Het Mice Research Articles

μ‐Opioid receptors mediate the effects of chronic ethanol binge drinking on the hippocampal neurogenic niche

Ethanol exposure and withdrawal alter the generation of new neurons in the adult hippocampus. The endogenous opioid system, particularly the μ-opioid receptor (MOR), can modulate neural progenitors and also plays a critical role in ethanol drinking and dependence. In the present study, we sought to determine whether MOR contributes to the effects of ethanol on the dentate gyrus (DG) neurogenic niche. MOR wild-type (WT), heterozygous (Het) and knockout (KO) littermates were subjected to voluntary ethanol drinking in repeated limited-access two-bottle choice (2BC) sessions. MOR deficiency did not alter progenitor proliferation, neuronal differentiation and maturation, apoptosis or microglia in ethanol-naïve mice. When exposed to five consecutive weeks of 2BC, MOR mutant mice exhibited a gene-dosage-dependent reduction of ethanol consumption compared with WT mice. Introducing a week of ethanol deprivation between each week of 2BC increased ethanol consumption in all genotypes and produced equivalent intakes in WT, Het and KO mice. Under the latter paradigm, ethanol drinking decreased progenitor proliferation and neuronal differentiation in the DG of WT mice. Interestingly, WT mice exhibited a strong negative correlation between ethanol intake and proliferation, which was disrupted in Het and KO mice. Moreover, MOR deficiency blocked the effect of ethanol on neuronal differentiation. MOR deficiency also protected against the neuroimmune response to ethanol drinking. Finally, chronic binge drinking induced a paradoxical decrease in apoptosis, which was independent of MOR. Altogether, our data suggest that MOR is implicated in some of the neuroplastic changes produced by chronic ethanol exposure in the DG.

An evaluation of the serotonin system and perseverative, compulsive, stereotypical, and hyperactive behaviors in dopamine transporter (DAT) knockout mice

Mice lacking the dopamine transporter (DAT) display major behavioral alterations that include hyperactivity, perseverative locomotor patterns, and reduced prepulse inhibition of the acoustic startle reflex. The objectives of this study were to investigate perseverative, compulsive, stereotypical, and hyperactive behaviors, as well as serotonin and its involvement with these behaviors, in DAT gene-altered mice. In the open field, mean turn angle and meandering were decreased in DAT knockout (DAT-KO) mice. DAT-KO mice displayed increased hyperactivity, increased velocity, less time immobile, and a failure to habituate over time in the open field unlike their DAT wildtype (DAT-WT) and heterozygous (DAT-HET) littermates. DAT-KO mice buried fewer marbles than DAT-WT and -HET mice in an assessment of compulsive-like behaviors, likely due to extreme hyperactivity and related inattention. Stereotypical head weaving was increased in untreated DAT-KO mice. Following administration of the 5-HT1A/7 agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), stereotypical head weaving and forepaw treading were increased more in DAT-KO mice than in DAT-WT or -HET mice. By contrast, head twitches induced by treatment with the 5-HT2A/2C agonist (±)-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI) were similar in mice of all three DAT genotypes. 5-HT1A autoreceptor function was intact in DAT-KO mice. Compared to DAT-WT mice, serotonin levels were increased in DAT-HET and -KO mice in frontal cortex and hippocampus, respectively, and serotonin turnover rates were increased ∼30 % in the striatum of DAT-KO mice. These findings extend and confirm prior behavioral and biochemical characterization of DAT-KO mice. Hyperactivity, stereotypy, and perseverative behaviors are increased in these mice, with brain-area specific increases in serotonin levels and serotonin turnover, and marked increases in postsynaptic 5-HT1A receptor-mediated stereotypic responses.

Transmembrane domain Nrg1 mutant mice show altered susceptibility to the neurobehavioural actions of repeated THC exposure in adolescence

Heavy cannabis abuse increases the risk of developing schizophrenia. Adolescents appear particularly vulnerable to the development of psychosis-like symptoms after cannabis use. To test whether the schizophrenia candidate gene neuregulin 1 (NRG1) modulates the effects of cannabinoids in adolescence, we tested male adolescent heterozygous transmembrane domain Nrg1 mutant (Nrg1 TM HET) mice and wild type-like littermates (WT) for their neurobehavioural response to repeated Δ(9)-tetrahydrocannabinol (THC, 10 mg/kg i.p. for 21 d starting on post-natal day 31). During treatment and 48 h after treatment withdrawal, we assessed several behavioural parameters relevant to schizophrenia. After behavioural testing we measured autoradiographic CB(1), 5-HT(2A) and NMDA receptor binding. The hyperlocomotor phenotype typical of Nrg1 mutants emerged after drug withdrawal and was more pronounced in vehicle than THC-treated Nrg1 TM HET mice. All mice were equally sensitive to THC-induced suppression of locomotion. However, mutant mice appeared protected against inhibiting effects of repeated THC on investigative social behaviours. Neither THC nor Nrg1 genotype altered prepulse inhibition. Repeated adolescent THC promoted differential effects on CB(1) and 5-HT(2A) receptor binding in the substantia nigra and insular cortex respectively, decreasing binding in WT while increasing it in Nrg1 TM HET mice. THC also selectively affected 5-HT(2A) receptor binding in several other regions in WT mice, whereas NMDA receptor binding was only affected in mutant mice. Overall, Nrg1 mutation does not appear to increase the induction of psychotomimetic symptoms by repeated adolescent THC exposure but may attenuate some of its actions on social behaviour and schizophrenia-relevant neurotransmitter receptor profiles.

An investigation into “two hit” effects of BDNF deficiency and young-adult cannabinoid receptor stimulation on prepulse inhibition regulation and memory in mice

Reduced brain-derived neurotrophic factor (BDNF) signaling has been shown in the frontal cortex and hippocampus in schizophrenia. The aim of the present study was to investigate whether a BDNF deficit would modulate effects of chronic cannabis intake, a well-described risk factor for schizophrenia development. BDNF heterozygous mice (HET) and wild-type controls were chronically treated during weeks 6, 7, and 8 of life with the cannabinoid receptor agonist, CP55,940 (CP). After a 2-week delay, there were no CP-induced deficits in any of the groups in short-term spatial memory in a Y-maze task or novel object recognition memory. Baseline prepulse inhibition (PPI) was lower but average startle was increased in BDNF HET compared to wild-type controls. Acute CP administration before the PPI session caused a marked increase in PPI in male HET mice pre-treated with CP but not in any of the other male groups. In females, there were small increases of PPI in all groups upon acute CP administration. Acute CP administration furthermore reduced startle and this effect was greater in HET mice irrespective of chronic CP pre-treatment. Analysis of the levels of [3H]CP55,940 binding by autoradiography revealed a significant increase in the nucleus accumbens of male BDNF HET mice previously treated with CP but not in any of the other groups or in the caudate nucleus. These results show that BDNF deficiency and chronic young-adult cannabinoid receptor stimulation do not interact in this model on learning and memory later in life. In contrast, male “two hit” mice, but not females, were hypersensitive to the effect of acute CP on sensorimotor gating. These effects may be related to a selective increase of [3H]CP55,940 binding in the nucleus accumbens, reflecting up-regulation of CB1 receptor density in this region. These data could be of relevance to our understanding of differential “two hit” neurodevelopmental mechanisms in schizophrenia.

Novel molecular changes induced by Nrg1 hypomorphism and Nrg1-cannabinoid interaction in adolescence: a hippocampal proteomic study in mice

Neuregulin 1 (NRG1) is linked to an increased risk of developing schizophrenia and cannabis dependence. Mice that are hypomorphic for Nrg1 (Nrg1 HET mice) display schizophrenia-relevant behavioral phenotypes and aberrant expression of serotonin and glutamate receptors. Nrg1 HET mice also display idiosyncratic responses to the main psychoactive constituent of cannabis, Δ9-tetrahydrocannabinol (THC). To gain traction on the molecular pathways disrupted by Nrg1 hypomorphism and Nrg1-cannabinoid interactions we conducted a proteomic study. Adolescent wildtype (WT) and Nrg1 HET mice were exposed to repeated injections of vehicle or THC and their hippocampi were submitted to 2D gel proteomics. Comparison of WT and Nrg1 HET mice identified proteins linked to molecular changes in schizophrenia that have not been previously associated with Nrg1. These proteins are involved in vesicular release of neurotransmitters such as SNARE proteins; enzymes impacting serotonergic neurotransmission, and proteins affecting growth factor expression. Nrg1 HET mice treated with THC expressed a distinct protein expression signature compared to WT mice. Replicating prior findings, THC caused proteomic changes in WT mice suggestive of greater oxidative stress and neurodegeneration. We have previously observed that THC selectively increased hippocampal NMDA receptor binding of adolescent Nrg1 HET mice. Here we observed outcomes consistent with heightened NMDA-mediated glutamatergic neurotransmission. This included differential expression of proteins involved in NMDA receptor trafficking to the synaptic membrane; lipid raft stabilization of synaptic NMDA receptors; and homeostatic responses to dampen excitotoxicity. These findings uncover novel proteins altered in response to Nrg1 hypomorphism and Nrg1-cannabinoid interactions that improves our molecular understanding of Nrg1 signaling and Nrg1-mediated genetic vulnerability to the neurobehavioral effects of cannabinoids.

462: Dysregulation of the peripheral circadian regulator Npas2 leads to altered fetal metabolic pathways in a knockout model

regulator Npas2 leads to altered fetal metabolic pathways in a knockout model Derek O’Neil, Hector Mendez-Figueroa, Toni-Ann Mistretta, Chunliu Su, Robert Lane, Kjersti Aagaard Baylor College of Medicine, Translational Biology and Molecular Medicine, Houston, TX, Baylor College of Medicine, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Houston, TX, Texas Children’s Hospital, Pathology, Houston, TX, University of Utah, Pediatrics, Salt Lake City, UT OBJECTIVE: In our non-human primate model of maternal high fat diet exposure, we previously identified the peripheral circadian domain gene Npas2 as a mediator of fetal metabolism which is epigenetically reprogrammed. As circadian activity and metabolic reprogramming is not well understood, we aimed to employ a murine gene knockout model to understand the role of Npas2 in regulating hepatic function throughout the lifespan of mice. STUDY DESIGN: Npas2 KO mice ( / ), Npas2 HET mice ( / ) and Npas2 WT mice ( / ) livers were harvested at day 2 (newborn) and at 25 weeks (adult) of life. Total RNA was extracted from hepatic tissue and converted to cDNA. Labeled cDNA was loaded onto the GeneChip® Mouse Exon 1.0 ST array (n 3/cohort). First pass analysis was performed using the Partek Genomics Suite software and GeneSpring GX 11. Genes meeting statistical and expression thresholds after controlling for multiple comparisons were then processed through Ingenuity Pathway Analysis, GSEA, and GeneSpring pathway analysis software. QPCR was performed on cDNA from newborn and adult Npas2 KO and wt mice to determine gene expression levels of genes pertaining to affected metabolic pathways. RESULTS: We identified 2891 genes that were dysregulated in KO mice compared to wt (F .05, fold-change 1.5 or -1.5). The primary dysregulated pathway in the Npas2 KO was the lipid and fatty acid metabolism pathway. Gene expression of Adra2a, Lpin1, Scd1, Sds, and Upp2 demonstrate significant (p .05) neonatal alterations that do not persist into adult life and revert to wt levels. Fgf1, Gpd1, Grb14, Gulo, and Hmgcs1 demonstrated significant (p .05) alterations in neonatal life that reversed expression levels in adult life. CONCLUSION: Npas2 primarily regulates the lipid and fatty acid metabolism pathway during early murine development. However, compensatory pathways are enacted in adult life, indicating a crucial role of Npas2 during fetal/neonatal development which had previously not been definitively described.

Gasc1 may affect skin carcinogenesis by regulating expression level of estrogen related gene

GASC1 encodes a histone demethylase catalyzing lysine (K) of histone, specifically K9 and K36 of histone H3. It has been reported that GASC1 is located within an amplicon at 9p23-24 frequently in esophageal cancer and overexpressed in undifferentiated ES cells and breast cancer. Since these reports suggest the involvement of GASC1 in carcinogenesis, we generated Gasc1 knockout mice to analyze the role of GASC1. It was shown that Het mice developed without any remarkable phenotype, while homozygous mice are embryonic lethal.

Systemic Ceramide Accumulation Causes Dysregulated Hematopoiesis

Abstract 3461Ceramide is a bioactive lipid that exists in all cells of the body. Its role in hematopoiesis has not been elucidated. We recently generated the first viable mouse model of systemic ceramide accumulation due to acid ceramidase (ACDase) deficiency (Farber disease). Mice homozygous for the ACDase P362R mutation (Hom) accumulate ceramide, have a reduced lifespan of 7–13 weeks, and are smaller than wild-type (WT) or heterozygous (Het) littermates. Peripheral blood analysis revealed that Hom ACDase-deficient mice had severe leukocytosis, mostly due to increased neutrophils. Lymphocyte and erythrocyte counts were normal. Hematopoietic and accessory organs were larger normalized to body weight in Hom compared to Het and WT animals: spleen (4× larger), thymus (3×), lymph nodes (LN) (8–12x), liver (1.5×). Kidneys and heart were not enlarged. IHC of the enlarged organs and bone marrow (BM) revealed massive infiltration by large, lipid-filled “foamy macrophages”. Infiltration was so severe that hematopoietic niches were disrupted: germinal center architecture was destroyed in the spleen of Hom ACDase mice, for example. In contrast, total cell numbers from the BM, spleen, thymus, and LN were reduced in Hom ACDase-deficient mice. Flow cytometry analyses revealed that T and B cell intermediates were most affected. By 9 weeks thymic CD4 CD8 double positive T cells were dramatically reduced to 5% of cells in Hom animals from 80% in WT. BM pro-B cells were reduced to 3% of cells in Hom (8% in WT) with pre-B cells reduced to <1% (14% in WT). Consequently, there were fewer IL-7 responsive cells. Responsiveness to LPS and anti-μ were similar in Hom and WT mice. In Dexter assays, an in vitro system where a monolayer of stromal cells supports myelopoiesis through direct cell contact, cultures from Hom ACDase-deficient mice failed to establish the typical foci formation seen in WT after successful attachment of myeloid precursors to the stroma. In cellulose based media, hematopoietic stem and progenitor cells (HSPC) were able to properly differentiate down the myeloid lineage: in colony-forming cell (CFC) assays, BM and spleen cells from 5, 7, and 9 week-old Hom, Het, and WT mice demonstrated similar differentiation capacities. Interestingly, there was an increase in the total number of CFC colonies in Hom ACDase mice at 5 weeks that normalized by 9 weeks. Additional CFC assays and IHC of the liver revealed extramedullary hematopoiesis, which may also contribute to the over-production of leukocytes and macrophages observed.In preliminary BMT experiments, lethally-irradiated Het animals transplanted with cells derived from Hom ACDase deficient mice lived as long as Het animals transplanted with WT cells (10 months; study end-point) and had normal peripheral blood cell counts. The reverse transplants of WT cells into Hom ACDase-deficient mice are underway. Together with the Dexter assay results these data suggest that the BM niche is unable to support and control HSPC differentiation. Briefly, to begin to elucidate the mechanism through which systemic ceramide accumulation is exerting its effects, cytokine analyses and ELISAs were performed on organs and serum. Serum cytokine analyses revealed an increase in MCP-1 and IL-12(p40) levels along with a trend towards an increase in MIP-1α and RANTES expression. No differences were observed in TNF-α, INF-γ, MIP-1β, eotaxin, IL-13, IL-12(p70), IL-10, IL-9, and IL-1b levels. MCP-1 levels were also higher in the organs of Hom ACDase-deficient mice than in WT animals; further, MCP-1 levels were higher in the enlarged organs of Hom mice than the serum, possibly creating a gradient to attract macrophages. Follow-up RT-PCR analyses revealed an increase in the expression of MCP-1 (in BM, LN, thymus, spleen), MIP-1α (LN, thymus, spleen), IL-6 (thymus, spleen), GM-CSF (thymus), iNOS (BM, spleen), and galectin-3 (LN, thymus, spleen) mRNA. No change was found in CCR2 (MCP-1 receptor), TGF-β, M-CSF, TNF-α, IL-1β, and IL-10 mRNA levels.This study reveals previously unappreciated consequences of ceramide accumulation on hematopoiesis. Our results suggest that ceramide accumulation due to ACDase deficiency perturbs hematopoiesis: the interaction between support cells and HSPC is disrupted and foamy macrophages infiltrate organs. HSPC themselves appear to be functional. Further studies will elucidate the mechanism through which ceramide hinders support cells from interacting with HSPCs. Disclosures:No relevant conflicts of interest to declare.

A Functional Thrombopoietin Receptor Is Required for Full Expression of Phenotype in a JAK2 V617F Transgenic Mouse Model of Polycythemia Vera

Abstract 427Polycythemia vera (PV) is a clonal stem cell disorder in which there is overproduction of red cells, white cells and platelets, extramedullary hematopoiesis and transformation to myelofibrosis. Enhanced hematopoiesis in PV is due to constitutive activation of JAK2, the cognate tyrosine kinase of type I cytokine receptors such as the erythropoietin and thrombopoietin receptors. Hematopoietic stem cells express the thrombopoietin receptor (Mpl) but paradoxically mice lacking the MPL gene exhibit only thrombocytopenia while maintaining the capacity to not only substantially elevate their platelet and leukocyte counts during marrow reproductive stress but also the number of multipotent and committed hematopoietic progenitor cells as well. Equally paradoxical, Mpl expression is impaired in PV, but how impaired expression of a master regulator of stem cell function could be associated with a global increase in hematopoiesis has been a conundrum. Recently, data were presented (Blood 119: 4625, 2012) suggesting that Mpl down regulation in PV was due to constitutive JAK2 activation, which abrogated the antiproliferative effects of the high plasma thrombopoietin (TPO) levels in PV, thus sustaining the PV phenotype. We have used a JAK2 V617F transgenic mouse model (V617Ftg) (Blood 111:5109, 2009) to examine the role of Mpl in the pathogenesis of the JAK2 V617F-positive PV phenotype and have observed that deletion of the MPL gene ameliorated the PV phenotype in this model, reducing the hemoglobin level, leukocyte and platelet counts and splenomegaly and reversing marrow fibrosis, while the presence of a single MPL gene was sufficient to restore the PV phenotype. However, total deletion of the MPL gene does not perfectly model human PV where Mpl is present but quantitatively reduced. Therefore, we examined the effect of manipulating Mpl behavior in vivo by modifying Mpl signaling while maintaining Mpl expression, first, by eliminating TPO production, and second, by expressing a functionally impaired Mpl receptor, and as a control, specifically impairing megakaryocyte differentiation alone. To this end, we crossbred the V617Ftg PV mouse with a TPO gene knockout mouse (TPO ko), with a mouse homozygous for the MPL mutation, C40R (C40R hom), which in the homozygous state causes thrombocytopenia, and finally, with an MKL1 knockout mouse (MKL1ko) that is thrombocytopenic due to impaired megakaryocyte maturation. Crossbreeding produced the three expected genotypes (JAK2 V617F/TPO ko, JAK2 V617F/C40R hom and JAK2 V617F/MKL1ko) without unexplained mortality or impoverished growth. We compared the blood counts, spleen weights and bone marrow and spleen histology of these three genotypes with each other and with JAK2 V617F/TPO heterozygous (het) mice over a 33 week period. The natural history of the V617Ftg PV mouse is one of initial erythrocytosis, thrombocytosis, leukocytosis, splenomegaly, and expansion of short term marrow repopulating cells (ST-HSC), common myeloid progenitor cells (CMP) and marrow and spleen committed hematopoietic progenitor cells (HPC)(CFU-GEMM, CFU-GM, BFU-E, CFU-E and CFU-MK), and myelofibrosis with a subsequent decline in erythrocytosis. Abrogating TPO production in the V617Ftg PV mouse normalized the platelet and leukocyte counts, delayed the development of erythrocytosis, reduced spleen size and ST-HSC, CMP, and all marrow and spleen committed HPC compartments and reversed marrow fibrosis. The V617Ftg PV - C40R MPL cross prevented thrombocytosis, erythrocytosis, leukocytosis and splenomegaly, and reduced all the marrow and spleen committed HPC compartments. By contrast, the V617Ftg PV-MKL1 ko cross slowed the rate of rise of the platelet count and hemoglobin level but had no effect on splenomegaly or marrow fibrosis, nor was there a reduction in any of the marrow or spleen committed HPC. Expression of one TPO gene by crossing the V617Ftg PV mouse with mice heterozygous for the TPO gene (JAK2 V617F/TPO het) restored the PV phenotype. In conclusion, while it is unquestioned that JAK2 V617F is necessary for expression of the PV phenotype, our data indicate that it alone is insufficient and requires not only the expression of a functional TPO receptor but also receptor activation by its cognate ligand TPO. This latter observation suggests that interfering with the TPO-Mpl interaction could be of therapeutic value in the treatment of human PV. Disclosures:No relevant conflicts of interest to declare.

Reduced Expression of STOP/MAP6 in Mice Leads to Cognitive Deficits

Background: STOP/MAP6 null (KO) mice recapitulate behavioral abnormalities related to positive and negative symptoms and cognitive deficits of schizophrenia. Here, we investigated whether decreased expression of STOP/MAP6 proteins in heterozygous mice (only one allele expressed) would result in abnormal behavior related to those displayed by STOP null mice. Methods: Using a comprehensive test battery, we investigated the behavioral phenotype of STOP heterozygous (Het) mice compared with STOP KO and wild type (WT) mice on animals raised either in standard conditions (controls) or submitted to maternal deprivation. Results: Control Het mice displayed prominent deficits in social interaction and learning, resembling KO mice. In contrast, they exhibited short-lasting locomotor hyperreactivity to acute mild stress and no impaired locomotor response to amphetamine, much like WT mice. Additionally, perinatal stress deteriorated Het mouse phenotype by exacerbating alterations related to positive symptoms such as their locomotor reactivity to acute mild stress and psychostimulant challenge. Conclusion: Results show that the dosage of susceptibility genes modulates their putative phenotypic contribution and that STOP expression has a high penetrance on cognitive abilities. Hence, STOP Het mice might be useful to investigate cognitive defects related to those observed in mental diseases and ultimately might be a valuable experimental model to evaluate preventive treatments.

Phosphodiesterase 11A (PDE11A) Expression in Mouse Tissues and Characterization of a PDE11A Mouse Knock-Out Model

Phosphodiesterases catalyze the hydrolysis of cyclic nucleotides and maintain physiologic levels of intracellular concentrations of cAMP and cGMP. Increased cAMP signaling has been associated with genetics disorders that lead to adrenocortical tumors and Cushing syndrome. Genetics defects in the phosphodiesterase 11A (PDE11A) are associated with increased levels of cAMP and bilateral adrenal hyperplasia; they also contribute to the development of adrenocortical, prostate, and testicular tumors in the general population. The aim of the present work is to study the expression of Pde11a in mouse tissues, as well as to understand better the Pde11a defect in a previously published Pde11a–/– knockout (KO) mouse model.1A colony of Pde11a–/– (KO), Pde11a+/– (Het) and wild type (wt) mice was studied in a complete phenotypical groß and microscopy pathological study. Molecular, immunohistochemical and biochemical studies were done to determine the function and expression of Pde11a in the tissues of normal and mutant mice. In normal mice higher PDE11A immunoreactivity(PDE11A-IR) was found in small intestine villi, thyroid follicular cells, prostate and spermatocytes; moderate PDE11A-IR in brain, liver hepatocytes, and exocrine pancreas and little, if any, in the pituitary gland. In mutant mice RNA studies showed a reduced Pde11a expression in steroidogenic tissues (adrenals and testis), lung, brain, and liver while prostate and kidney presented comparable expression with normal mice. PDE11A protein expression and activity assays did not show differences among three groups. Pathological studies showed differences: Adrenal subcapsular hyperplasia and foamy cells were more frequent in Het(37.7% and 30.2% respectively, p<0.05) than in wt group (26.3% and 15.8% respectively); Spinal epidermoid cyst in Het (15%) and wt mice (10%); kidney-pelvis interstitial lymphocytes only in KO mice (10.7%, P<0.05). KO mice showed increased absolute and organ specific weight. This study show a clear profile of Pde11a protein expression in endocrine and other mouse tissues, as well as convincing evidence of only partial Pde11a suppression in the previously published KO mouse. These mice developed adrenal hyperplasia and other abnormalities like humans with heterozygote PDE11A defects and partial inactivation of the enzyme. These data support the involvement of PDE11A in the pathogenesis of adrenocortical hyperplasia, but also provide significant insight into a previously reported mouse model.

G.P.122 Heterozygous LmnadelK32 mutant mice showed alterations of the ubiquitin–proteasome system and developed dilated cardiomyopathy

Abstract Mutations of LMNA gene give rise to a range of muscular dystrophies associated with dilated cardiomyopathy (DCM) and conduction and/or rhythm defects. LMNA encodes lamin A/C, nuclear envelope proteins that form a meshwork beneath the inner nuclear membrane. The pathomechanisms linking LMNA mutations to muscular and cardiac diseases remain to be elucidated. We created the LmnaΔK32 knock-in mouse, mutation associated with congenital muscular dystrophy in humans. The heterozygous LmnaΔK32/+ mice (Het) showed neither skeletal muscle nor rhythm/conduction defects. But they progressively developed cardiac dysfunction and DCM, and died between 35 and 70 weeks of age. Lamin A/C showed similar nuclear rim localization in Het and Wt hearts. The evolution of DCM was associated with a modulation of cardiac lamin A/C protein level. Before the development of DCM, lamin A/C protein level was 50% lower in Het than Wt hearts whereas it did not differ between the two groups thereafter. This difference in lamin A/C cardiac content was due to ΔK32-lamin degradation via the ubiquitin–proteasome system (UPS), leading to very low level of Δ K32-lamin in Het hearts (7.5% of total lamin A/C content). The function of the UPS was assessed in vivo by crossing our mice with UbG76V-GFP transgenic mice, expressing a UPS reporter substrate. Levels of UbG76V-GFP proteins were 2-fold higher in Het than in Wt mice, showing the global impairment of UPS in the heart of Het mice. After gene transfer in rat engineered heart tissue and treatment with proteasome inhibitor, ΔK32-lamin formed nuclear aggregates and impaired contractile function, suggesting a poison peptide effect. The LmnaΔK32/+ mice are the first knock-in Lmna model with a cardiac-specific phenotype at the heterozygous state. They represent a valuable tool for studying dominant LMNA-related DCM. Our data provide evidence that degradation of ΔK32-lamin A/C is a protective process, limiting the dominant negative effect on cardiac function.

Adolescent neuregulin 1 heterozygous mice display enhanced behavioural sensitivity to methamphetamine

Methamphetamine use triggers psychosis in genetically vulnerable individuals, however the exact nature of this genetic predisposition requires elucidation. In addition, adolescence may be a particular period of neurodevelopmental vulnerability to the actions of methamphetamine; interestingly, this period coincides with a higher likelihood of onset of schizophrenia and drug experimentation. In the current study we investigated whether adolescent mice heterozygous for the schizophrenia susceptibility gene neuregulin 1 (Nrg1 HET mice) exhibit altered behavioural responses to methamphetamine (0.6 or 2.4mg/kg) in schizophrenia-relevant paradigms. The responses measured were locomotor activity in the open field test and sensorimotor gating function in the prepulse inhibition of startle paradigm (PPI). Adolescent Nrg1 HET mice displayed a subtle, transient, novelty-induced baseline locomotor hyperactivity over days, and a selective PPI deficit at the prepulse intensity-interstimulus interval (ISI) combination of 82dB-64ms. Adolescent Nrg1 HET mice were more sensitive to the locomotor stimulatory effects of an acute, low-dose of methamphetamine (0.6mg/kg) relative to wild-type (WT) controls. The augmented response to acute methamphetamine observed in Nrg1 HET mice disappeared with repeated, daily dosing over 7days. Methamphetamine did not affect average PPI (total or across different prepulse intensities), however 0.6mg/kg methamphetamine triggered a PPI deficit selectively in Nrg1 HET mice but not WT mice at 82dB-256ms. Our results show that locomotor hyperactivity in Nrg1 HET mice, albeit subtle, can manifest much earlier than previously reported and that Nrg1 may confer vulnerability to the acute actions of methamphetamine, a drug known to trigger psychotic reactions in humans.

Menin liver-specific hemizygous mice challenged with high fat diet show increased weight gain and markers of metabolic impairment

OBJECTIVE:The menin tumor suppressor protein is abundantly expressed in the liver, although no function has been identified because of lack of tumor development in multiple endocrine neoplasia type 1 (Men1) null livers. We examine the phenotype of mice lacking one functional allele of Men1 (consistent with the phenotype in humans with MEN1 syndrome) challenged with high fat diet (HFD) to elucidate a metabolic function for hepatic menin.METHODS:In this study, we challenged mice harboring a liver-specific hemizygous deletion of Men1 (HETs) alongside wild-type (WT) counterparts with HFD for 3 months and monitored the severity of metabolic changes. We demonstrate that the HET mice challenged with HFD for 3 months show an increased weight gain with decreased glucose tolerance compared with WT counterparts. Along with these changes, there was a more severe serum hormone profile involving increased serum insulin, glucose and glucagon, all hallmarks of the type 2 diabetic phenotype. In concert with increased serum hormones, we found that these mice have significantly increased liver triglycerides coupled with increased liver steatosis and inflammatory markers. Quantitative real-time PCR and western blotting studies show increases in enzymes involved with lipogenesis and hepatic glucose production.CONCLUSION:We conclude that hepatic menin is required for regulation of diet-induced metabolism, and our studies indicate a protective role for the Men1 gene in the liver when challenged with HFD.

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

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

Contribution of β2 subunit containing nicotinic acetylcholine receptors to anxiety‐like behaviors in male mice using a novel model of anxiety

To test the hypothesis that inhibition of beta 2 subunit containing nicotinic acetylcholine receptors (β 2*nAChRs) promotes anxiolysis, we compared β2*nAChR null mutant (β2KO) and heterozygous (Het) mice to wild type (WT) littermate controls in the light‐dark assay and a novel bottle inversion assay (BIA) where water access was restricted. Behavioral measures included bottle‐interaction, rearing, digging, and social pursuit of a cage mate. In support of our hypothesis, there was a non‐significant trend for beta2KOs and Hets to show shorter latencies than WT mice to leave the dark chamber. BIA revealed a significant reduction in social pursuits of β2KOs and Hets in comparison to their WT counterparts (p's &lt; 0.05). There was a similar reduction of this behavior following injection of 1 mg/kg midazolam in WT (p &lt; 0.05) mice, validating social pursuit as a measure of anxiety‐like behavior in this task. These findings suggest that inhibition of β2*nAChRs may promote anxiolysis and identify β2*nAChRs as a potential therapeutic target for anxiety disorder. This research was supported by a Jeffress Memorial Trust research grant J‐951 and R01 DA031289, P50 DA005274, R25 GM090084, and T32 DA00702734.β

Role of NMDA receptor subtypes in regulation of central amygdala

We have recently shown that mice lacking the NMDA receptor GluN2C (GluN2C KO) subunit exhibit deficits in contextual and cued fear acquisition (Hillman et al., 2011). The central amygdala (CeA) is composed of lateral (CEl) and medial (CEm) subnuclei which are involved in fear acquisition and expression respectively. Furthermore, CEloff neurons are characterized as PKCδ‐positive, tonically inhibit the CEm resulting in regulation of fear expression. We found that in comparison to wildtype, GluN2C het and KO mice have over two‐fold pERK stained neurons in the CEl which co‐localizes with PKCδ. Additionally increased frequency of sIPSCs was observed in CEm of GluN2C KO versus wildtype mice. We sought to determine if potentiation of GluN2Ccontaining receptors would alleviate fear acquisition deficit in GluN2C heterozygous mice. D‐cycloserine, which has two‐fold efficacy at GluN2C‐containing receptors compared to glycine, injected prior to fear conditioning increased fear acquisition in GluN2C heterozygous animals while it had no effect on knockout counterparts. A potential role of GluN2C receptors expressed in mediodorsal thalamus was further identified in regulating CEl activity. The findings presented herein show that GluN2C subunit may regulate basal CeA activity which may have important implication for anxiety and fear disorders.

Anti‐Phase Expression of the BK Channel (Kcnma1) Alters Circadian Locomotor Activity in Mice

Circadian rhythmicity, driven by the suprachiasmatic nucleus (SCN) of the hypothalamus, is essential to the survival or fitness of all organisms. The BK Ca2+ and voltage‐activated K+ channel is expressed in a circadian manner in the SCN, peaking at night, and genetic deletion of the BK channel (Kcnma1−/−) disrupts circadian behavioral rhythms. To determine whether the nighttime‐phased expression pattern was essential for normal circadian rhythmicity, we analyzed wheel running activity of transgenic mice that expressed the BK channel opposite to its normal phase in the SCN (Per1:BKR207Q ). We predicted this alteration of BK phase by expression of one or two copies (1C, 2C) of the Per1:BKR207Q transgene, or one copy of Per1:BKR207Q with one deleted endogenous copy of Kcnma1 (1C; BK het) would disrupt circadian wheel rhythms. In contrast, 1C, 2C, and 1C; BK het mice all had relatively normal baseline circadian rhythms. However, 2C mice had a longer active interval compared to WT, suggesting that nighttime‐phased expression of the BK channel is important for restricting locomotor activity. Additionally, 2C mice had a larger light‐pulse induced phase delays, suggesting these mice were more sensitive to phase‐shifting stimuli than WT. A better understanding of the neural encoding of circadian rhythms may impact the treatment of sleep and other disorders of circadian rhythmicity.This work was funded by an APS summer research fellowship to (B.N.W.) and NSF (IOS‐0956237) and NHLBI (R01HL102758) to A.L.M.

The response of neuregulin 1 mutant mice to acute restraint stress

Stress plays a role in the development and severity of psychotic symptoms and there may be a genetic component to stress vulnerability in schizophrenia. Using an established mouse model for schizophrenia, we investigated the behavioural and endocrine response of Nrg1 transmembrane domain mutant mice (Nrg1 HET) and wild type-like (WT) littermates to acute restraint stress. Animals were screened at 3–4 months and 6–7 months of age (before and after onset of hyperlocomotion) for open field behaviour and serum corticosterone levels. In younger mice, stress reduced locomotive and explorative measures and increased anxiety-like behaviour regardless of genotype. Older Nrg1 mutants were less susceptible to the effects of stress on anxiety-related behaviours. All mice responded to restraint stress with robust increases in serum corticosterone. Importantly, the stress-induced increase in corticosterone was more pronounced in Nrg1 mutant than WT mice at the younger but not the older age. Our results suggest that transmembrane domain Nrg1 has only a moderate effect on the acute stress response of mice. The behavioural differences detected between WT and Nrg1 HET mice at the older age were evident without parallel modifications to the glucocorticoid system.

Basic fibroblast growth factor in an animal model of spontaneous mammary tumor progression

Although basic fibroblast growth factor (FGF2) was the first pro-angiogenic molecule discovered, it has numerous activities on the growth and differentiation of non-vascular cell types. FGF2 is both stimulatory and inhibitory, depending on the cell type evaluated, the experimental design used and the context in which it is tested. Here, we investigated the effects of manipulating endogenous FGF2 on the development of mammary cancer to determine whether its endogenous contribution in vivo is pro- or anti-tumorigenic. Specifically, we examined the effects of FGF2 gene dosing in a cross between a spontaneous breast tumor model (PyVT+ mice) and FGF2-/- (FGF KO) mice. Using these mice, the onset and progression of mammary tumors was determined. As predicted, female FGF2 WT mice developed mammary tumors starting around 60 days after birth and by 80 days, 100% of FGF2 WT female mice had mammary tumors. In contrast, 80% of FGF2 KO female mice had no palpable tumors until nearly three weeks later (85 days) at times when 100% of the WT cohort was tumor positive. All FGF KO mice were tumor-bearing by 115 days. When we compared the onset of mammary tumor development and the tumor progression curves between FGF het and FGF KO mice, we observed a difference, which suggested a gene dosing effect. Analysis of the tumors demonstrated that there were significant differences in tumor size depending on FGF2 status. The delay in tumor onset supports a functional role for FGF2 in mammary tumor progression, but argues against an essential role for FGF2 in overall mammary tumor progression.