cardiac function and is regulated by the Notch3/RBPJκ pathway. © 2016 American Heart Association, Inc.

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.

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

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

The role of carbonic anhydrase VI in bitter taste perception: evidence from the Car6−/− mouse model

PubMed Central

2014-01-01

Background Carbonic anhydrase VI (CA VI) is a secretory isozyme of the α-CA gene family. It is highly expressed in the salivary and mammary glands and secreted into saliva and milk. Although CA VI was first described as a gustatory protein, its exact functional roles have remained enigmatic. Interestingly, polymorphism of the CA6 gene was recently linked to bitter taste perception in humans. In this study, we compared the preference of Car6−/− and wild-type mice for different taste modalities in an IntelliCage monitoring environment. Morphologies of taste buds, tongue papillae, and von Ebner’s glands were evaluated by light microscopy. Cell proliferation and rate of apoptosis in tongue specimens were examined by Ki67 immunostaining and fluorescent DNA fragmentation staining, respectively. Results The behavioral follow up of the mice in an IntelliCage system revealed that Car6−/− mice preferred 3 μM quinine (bitter) solution, whereas wild type mice preferred water. When the quinine concentration increased, both groups preferentially selected water. Histological analysis, Ki67 immunostaining and detection of apoptosis did not reveal any significant changes between tongue specimens of the knockout and wild type mice. Conclusions Our knockout mouse model confirms that CA VI is involved in bitter taste perception. CA VI may be one of the factors which contribute to avoidance of bitter, potentially harmful, substances. PMID:25134447

Grainyhead-like 3 (Grhl3) deficiency in brain leads to altered locomotor activity and decreased anxiety-like behaviors in aged mice.

PubMed

Dworkin, Sebastian; Auden, Alana; Partridge, Darren D; Daglas, Maria; Medcalf, Robert L; Mantamadiotis, Theo; Georgy, Smitha R; Darido, Charbel; Jane, Stephen M; Ting, Stephen B

2017-06-01

The highly conserved Grainyhead-like (Grhl) family of transcription factors, comprising three members in vertebrates (Grhl1-3), play critical regulatory roles during embryonic development, cellular proliferation, and apoptosis. Although loss of Grhl function leads to multiple neural abnormalities in numerous animal models, a comprehensive analysis of Grhl expression and function in the mammalian brain has not been reported. Here they show that only Grhl3 expression is detectable in the embryonic mouse brain; particularly within the habenula, an organ known to modulate repressive behaviors. Using both Grhl3-knockout mice (Grhl3 -/- ), and brain-specific conditional deletion of Grhl3 in adult mice (Nestin-Cre/Grhl3 flox/flox ), they performed histological expression analyses and behavioral tests to assess long-term effects of Grhl3 loss on motor co-ordination, spatial memory, anxiety, and stress. They found that complete deletion of Grhl3 did not lead to noticeable structural or cell-intrinsic defects in the embryonic brain; however, aged Grhl3 conditional knockout (cKO) mice showed enlarged lateral ventricles and displayed marked changes in motor function and behaviors suggestive of decreased fear and anxiety. They conclude that loss of Grhl3 in the brain leads to significant alterations in locomotor activity and decreased self-inhibition, and as such, these mice may serve as a novel model of human conditions of impulsive behavior or hyperactivity. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 775-788, 2017. © 2017 Wiley Periodicals, Inc.

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.

Mitochondrial gene expression and increased oxidative metabolism: role in increased lifespan of fat-specific insulin receptor knock-out mice

PubMed Central

Katic, Masa; Kennedy, Adam R.; Leykin, Igor; Norris, Andrew; McGettrick, Aileen; Gesta, Stephane; Russell, Steven J.; Bluher, Matthias; Maratos-Flier, Eleftheria; Kahn, C. Ronald

2009-01-01

Summary Caloric restriction, leanness and decreased activity of insulin/insulin-like growth factor 1 (IGF-1) receptor signaling are associated with increased longevity in a wide range of organisms from Caenorhabditis elegans to humans. Fat-specific insulin receptor knock-out (FIRKO) mice represent an interesting dichotomy, with leanness and increased lifespan, despite normal or increased food intake. To determine the mechanisms by which a lack of insulin signaling in adipose tissue might exert this effect, we performed physiological and gene expression studies in FIRKO and control mice as they aged. At the whole body level, FIRKO mice demonstrated an increase in basal metabolic rate and respiratory exchange ratio. Analysis of gene expression in white adipose tissue (WAT) of FIRKO mice from 6 to 36 months of age revealed persistently high expression of the nuclear-encoded mitochondrial genes involved in glycolysis, tricarboxylic acid cycle, β-oxidation and oxidative phosphorylation as compared to expression of the same genes in WAT from controls that showed a tendency to decline in expression with age. These changes in gene expression were correlated with increased cytochrome c and cytochrome c oxidase subunit IV at the protein level, increased citrate synthase activity, increased expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and PGC-1β, and an increase in mitochondrial DNA in WAT of FIRKO mice. Together, these data suggest that maintenance of mitochondrial activity and metabolic rates in adipose tissue may be important contributors to the increased lifespan of the FIRKO mouse. PMID:18001293

Maternal eNOS deficiency determines a fatty liver phenotype of the offspring in a sex dependent manner

PubMed Central

Hocher, Berthold; Haumann, Hannah; Rahnenführer, Jan; Reichetzeder, Christoph; Kalk, Philipp; Pfab, Thiemo; Tsuprykov, Oleg; Winter, Stefan; Hofmann, Ute; Li, Jian; Püschel, Gerhard P.; Lang, Florian; Schuppan, Detlef; Schwab, Matthias; Schaeffeler, Elke

2016-01-01

ABSTRACT Maternal environmental factors can impact on the phenotype of the offspring via the induction of epigenetic adaptive mechanisms. The advanced fetal programming hypothesis proposes that maternal genetic variants may influence the offspring's phenotype indirectly via epigenetic modification, despite the absence of a primary genetic defect. To test this hypothesis, heterozygous female eNOS knockout mice and wild type mice were bred with male wild type mice. We then assessed the impact of maternal eNOS deficiency on the liver phenotype of wild type offspring. Birth weight of male wild type offspring born to female heterozygous eNOS knockout mice was reduced compared to offspring of wild type mice. Moreover, the offspring displayed a sex specific liver phenotype, with an increased liver weight, due to steatosis. This was accompanied by sex specific differences in expression and DNA methylation of distinct genes. Liver global DNA methylation was significantly enhanced in both male and female offspring. Also, hepatic parameters of carbohydrate metabolism were reduced in male and female offspring. In addition, male mice displayed reductions in various amino acids in the liver. Maternal genetic alterations, such as partial deletion of the eNOS gene, can affect liver metabolism of wild type offspring without transmission of the intrinsic defect. This occurs in a sex specific way, with more detrimental effects in females. This finding demonstrates that a maternal genetic defect can epigenetically alter the phenotype of the offspring, without inheritance of the defect itself. Importantly, these acquired epigenetic phenotypic changes can persist into adulthood. PMID:27175980

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

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

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

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

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

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

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.

Effect of gravity change on the production of thrombopoietic growth factors.

PubMed

Fuse, A; Aoki, Y; Sato, T; Kita, M; Yamamoto, T; Toriizuka, K; Sunohara, M; Takeoka, H

2001-10-01

It is reported that the stay in the space develops anemia, thrombocytopenia, and altered function and structure of red blood cell. The mechanism of these abnormalities was not clarified yet. The cloning of the thrombopoietin (TPO), followed by the analysis of TPO and c-mpl (its cellular receptor) knockout mice confirmed its role as the primary regulator of thrombopoiesis. TPO has been shown to stimulate both megakaryocyte colony growth from marrow progenitor cells and the maturation of immature megakaryocyte to form functional platelet. This process includes the massive cytoskeletal rearrangement, such as proplatelet formation and fragmentation of proplatelet. In this study we have focused on the production of thrombopoietic growth factors in mice those were exposed to gravity change by parabolic flight (PF).

Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

NASA Technical Reports Server (NTRS)

Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

2002-01-01

Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

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.

Postnatal Loss of Mef2c Results in Dissociation of Effects on Synapse Number and Learning and Memory.

PubMed

Adachi, Megumi; Lin, Pei-Yi; Pranav, Heena; Monteggia, Lisa M

2016-07-15

Myocyte enhancer factor 2 (MEF2) transcription factors play critical roles in diverse cellular processes during central nervous system development. Studies attempting to address the role of MEF2 in brain have largely relied on overexpression of a constitutive MEF2 construct that impairs memory formation or knockdown of MEF2 function that increases spine numbers and enhances memory formation. Genetic deletion of individual MEF2 isoforms in brain during embryogenesis demonstrated that Mef2c loss negatively regulates spine numbers resulting in learning and memory deficits, possibly as a result of its essential role in development. To investigate MEF2C function in brain further, we genetically deleted Mef2c during postnatal development in mice. We characterized these conditional Mef2c knockout mice in an array of behavioral paradigms and examined the impact of postnatal loss of Mef2c on long-term potentiation. We observed increased spine numbers in hippocampus of the conditional Mef2c knockout mice. However, the postnatal loss of Mef2c did not impact learning and memory, long-term potentiation, or social and repetitive behaviors. Our findings demonstrate a critical role for MEF2C in the regulation of spine numbers with a dissociation of learning and memory, synaptic plasticity, and measures of autism-related behaviors in postnatal brain. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Activation of microglia induces symptoms of Parkinson’s disease in wild-type, but not in IL-1 knockout mice

PubMed Central

2013-01-01

Background Parkinson’s disease (PD) is an age-related progressive neurodegenerative disorder caused by selective loss of dopaminergic neurons from the substantia nigra (SN) to the striatum. The initial factor that triggers neurodegeneration is unknown; however, inflammation has been demonstrated to be significantly involved in the progression of PD. The present study was designed to investigate the role of the pro-inflammatory cytokine interleukin-1 (IL-1) in the activation of microglia and the decline of motor function using IL-1 knockout (KO) mice. Methods Lipopolysaccharide (LPS) was stereotaxically injected into the SN of mice brains as a single dose or a daily dose for 5 days (5 mg/2 ml/injection, bilaterally). Animal behavior was assessed with the rotarod test at 2 hr and 8, 15 and 22 days after the final LPS injection. Results LPS treatment induced the activation of microglia, as demonstrated by production of IL-1β and tumor necrosis factor (TNF) α as well as a change in microglial morphology. The number of cells immunoreactive for 4-hydroxynonenal (4HNE) and nitrotyrosine (NT), which are markers for oxidative insults, increased in the SN, and impairment of motor function was observed after the subacute LPS treatment. Cell death and aggregation of α-synuclein were observed 21 and 30 days after the final LPS injection, respectively. Behavioral deficits were observed in wild-type and TNFα KO mice, but IL-1 KO mice behaved normally. Tyrosine hydroxylase (TH) gene expression was attenuated by LPS treatment in wild-type and TNFα KO mice but not in IL-1 KO mice. Conclusions The subacute injection of LPS into the SN induces PD-like pathogenesis and symptoms in mice that mimic the progressive changes of PD including the aggregation of α-synuclein. LPS-induced dysfunction of motor performance was accompanied by the reduced gene expression of TH. These findings suggest that activation of microglia by LPS causes functional changes such as dopaminergic neuron attenuation in an IL-1-dependent manner, resulting in PD-like behavioral impairment. PMID:24289537

The antioxidant and anti-inflammatory activities of tocopherols are independent of Nrf2 in mice.

PubMed

Li, Guangxun; Lee, Mao-Jung; Liu, Anna Ba; Yang, Zhihong; Lin, Yong; Shih, Weichung Joe; Yang, Chung S

2012-04-01

The present study investigated the antioxidant and anti-inflammatory actions of tocopherols in mice and determined whether the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is involved in these activities. A mixture of tocopherols (γ-TmT) that is rich in γ-tocopherol was used. Nrf2 knockout (Nrf2 -/-) and wild-type mice were maintained on 0.03, 0.1, or 0.3% γ-TmT-enriched diet starting 2 weeks before the administration of dextran sulfate sodium (DSS) in drinking water (for 1 week, to induce colonic inflammation), until the termination of the experiment at 3 days after the DSS treatment. Dietary γ-TmT dose dependently lowered the levels of 8-oxo-deoxyguanosine, nitrotyrosine, inflammation index, and leukocyte infiltration in colon tissues, as well as 8-isoprostane and prostaglandin E2 in the serum, in both Nrf2 (-/-) and wild-type mice. No significant difference on the inhibitory actions of γ-TmT between the Nrf2 (-/-) and the wild-type mice was observed. The γ-TmT treatment significantly increased the serum levels of γ- and δ-tocopherols. Interestingly, the serum levels of tocopherol metabolites, specifically the γ- and δ-forms of carboxymethylbutyl hydroxychroman and carboxyethyl hydroxychroman, in Nrf2 (-/-) mice were significantly higher than those in wild-type mice. These findings suggest that the antioxidant and anti-inflammatory activities of γ-TmT in the colon are mostly due to the direct action of tocopherols in trapping reactive oxygen and nitrogen species, independent of the antioxidant enzymes and anti-inflammatory proteins that are regulated by Nrf2; however, Nrf2 knockout appears to affect the serum levels of tocopherol metabolites. Copyright © 2011. Published by Elsevier Inc.

Arrest of B16 Melanoma Cells in the Mouse Pulmonary Microcirculation Induces Endothelial Nitric Oxide Synthase-Dependent Nitric Oxide Release that Is Cytotoxic to the Tumor Cells

PubMed Central

Qiu, Hongming; Orr, F.William; Jensen, Derrek; Wang, Hui Helen; McIntosh, Alan R.; Hasinoff, Brian B.; Nance, Dwight M.; Pylypas, Susan; Qi, Ke; Song, Chun; Muschel, Ruth J.; Al-Mehdi, Abu-Bakr

2003-01-01

Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by NG-nitro-l-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only ∼55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis. PMID:12547699

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

Native-like aggregates of Factor VIII (FVIII) are immunogenic von Willebrand Factor deficient and hemophilia A mice

PubMed Central

Pisal, Dipak S.; Kosloski, Matthew P.; Middaugh, C. Russell; Bankert, Richard B.; Balu-Iyer, Sathy V.

2013-01-01

The administration of recombinant Factor VIII (FVIII) is the first line therapy for Hemophilia A (HA), but 25–35% of patients develop an inhibitory antibody response. In general, the presence of aggregates contributes to unwanted immunogenic responses against therapeutic proteins. FVIII has been shown to form both native-like and non-native aggregates. Previously, we showed that non-native aggregates of FVIII are less immunogenic compared to the native protein. Here we investigated the effect of native-like aggregates of FVIII on immunogenicity in HA and von Willebrand Factor knockout (vWF−/−) mice. Mice immunized with native-like aggregates showed significantly higher inhibitory antibody titers compared to animals that received native FVIII. Following re-stimulation in vitro with native FVIII, the activation of CD4+ T cells isolated from mice immunized with native-like aggregates is ~4 fold higher than mice immunized with the native protein. Furthermore, this is associated with increases in the secretion of pro-inflammatory cytokines IL-6 and IL-17 in the native-like aggregate treatment group. The results indicate that the native-like aggregates of FVIII are more immunogenic than native FVIII for both the B cell and T cell responses. PMID:22388918

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.

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

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

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

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

Defective heat shock factor 1 inhibits the growth of fibrosarcoma derived from simian virus 40/T antigen-transformed MEF cells

PubMed Central

JIANG, QIYING; ZHANG, ZHI; LI, SHULIAN; WANG, ZHAOYANG; MA, YUANFANG; HU, YANZHONG

2015-01-01

Heat shock factor 1 (Hsf1) serves an important role in regulating the proliferation of human tumor cell lines in vitro and tissue specific tumorigenesis in certain mouse models. However, its role in viral-oncogenesis remains to be fully elucidated. In the current study, the role of Hsf1 in fibroblastoma derived from simian virus 40/T antigen (SV40/TAG)-transformed mouse embryonic fibroblast (MEF) cell lines was investigated. Knockout of Hsf1 inhibited MEF cell proliferation in vitro and fibroblastoma growth and metastasis to the lungs in vivo in nude mice. Knockout of Hsf1 increased the protein expression levels of p53 and phosphorylated retinoblastoma protein (pRb), however reduced the expression of heat shock protein 25 (Hsp25) in addition to the expression of the angiogenesis markers vascular endothelial growth factor, cluster of differentiation 34 and factor VIII related antigen. Furthermore, immunoprecipitation indicated that knockout of Hsf1 inhibited the association between SV40/TAG and p53 or pRb. These data suggest that Hsf1 is involved in the regulation of SV40/TAG-derived fibroblastoma growth and metastasis by modulating the association between SV40/TAG and tumor suppressor p53 and pRb. The current study provides further evidence that Hsf1 may be a novel therapeutic target in the treatment of cancer. PMID:26352782

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.

Macrophage activation by heparanase is mediated by TLR-2 and TLR-4 and associates with plaque progression.

PubMed

Blich, Miry; Golan, Amnon; Arvatz, Gil; Sebbag, Anat; Shafat, Itay; Sabo, Edmond; Cohen-Kaplan, Victoria; Petcherski, Sirouch; Avniel-Polak, Shani; Eitan, Amnon; Hammerman, Haim; Aronson, Doron; Axelman, Elena; Ilan, Neta; Nussbaum, Gabriel; Vlodavsky, Israel

2013-02-01

Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages. Highly purified heparanase was added to mouse peritoneal macrophages and macrophage-like J774 cells, and the levels of tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll-like receptor-2 and Toll-like receptor-4 knockout mice were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction, stable angina, and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with stable angina or acute myocardial infarction. Addition or overexpression of heparanase variants resulted in marked increase in tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 levels. Mouse peritoneal macrophages harvested from Toll-like receptor-2 or Toll-like receptor-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute myocardial infarction, compared with patients with stable angina and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared with specimens of stable plaque and controls. Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression toward vulnerability.

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.

Translational Mouse Models of Autism: Advancing Toward Pharmacological Therapeutics

PubMed Central

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

2016-01-01

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

Disruption of the midkine gene (Mdk) resulted in altered expression of a calcium binding protein in the hippocampus of infant mice and their abnormal behaviour.

PubMed

Nakamura, E; Kadomatsu, K; Yuasa, S; Muramatsu, H; Mamiya, T; Nabeshima, T; Fan, Q W; Ishiguro, K; Igakura, T; Matsubara, S; Kaname, T; Horiba, M; Saito, H; Muramatsu, T

1998-12-01

Midkine (MK) is a growth factor implicated in the development and repair of various tissues, especially neural tissues. However, its in vivo function has not been clarified. Knockout mice lacking the MK gene (Mdk) showed no gross abnormalities. We closely analysed postnatal brain development in Mdk(-/-) mice using calcium binding proteins as markers to distinguish neuronal subpopulations. Intense and prolonged calretinin expression was found in the dentate gyrus granule cell layer of the hippocampus of infant Mdk(-/-) mice. In infant Mdk(+/+) mice, calretinin expression in the granule cell layer was weaker, and had disappeared by 4 weeks after birth, when calretinin expression still persisted in Mdk(-/-) mice. Furthermore, 4 weeks after birth, Mdk(-/-) mice showed a deficit in their working memory, as revealed by a Y-maze test, and had an increased anxiety, as demonstrated by the elevated plus-maze test. Midkine plays an important role in the regulation of postnatal development of the hippocampus.

Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice

PubMed Central

Hattori, Satoko; Takao, Keizo; Tanda, Koichi; Toyama, Keiko; Shintani, Norihito; Baba, Akemichi; Hashimoto, Hitoshi; Miyakawa, Tsuyoshi

2012-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1). Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory. PMID:23060763

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...

Targeted Disruption of Pancreatic-Derived Factor (PANDER, FAM3B) Impairs Pancreatic β-Cell Function

PubMed Central

Robert-Cooperman, Claudia E.; Carnegie, Jason R.; Wilson, Camella G.; Yang, Jichun; Cook, Joshua R.; Wu, Jianmei; Young, Robert A.; Wolf, Bryan A.; Burkhardt, Brant R.

2010-01-01

OBJECTIVE Pancreatic-derived factor (PANDER, FAM3B) is a pancreatic islet-specific cytokine-like protein that is secreted from β-cells upon glucose stimulation. The biological function of PANDER is unknown, and to address this we generated and characterized a PANDER knockout mouse. RESEARCH DESIGN AND METHODS To generate the PANDER knockout mouse, the PANDER gene was disrupted and its expression was inhibited by homologous recombination via replacement of the first two exons, secretion signal peptide and transcriptional start site, with the neomycin gene. PANDER−/− mice were then phenotyped by a number of in vitro and in vivo tests to evaluate potential effects on glucose regulation, insulin sensitivity, and β-cell morphology and function. RESULTS Glucose tolerance tests demonstrated significantly higher blood glucose levels in PANDER−/− versus wild-type male mice. To identify the mechanism of the glucose intolerance, insulin sensitivity and pancreatic β-cell function were examined. Hyperinsulinemic-euglycemic clamps and insulin tolerance testing showed similar insulin sensitivity for both the PANDER−/− and wild-type mice. The in vivo insulin response following intraperitoneal glucose injection surprisingly produced significantly higher insulin levels in the PANDER−/− mice, whereas insulin release was blunted with arginine administration. Islet perifusion and calcium imaging studies showed abnormal responses of the PANDER−/− islets to glucose stimulation. In contrast, neither islet architecture nor insulin content was impacted by the loss of PANDER. Interestingly, the elevated insulin levels identified in vivo were attributed to decreased hepatic insulin clearance in the PANDER−/− islets. Taken together, these results demonstrated decreased pancreatic β-cell function in the PANDER−/− mouse. CONCLUSIONS These results support a potential role of PANDER in the pancreatic β-cell for regulation or facilitation of insulin secretion. PMID:20566664

Fibroblast growth factor 21 is not required for glucose homeostasis, ketosis and tumour suppression associated to ketogenic diets in mice

PubMed Central

Stemmer, Kerstin; Zani, Fabio; Habegger, Kirk M.; Neff, Christina; Kotzbeck, Petra; Bauer, Michaela; Yalamanchilli, Suma; Azad, Ali; Lehti, Maarit; Martins, Paulo J.F.; Müller, Timo D.; Pfluger, Paul T.; Seeley, Randy J.

2016-01-01

AIMS/HYPOTHESIS Ketogenic diets (KDs) increasingly gained attention as effective means for weight loss and potential adjunctive treatment of cancer. Metabolic benefits of KDs are regularly ascribed towards enhanced hepatic secretion of fibroblast growth factor (FGF) 21, and its systemic effects on fatty acid oxidation, energy expenditure and body weight. Ambiguous data from Fgf21 knockout strains and low FGF21 concentrations reported for humans in ketosis have nevertheless cast doubt regarding the endogenous function of FGF21. We here aimed to elucidate the causal role of FGF21 in mediating therapeutic benefits of KDs on metabolism and cancer. METHODS We established a dietary model of increased vs. decreased FGF21 by feeding C57BL/6J mice with KDs, either depleted or enriched with protein, respectively. We furthermore used wild type and Fgf21 knockout mice that were subjected to the respective diets, and monitored energy and glucose homeostasis as well as tumor growth after transplantation of Lewis-Lung-Carcinoma cells. RESULTS Hepatic and circulating but not adipose tissue FGF21 levels were profoundly increased by protein starvation and independent of the state of ketosis. We demonstrate that endogenous FGF21 is not essential for the maintenance of normoglycemia upon protein and carbohydrate starvation and is dispensable for the effects of KDs on energy expenditure. Furthermore, the tumor-suppressing effects of KDs were independent from FGF21, and rather driven by concomitant protein and carbohydrate starvation. CONCLUSION/INTERPRETATION Our data indicate that multiple systemic effects of KDs exposure in mice that were previously ascribed towards increased FGF21 secretion are rather a consequence of protein malnutrition. PMID:26099854

BDNF — a key transducer of antidepressant effects

PubMed Central

Björkholm, Carl; Monteggia, Lisa M.

2016-01-01

How do antidepressants elicit an antidepressant response? Here, we review accumulating evidence that the neurotrophin brain-derived neurotrophic factor (BDNF) serves as a transducer, acting as the link between the antidepressant drug and the neuroplastic changes that result in the improvement of the depressive symptoms. Over the last decade several studies have consistently highlighted BDNF as a key player in antidepressant action. An increase in hippocampal and cortical expression of BDNF mRNA parallels the antidepressant-like response of conventional antidepressants such as SSRIs. Subsequent studies showed that a single bilateral infusion of BDNF into the ventricles or directly into the hippocampus is sufficient to induce a relatively rapid and sustained antidepressant-like effect. Importantly, the antidepressant-like response to conventional antidepressants is attenuated in mice where the BDNF signaling has been disrupted by genetic manipulations. Low dose ketamine, which has been found to induce a rapid antidepressant effect in patients with treatment-resistant depression, is also dependent on increased BDNF signaling. Ketamine transiently increases BDNF translation in hippocampus, leading to enhanced synaptic plasticity and synaptic strength. Ketamine has been shown to increase BDNF translation by blocking NMDA receptor activity at rest, thereby inhibiting calcium influx and subsequently halting eukaryotic elongation factor 2 (eEF2) kinase leading to a desuppression of protein translation, including BDNF translation. The antidepressant-like response of ketamine is abolished in BDNF and TrkB conditional knockout mice, eEF2 kinase knockout mice, in mice carrying the BDNF met/met allele, and by intra-cortical infusions of BDNF-neutralizing antibodies. In summary, current data suggests that conventional antidepressants and ketamine mediate their antidepressant-like effects by increasing BDNF in forebrain regions, in particular the hippocampus, making BDNF an essential determinant of antidepressant efficacy. PMID:26519901

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

Sundar, Isaac K.; Hwang, Jae-Woong; Wu, Shaoping

Research highlights: {yields} Vitamin D deficiency is linked to accelerated decline in lung function. {yields} Levels of vitamin D receptor (VDR) are decreased in lungs of patients with COPD. {yields} VDR knock-out mouse showed increased lung inflammation and emphysema. {yields} This was associated with decline in lung function and increased MMPs. {yields} VDR knock-out mouse model is useful for studying the mechanisms of lung diseases. -- Abstract: Deficiency of vitamin D is associated with accelerated decline in lung function. Vitamin D is a ligand for nuclear hormone vitamin D receptor (VDR), and upon binding it modulates various cellular functions. Themore » level of VDR is reduced in lungs of patients with chronic obstructive pulmonary disease (COPD) which led us to hypothesize that deficiency of VDR leads to significant alterations in lung phenotype that are characteristics of COPD/emphysema associated with increased inflammatory response. We found that VDR knock-out (VDR{sup -/-}) mice had increased influx of inflammatory cells, phospho-acetylation of nuclear factor-kappaB (NF-{kappa}B) associated with increased proinflammatory mediators, and up-regulation of matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MMP-12 in the lung. This was associated with emphysema and decline in lung function associated with lymphoid aggregates formation compared to WT mice. These findings suggest that deficiency of VDR in mouse lung can lead to an early onset of emphysema/COPD because of chronic inflammation, immune dysregulation, and lung destruction.« less

Induction of the plasticity-associated immediate early gene Arc by stress and hallucinogens: role of brain-derived neurotrophic factor.

PubMed

Benekareddy, Madhurima; Nair, Amrita R; Dias, Brian G; Suri, Deepika; Autry, Anita E; Monteggia, Lisa M; Vaidya, Vidita A

2013-03-01

Exposure to stress and hallucinogens in adulthood evokes persistent alterations in neurocircuitry and emotional behaviour. The structural and functional changes induced by stress and hallucinogen exposure are thought to involve transcriptional alterations in specific effector immediate early genes. The immediate early gene, activity regulated cytoskeletal-associated protein (Arc), is important for both activity and experience dependent plasticity. We sought to examine whether trophic factor signalling through brain-derived neurotrophic factor (BDNF) contributes to the neocortical regulation of Arc mRNA in response to distinct stimuli such as immobilization stress and the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI). Acute exposure to either immobilization stress or DOI induced Arc mRNA levels within the neocortex. BDNF infusion into the neocortex led to a robust up-regulation of local Arc transcript expression. Further, baseline Arc mRNA expression in the neocortex was significantly decreased in inducible BDNF knockout mice with an adult-onset, forebrain specific BDNF loss. The induction of Arc mRNA levels in response to both acute immobilization stress or a single administration of DOI was significantly attenuated in the inducible BDNF knockout mice. Taken together, our results implicate trophic factor signalling through BDNF in the regulation of cortical Arc mRNA expression, both under baseline conditions and following stress and hallucinogen exposure. These findings suggest the possibility that the regulation of Arc expression via BDNF provides a molecular substrate for the structural and synaptic plasticity observed following stimuli such as stress and hallucinogens.

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

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.

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

Involvement of prostaglandins and histamine in nickel wire-induced acute inflammation in mice.

PubMed

Hirasawa, Noriyasu; Goi, Yoshiaki; Tanaka, Rina; Ishihara, Kenji; Ohtsu, Hiroshi; Ohuchi, Kazuo

2010-06-15

The irritancy of Nickel (Ni) ions has been well documented clinically. However, the chemical mediators involved in the acute inflammation induced by solid Ni are not fully understood. We used the Ni wire-implantation model in mice and examined roles of prostaglandins and histamine in plasma leakage in the acute phase. The subcutaneous implantation of a Ni wire into the back of mice induced plasma leakage from 8 to 24 h and tissue necrosis around the wire at 3 days, whereas the implantation of an aluminum wire induced no such inflammatory responses. An increase in the mRNA for cyclooxygenase (COX)-2 and HDC in cells around the Ni wire was detected 4 h after the implantation. The leakage of plasma at 8 h was inhibited by indomethacin in a dose-dependent manner. Dexamethasone and the p38 MAP kinase inhibitor SB203580 also inhibited the exudation of plasma consistent with the inhibition of the expression of COX-2 mRNA. Furthermore, plasma leakage was partially but siginificantly reduced in histamine H1 receptor knockout mice and histidine decarboxylase (HDC) knockout mice but not in H2 receptor knockout mice. These results suggested that the Ni ions released from the wire induced the expression of COX-2 and HDC, resulting in an increase in vascular permeability during the acute phase of inflammation. (c) 2009 Wiley Periodicals, Inc.

Lethal Crimean-Congo hemorrhagic fever virus infection in interferon α/β receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy.

PubMed

Zivcec, Marko; Safronetz, David; Scott, Dana; Robertson, Shelly; Ebihara, Hideki; Feldmann, Heinz

2013-06-15

Crimean-Congo hemorrhagic fever (CCHF) is a widely distributed viral hemorrhagic fever characterized by rapid onset of flu-like symptoms often followed by hemorrhagic manifestations. CCHF virus (CCHFV), a bunyavirus in the Nairovirus genus, is capable of infecting a wide range of mammalian hosts in nature but so far only causes disease in humans. Recently, immunocompromised mice have been reported as CCHF disease models, but detailed characterization is lacking. Here, we closely followed infection and disease progression in CCHFV-infected interferon α/β receptor knockout (IFNAR(-/-)) mice and age-matched wild-type (WT) mice. WT mice quickly clear CCHFV without developing any disease signs. In contrast, CCHFV infected IFNAR(-/-) mice develop an acute fulminant disease with high viral loads leading to organ pathology (liver and lymphoid tissues), marked proinflammatory host responses, severe thrombocytopenia, coagulopathy, and death. Disease progression closely mimics hallmarks of human CCHF disease, making IFNAR(-/-) mice an excellent choice to assess medical countermeasures.

Knockout of Foxp2 disrupts vocal development in mice

PubMed Central

Castellucci, Gregg A.; McGinley, Matthew J.; McCormick, David A.

2016-01-01

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. PMID:26980647

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.

MRI of Retinal Free Radical Production With Laminar Resolution In Vivo

PubMed Central

Berkowitz, Bruce A.; Lewin, Alfred S.; Biswal, Manas R.; Bredell, Bryce X.; Davis, Christopher; Roberts, Robin

2016-01-01

Purpose Recent studies have suggested the hypothesis that quench-assisted 1/T1 magnetic resonance imaging (MRI) measures free radical production with laminar resolution in vivo without the need of a contrast agent. Here, we test this hypothesis further by examining the spatial and detection sensitivity of quench-assisted 1/T1 MRI to strain, age, or retinal cell layer-specific genetic manipulations. Methods We studied: adult wild-type mice; mice at postnatal day 7 (P7); cre dependent retinal pigment epithelium (RPE)-specific MnSOD knockout mice; doxycycline-treated Sod2flox/flox mice lacking the cre transgene; and α-transducin knockout (Gnat1−/−) mice on a C57Bl/6 background. Transretinal 1/T1 profiles were mapped in vivo in the dark without or with antioxidant treatment, or followed by light exposure. We calibrated profiles spatially using optical coherence tomography. Results Dark-adapted RPE-specific MnSOD knockout mice had greater than normal 1/T1 in the RPE and outer nuclear layers that was corrected to wild-type levels by antioxidant treatment. Dark and light Gnat1−/− mice also had greater than normal outer retinal 1/T1 values. In adult wild-type mice, dark values of 1/T1 in the ellipsoid region and in the outer segment were suppressed by 13 minutes of light. By 29 minutes of light, 1/T1 reduction extended to the outer nuclear layer. Gnat1−/− mice demonstrated a faster light-evoked suppression of 1/T1 values in the outer retina. In P7 mice, transretinal 1/T1 profiles were the same in dark and light. Conclusions Quench-assisted MRI has the laminar resolution and detection sensitivity to evaluate normal and pathologic production of free radicals in vivo. PMID:26886890

Norepinephrine Transporter Heterozygous Knockout Mice Exhibit Altered Transport and Behavior

PubMed Central

Fentress, HM; Klar, R; Krueger, JK; Sabb, T; Redmon, SN; Wallace, NM; Shirey-Rice, JK; Hahn, MK

2013-01-01

The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically-driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET+/−), demonstrating that they display an ~50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity, assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface-resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET+/− mouse establishes an activated state of existing, surface NET proteins. NET+/− mice exhibit increased anxiety in the open field and light-dark box and display deficits in reversal learning in the Morris Water Maze. These data suggest recovery of near basal activity in NET+/− mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET+/− mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders. PMID:24102798

Lack of Neuropathy-Related Phenotypes in Hint1 Knockout Mice

PubMed Central

Seburn, Kevin L.; Morelli, Kathryn H.; Jordanova, Albena; Burgess, Robert W.

2014-01-01

Mutations in HINT1, the gene encoding histidine triad nucleotide-binding protein 1 (HINT1), cause a recessively inherited peripheral neuropathy that involves primarily motor dysfunction and is usually associated with neuromyotonia, i.e. prolonged muscle contraction resulting from hyperexcitability of the peripheral nerve. Because these mutations are hypothesized to cause loss of function, we analyzed Hint1 knockout mice for their relevance as a disease model. Mice lacking Hint1 were normal in appearance and in behavioral tests or motor performance, although they moved slower and for a smaller fraction of time than wild-type (WT) mice in an open field arena. Muscles, neuromuscular junctions, and nodes of Ranvier are anatomically normal and did not show evidence of degeneration or regeneration. Axon numbers and myelination in peripheral nerves were normal at 4 and 13 months of age. Axons were slightly smaller than those in WT mice at 4 months of age, but this did not cause a decrease in conduction velocity, and no differences in axon diameters were detected at 13 months. Using electromyography, we were unable to detect neuromyotonia, even using supra-physiological stimuli and stressors such as reduced temperature or 3,4 diaminopyridine to block potassium channels. Therefore, we conclude that Hint1 knockout mice may be useful for studying the biochemical activities of HINT1, but these mice do not provide a disease model or a means for investigating the basis of HINT1-associated neuropathy and neuromyotonia. PMID:24918641

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.

Stress-induced analgesia and morphine responses are changed in catechol-O-methyltransferase-deficient male mice.

PubMed

Kambur, Oleg; Männistö, Pekka T; Viljakka, Kaarin; Reenilä, Ilkka; Lemberg, Kim; Kontinen, Vesa K; Karayiorgou, Maria; Gogos, Joseph A; Kalso, Eija

2008-10-01

Catechol-O-methyltransferase (COMT) polymorphisms modulate pain and opioid analgesia in human beings. It is not clear how the effects of COMT are mediated and only few relevant animal studies have been performed. Here, we used old male Comt gene knock-out mice as an animal model to study the effects of COMT deficiency on nociception that was assessed by the hot plate and tail flick tests. Stress-induced analgesia was achieved by forced swim. Morphine antinociception was measured after 10 mg/kg of morphine subcutaneously. Morphine tolerance was produced with subcutaneous morphine pellets and withdrawal provoked with subcutaneous naloxone. In the hot plate test, morphine-induced antinociception was significantly greater in the COMT knock-out mice, compared to the wild-type mice. This may be due to increased availability of opioid receptors as suggested by previous human studies. In the tail flick test, opioid-mediated stress-induced analgesia was absent and morphine-induced analgesia was decreased in COMT knock-out mice. In the hot plate test, stress-induced analgesia developed to all mice regardless of the COMT genotype. There were no differences between the genotypes in the baseline nociceptive thresholds, morphine tolerance and withdrawal. Our findings show, for the first time, the importance of COMT activity in stress- and morphine-induced analgesia in mice. COMT activity seems to take part in the modulation of nociception not only in the brain, as suggested earlier, but also at the spinal/peripheral level.

TAM receptor knockout mice are susceptible to retinal autoimmune induction.

PubMed

Ye, Fei; Li, Qiutang; Ke, Yan; Lu, Qingjun; Han, Lixia; Kaplan, Henry J; Shao, Hui; Lu, Qingxian

2011-06-16

TAM receptors are expressed mainly by dendritic cells and macrophages in the immune system, and mice lacking TAM receptors develop systemic autoimmune diseases because of inefficient negative control of the cytokine signaling in those cells. This study aims to test the susceptibility of the TAM triple knockout (tko) mice to the retina-specific autoantigen to develop experimental autoimmune uveoretinitis (EAU). TAM tko mice that were or were not immunized with interphotoreceptor retinoid-binding protein (IRBP) peptides were evaluated for retinal infiltration of the macrophages and CD3(+) T cells by immunohistochemistry, spontaneous activation of CD4(+) T cells, and memory T cells by flow cytometry and proliferation of IRBP-specific CD4(+) T cells by [(3)H]thymidine incorporation assay. Ocular inflammation induced by IRBP peptide immunization and specific T cell transfer were observed clinically by funduscopy and confirmed by histology. Tko mice were found to have less naive, but more activated, memory T cells, among which were exhibited high sensitivity to ocular IRBP autoantigens. Immunization with a low dose of IRBP and adoptive transfer of small numbers of IRBP-specific T cells from immunized tko mice caused the infiltration of lymphocytes, including CD3(+) T cells, into the tko retina. Mice without TAM receptor spontaneously develop IRBP-specific CD4(+) T cells and are more susceptible to retinal autoantigen immunization. This TAM knockout mouse line provides an animal model with which to study the role of antigen-presenting cells in the development of T cell-mediated uveitis.

Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury.

PubMed

Nakamura, Yuka; Fujita, Yuki; Ueno, Masaki; Takai, Toshiyuki; Yamashita, Toshihide

2011-01-21

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.

Dietary xenosterols lead to infertility and loss of abdominal adipose tissue in sterolin-deficient mice[S

PubMed Central

Solca, Curzio; Tint, G. Stephen; Patel, Shailendra B.

2013-01-01

The investigation of the human disease sitosterolemia (MIM 210250) has shed light not only on the pathways by which dietary sterols may traffic but also on how the mammalian body rids itself of cholesterol and defends against xenosterols. Two genes, ABCG5 and ABCG8, located at the sitosterolemia locus, each encodes a membrane-bound ABC half-transporter and constitutes a functional unit whose activity has now been shown to account for biliary and intestinal sterol excretion. Knockout mice deficient in Abcg5 or Abcg8 recapitulate many of the phenotypic features of sitosterolemia. During the course of our studies to characterize these knockout mice, we noted that these mice, raised on normal rodent chow, exhibited infertility as well as loss of abdominal fat. We show that, although sitosterolemia does not lead to any structural defects or to any overt endocrine defects, fertility could be restored if xenosterols are specifically blocked from entry and that the loss of fat is also reversed by a variety of maneuvers that limit xenosterol accumulation. These studies show that xenosterols may have a significant biological impact on normal mammalian physiology and that the Abcg5 or Abcg8 knockout mouse model may prove useful in investigating the role of xenosterols on mammalian physiology. PMID:23180829

p53-upregulated-modulator-of-apoptosis (PUMA) deficiency affects food intake but does not impact on body weight or glucose homeostasis in diet-induced obesity.

PubMed Central

Litwak, Sara A.; Loh, Kim; Stanley, William J.; Pappas, Evan G.; Wali, Jibran A.; Selck, Claudia; Strasser, Andreas; Thomas, Helen E.; Gurzov, Esteban N.

2016-01-01

BCL-2 proteins have been implicated in the control of glucose homeostasis and metabolism in different cell types. Thus, the aim of this study was to determine the role of the pro-apoptotic BH3-only protein, p53-upregulated-modulator-of-apoptosis (PUMA), in metabolic changes mediated by diet-induced obesity, using PUMA deficient mice. At 10 weeks of age, knockout and wild type mice either continued consuming a low fat chow diet (6% fat), or were fed with a high fat diet (23% fat) for 14–17 weeks. We measured body composition, glucose and insulin tolerance, insulin response in peripheral tissues, energy expenditure, oxygen consumption, and respiratory exchange ratio in vivo. All these parameters were indistinguishable between wild type and knockout mice on chow diet and were modified equally by diet-induced obesity. Interestingly, we observed decreased food intake and ambulatory capacity of PUMA knockout mice on high fat diet. This was associated with increased adipocyte size and fasted leptin concentration in the blood. Our findings suggest that although PUMA is dispensable for glucose homeostasis in lean and obese mice, it can affect leptin levels and food intake during obesity. PMID:27033313

p53-upregulated-modulator-of-apoptosis (PUMA) deficiency affects food intake but does not impact on body weight or glucose homeostasis in diet-induced obesity.

PubMed

Litwak, Sara A; Loh, Kim; Stanley, William J; Pappas, Evan G; Wali, Jibran A; Selck, Claudia; Strasser, Andreas; Thomas, Helen E; Gurzov, Esteban N

2016-04-01

BCL-2 proteins have been implicated in the control of glucose homeostasis and metabolism in different cell types. Thus, the aim of this study was to determine the role of the pro-apoptotic BH3-only protein, p53-upregulated-modulator-of-apoptosis (PUMA), in metabolic changes mediated by diet-induced obesity, using PUMA deficient mice. At 10 weeks of age, knockout and wild type mice either continued consuming a low fat chow diet (6% fat), or were fed with a high fat diet (23% fat) for 14-17 weeks. We measured body composition, glucose and insulin tolerance, insulin response in peripheral tissues, energy expenditure, oxygen consumption, and respiratory exchange ratio in vivo. All these parameters were indistinguishable between wild type and knockout mice on chow diet and were modified equally by diet-induced obesity. Interestingly, we observed decreased food intake and ambulatory capacity of PUMA knockout mice on high fat diet. This was associated with increased adipocyte size and fasted leptin concentration in the blood. Our findings suggest that although PUMA is dispensable for glucose homeostasis in lean and obese mice, it can affect leptin levels and food intake during obesity.

INDUCTION OF MAMMARY GLAND DEVELOPMENT IN ESTROGEN RECEPTOR-ALPHA KNOCKOUT MICE

EPA Science Inventory

Mammary glands from the estrogen receptor knockout ( ERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted Er signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute t...

Class IA phosphoinositide 3-kinase regulates heart size and physiological cardiac hypertrophy.

PubMed

Luo, Ji; McMullen, Julie R; Sobkiw, Cassandra L; Zhang, Li; Dorfman, Adam L; Sherwood, Megan C; Logsdon, M Nicole; Horner, James W; DePinho, Ronald A; Izumo, Seigo; Cantley, Lewis C

2005-11-01

Class I(A) phosphoinositide 3-kinases (PI3Ks) are activated by growth factor receptors, and they regulate, among other processes, cell growth and organ size. Studies using transgenic mice overexpressing constitutively active and dominant negative forms of the p110alpha catalytic subunit of class I(A) PI3K have implicated the role of this enzyme in regulating heart size and physiological cardiac hypertrophy. To further understand the role of class I(A) PI3K in controlling heart growth and to circumvent potential complications from the overexpression of dominant negative and constitutively active proteins, we generated mice with muscle-specific deletion of the p85alpha regulatory subunit and germ line deletion of the p85beta regulatory subunit of class I(A) PI3K. Here we show that mice with cardiac deletion of both p85 subunits exhibit attenuated Akt signaling in the heart, reduced heart size, and altered cardiac gene expression. Furthermore, exercise-induced cardiac hypertrophy is also attenuated in the p85 knockout hearts. Despite such defects in postnatal developmental growth and physiological hypertrophy, the p85 knockout hearts exhibit normal contractility and myocardial histology. Our results therefore provide strong genetic evidence that class I(A) PI3Ks are critical regulators for the developmental growth and physiological hypertrophy of the heart.

Impaired spine formation and learning in GPCR kinase 2 interacting protein-1 (GIT1) knockout mice.

PubMed

Menon, Prashanthi; Deane, Rashid; Sagare, Abhay; Lane, Steven M; Zarcone, Troy J; O'Dell, Michael R; Yan, Chen; Zlokovic, Berislav V; Berk, Bradford C

2010-03-04

The G-protein coupled receptor (GPCR)-kinase interacting proteins 1 and 2 (GIT1 and GIT2) are scaffold proteins with ADP-ribosylating factor GTPase activity. GIT1 and GIT2 control numerous cellular functions and are highly expressed in neurons, endothelial cells and vascular smooth muscle cells. GIT1 promotes dendritic spine formation, growth and motility in cultured neurons, but its role in brain in vivo is unknown. By using global GIT1 knockout mice (GIT1 KO), we show that compared to WT controls, deletion of GIT1 results in markedly reduced dendritic length and spine density in the hippocampus by 36.7% (p<0.0106) and 35.1% (p<0.0028), respectively. This correlated with their poor adaptation to new environments as shown by impaired performance on tasks dependent on learning. We also studied the effect of GIT1 gene deletion on brain microcirculation. In contrast to findings in systemic circulation, GIT1 KO mice had an intact blood-brain barrier and normal regional cerebral blood flow as determined with radiotracers. Thus, our data suggest that GIT1 plays an important role in brain in vivo by regulating spine density involved in synaptic plasticity that is required for processes involved in learning. 2009 Elsevier B.V. All rights reserved.

A muscle-specific knockout implicates nuclear receptor coactivator MED1 in the regulation of glucose and energy metabolism.

PubMed

Chen, Wei; Zhang, Xiaoting; Birsoy, Kivanc; Roeder, Robert G

2010-06-01

As conventional transcriptional factors that are activated in diverse signaling pathways, nuclear receptors play important roles in many physiological processes that include energy homeostasis. The MED1 subunit of the Mediator coactivator complex plays a broad role in nuclear receptor-mediated transcription by anchoring the Mediator complex to diverse promoter-bound nuclear receptors. Given the significant role of skeletal muscle, in part through the action of nuclear receptors, in glucose and fatty acid metabolism, we generated skeletal muscle-specific Med1 knockout mice. Importantly, these mice show enhanced insulin sensitivity and improved glucose tolerance as well as resistance to high-fat diet-induced obesity. Furthermore, the white muscle of these mice exhibits increased mitochondrial density and expression of genes specific to type I and type IIA fibers, indicating a fast-to-slow fiber switch, as well as markedly increased expression of the brown adipose tissue-specific UCP-1 and Cidea genes that are involved in respiratory uncoupling. These dramatic results implicate MED1 as a powerful suppressor in skeletal muscle of genetic programs implicated in energy expenditure and raise the significant possibility of therapeutical approaches for metabolic syndromes and muscle diseases through modulation of MED1-nuclear receptor interactions.

MOXI Is a Mitochondrial Micropeptide That Enhances Fatty Acid β-Oxidation.

PubMed

Makarewich, Catherine A; Baskin, Kedryn K; Munir, Amir Z; Bezprozvannaya, Svetlana; Sharma, Gaurav; Khemtong, Chalermchai; Shah, Akansha M; McAnally, John R; Malloy, Craig R; Szweda, Luke I; Bassel-Duby, Rhonda; Olson, Eric N

2018-06-26

Micropeptide regulator of β-oxidation (MOXI) is a conserved muscle-enriched protein encoded by an RNA transcript misannotated as non-coding. MOXI localizes to the inner mitochondrial membrane where it associates with the mitochondrial trifunctional protein, an enzyme complex that plays a critical role in fatty acid β-oxidation. Isolated heart and skeletal muscle mitochondria from MOXI knockout mice exhibit a diminished ability to metabolize fatty acids, while transgenic MOXI overexpression leads to enhanced β-oxidation. Additionally, hearts from MOXI knockout mice preferentially oxidize carbohydrates over fatty acids in an isolated perfused heart system compared to wild-type (WT) animals. MOXI knockout mice also exhibit a profound reduction in exercise capacity, highlighting the role of MOXI in metabolic control. The functional characterization of MOXI provides insight into the regulation of mitochondrial metabolism and energy homeostasis and underscores the regulatory potential of additional micropeptides that have yet to be identified. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Dlgap1 knockout mice exhibit alterations of the postsynaptic density and selective reductions in sociability.

PubMed

Coba, M P; Ramaker, M J; Ho, E V; Thompson, S L; Komiyama, N H; Grant, S G N; Knowles, J A; Dulawa, S C

2018-02-02

The scaffold protein DLGAP1 is localized at the post-synaptic density (PSD) of glutamatergic neurons and is a component of supramolecular protein complexes organized by PSD95. Gain-of-function variants of DLGAP1 have been associated with obsessive-compulsive disorder (OCD), while haploinsufficient variants have been linked to autism spectrum disorder (ASD) and schizophrenia in human genetic studies. We tested male and female Dlgap1 wild type (WT), heterozygous (HT), and knockout (KO) mice in a battery of behavioral tests: open field, dig, splash, prepulse inhibition, forced swim, nest building, social approach, and sucrose preference. We also used biochemical approaches to examine the role of DLGAP1 in the organization of PSD protein complexes. Dlgap1 KO mice were most notable for disruption of protein interactions in the PSD, and deficits in sociability. Other behavioral measures were largely unaffected. Our data suggest that Dlgap1 knockout leads to PSD disruption and reduced sociability, consistent with reports of DLGAP1 haploinsufficient variants in schizophrenia and ASD.