Single-minded 1 (Sim1) encodes a transcription factor essential for formation of the hypothalamic paraventricular nucleus (PVN). Sim1 haploinsufficiency is associated with hyperphagic obesity and ...increased linear growth in humans and mice, similar to the phenotype of melanocortin 4 receptor (Mc4r) mutations. PVN neurons in Sim1+/− mice are hyporesponsive to the melanocortin agonist melanotan II. PVN neuropeptides oxytocin (Oxt), TRH and CRH inhibit feeding when administered centrally. Consequently, we hypothesized that altered PVN neuropeptide expression mediates the hyperphagia of Sim1+/− mice. To test this hypothesis, we measured hypothalamic expression of PVN neuropeptides in Sim1+/− and wild-type mice. Oxt mRNA and peptide were decreased by 80% in Sim1+/− mice, whereas TRH, CRH, arginine vasopressin (Avp), and somatostatin mRNAs were decreased by 20–40%. Sim1+/− mice also showed abnormal regulation of Oxt but not CRH mRNA in response to feeding state. A selective Mc4r agonist activated PVN Oxt neurons in wild-type mice, supporting involvement of these neurons in melanocortin feeding circuits. To test whether Oxt itself regulates feeding, we measured the effects of central administration of an Oxt receptor antagonist or repeated doses of Oxt on food intake of Sim1+/− and wild-type mice. Sim1+/− mice were hypersensitive to the orexigenic effect of the Oxt receptor antagonist. Oxt decreased the food intake and weight gain of Sim1+/− mice at a dose that did not affect wild-type mice. Our results support the importance of Oxt neurons in feeding regulation and suggest that reduced Oxt neuropeptide is one mechanism mediating the hyperphagic obesity of Sim1+/− mice.
Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA ...repeats in intron 1 of FXN, while some are compound heterozygotes with an expanded GAA tract in one allele and a missense or nonsense mutation in the other. A missense mutation, changing a glycine to valine at position 130 (G130V), is prevalent among the clinical variants. We and others have demonstrated that levels of mature FXN protein in FRDA G130V samples are reduced below those detected in samples harboring homozygous repeat expansions. Little is known regarding expression and function of endogenous FXN-G130V protein due to lack of reagents and models that can distinguish the mutant FXN protein from the wild-type FXN produced from the GAA-expanded allele. We aimed to determine the effect of the G130V (murine G127V) mutation on Fxn expression and to define its multi-system impact in vivo. We used CRISPR/Cas9 to introduce the G127V missense mutation in the Fxn coding sequence and generated homozygous mice (FxnG127V/G127V). We also introduced the G127V mutation into a GAA repeat expansion FRDA mouse model (FxnGAA230/KO; KIKO) to generate a compound heterozygous strain (FxnG127V/GAA230). We performed neurobehavioral tests on cohorts of WT and Fxn mutant animals at three-month intervals for one year, and collected tissue samples to analyze molecular changes during that time. The endogenous Fxn G127V protein is detected at much lower levels in all tissues analyzed from FxnG127V/G127V mice compared to age and sex-matched WT mice without differences in Fxn transcript levels. FxnG127V/G127V mice are significantly smaller than WT counterparts, but perform similarly in most neurobehavioral tasks. RNA sequencing analysis revealed reduced expression of genes in oxidative phosphorylation and protein synthesis, underscoring the metabolic consequences in our mouse model expressing extremely low levels of Fxn. Results of these studies provide insight into the unique pathogenic mechanism of the FXN G130V mechanism and the tolerable limit of Fxn/FXN expression in vivo.
•Extremely low Fxn levels are detected in CNS and heart tissues of Fxn G127V mice.•Fxn G127V mice are small and develop a hunched posture.•Endurance and activity are reduced in adult Fxn G127V mice.•Motor coordination is not affected in adult Fxn G127V mice.
Brain-derived neurotrophic factor (BDNF) regulates neuronal development and function. However, it has been difficult to discern its role in the adult brain in influencing complex behavior. Here, we ...use a recently developed inducible knockout system to show that deleting BDNF in broad forebrain regions of adult mice impairs hippocampal-dependent learning and long-term potentiation. We use the inducible nature of this system to show that the loss of BDNF during earlier stages of development causes hyperactivity and more pronounced hippocampal-dependent learning deficits. We also demonstrate that the loss of forebrain BDNF attenuates the actions of desipramine, an antidepressant, in the forced swim test, suggesting the involvement of BDNF in antidepressant efficacy. These results establish roles for BDNF in the adult, and demonstrate the strength of this inducible knockout system in studying gene function in the adult brain.
Mutations in the methyl-CpG binding protein 2 (MeCP2) gene cause Rett syndrome (RTT), a neurodevelopmental disorder that is accompanied by a broad array of behavioral phenotypes, mainly affecting ...females. Methyl-CpG binding protein 2 is a transcriptional repressor that is widely expressed in all tissues.
To investigate whether the postnatal loss of MeCP2 in the forebrain is sufficient to produce the behavioral phenotypes observed in RTT, we have generated conditional MeCP2 knockout mice.
These mice display behavioral abnormalities similar to RTT phenotypes, including hindlimb clasping, impaired motor coordination, increased anxiety, and abnormal social behavior with other mice. These mice, however, have normal locomotor activity and unimpaired context-dependent fear conditioning, suggesting that the behavioral deficits observed are the result of loss of MeCP2 function in postnatal forebrain and not the result of generalized global deficits.
These data highlight the important role of MeCP2 in the forebrain and suggest that even partial loss of MeCP2 expression in these brain regions is sufficient to recapitulate features of RTT.
Single-minded 1 (SIM1) is one of only six genes implicated in human monogenic obesity. Haploinsufficiency of this hypothalamic transcription factor is associated with hyperphagic obesity and ...increased linear growth in both humans and mice. Additionally, Sim1 heterozygous mice show enhanced hyperphagia and obesity in response to a high-fat diet. Thus the phenotype of Sim1 haploinsufficiency is similar to that of agouti yellow (Ay), and melanocortin 4 receptor (Mc4r) knockout mice, both of which are defective in hypothalamic melanocortin signaling. Sim1 and Mc4r are both expressed in the paraventricular nucleus (PVN). Here we report that Sim1 heterozygous mice, which have normal energy expenditure, are hyperphagic despite having elevated hypothalamic proopiomelanocortin (Pomc) expression. In response to the melanocortin agonist melanotan-2 (MTII) they exhibit a blunted suppression of feeding yet increase their energy expenditure normally. They also fail to activate PVN neurons in response to the drug at a dose that induces robust c-Fos expression in a subset of Sim1 PVN neurons in wild-type mice. The resistance to melanocortin signaling in Sim1 heterozygotes is not due to a reduced number of Sim1 neurons in the PVN. Hypothalamic Sim1 gene expression is induced by leptin and MTII treatment. Our results demonstrate that Sim1 heterozygotes are resistant to hypothalamic melanocortin signaling and suggest that Sim1-expressing PVN neurons regulate feeding, but not energy expenditure, in response to melanocortin signaling.
Single-minded 1 (SIM1) mutations are associated with obesity in mice and humans. Haploinsufficiency of mouse Sim1 causes hyperphagic obesity with increased linear growth and enhanced sensitivity to a ...high-fat diet, a phenotype similar to that of agouti yellow and melanocortin 4 receptor knockout mice. To investigate the effects of increased Sim1 dosage, we generated transgenic mice that overexpress human SIM1 and examined their phenotype. Compared with wild-type mice, SIM1 transgenic mice had no obvious phenotype on a low-fat chow diet but were resistant to diet-induced obesity on a high-fat diet due to reduced food intake with no change in energy expenditure. The SIM1 transgene also completely rescued the hyperphagia and partially rescued the obesity of agouti yellow mice, in which melanocortin signaling is abrogated. Our results indicate that the melanocortin 4 receptor signals through Sim1 or its transcriptional targets in controlling food intake but not energy expenditure.
Abstract only Duchenne muscular dystrophy (DMD) is caused by mutations that abolish dystrophin expression. Dilated cardiomyopathy, the leading cause of death in DMD patients, is characterized by the ...development of severe subepicardial fibrosis that precedes cardiac dysfunction. While the cellular mechanisms governing this process remain unknown, a recent study in mdx/mTR mice suggested that inflammation, in response to cardiomyocyte damage caused by dystrophin deficiency, activates the epicardium to undergo epithelial-to-mesenchymal transition into fibroblasts, thereby contributing to subepicardial fibrosis. Whether dystrophin plays an epicardial-autonomous role in this process has not been investigated. The DMD gene produces at least eight dystrophin isoforms from seven independent, tissue-specific promoters ( Fig. 1 ). We used an immortalized mouse embryonic epicardial cell line to determine which isoforms are present. We found transcripts for the full-length muscle isoform of dystrophin (Dp427m) in addition to the ubiquitous, more abundant Dp71 and Dp40 isoforms. RT-PCR and western blot analysis revealed the presence of three different Dp71 splice variants. Immunofluorescence and subcellular fractionation determined that Dp71 localizes primarily to the nucleus. Studies are ongoing to understand the role of Dp71 in epicardial cell proliferation and gene expression, as well as defining changes in dystrophin expression during the transition from the activated epicardium to myofibroblasts. Further investigation is needed to determine the role of these shorter isoforms in DMD pathology and how gene editing of full-length dystrophin may impact their function.
Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here ...we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.
Duchenne muscular dystrophy (DMD) is a recessive X-linked neuromuscular disorder characterized by progressive muscle degeneration with DMD-associated cardiomyopathy being the primary mode of death. ...Vasoactive intestinal peptide (VIP) is a 28 amino acid neuropeptide with biological effects mediated by G protein-coupled receptors. Utilizing a genetically modified form of VIP (PB1046) targeting cardiomyocytes, we hypothesized that augmentation of VIP signaling prevents the development of DMD-associated cardiomyopathy through inhibition of NF-κB. Either PB1046 (1.5 mg/kg) or a Placebo (saline) was injected subcutaneously every other day in three mouse modelsDMD, DMD, and wild type mice starting at 4 weeks of age for a total of 8 weeks. Cardiac function was assessed by weekly echocardiography. The cardiac tissues were collected at 14 weeks of age for histological and molecular analyses. Drug-treated DMD mice showed preservation of cardiac function (fractional shortening 61%±0.4 vs 45%±1.3, p<0.01; n=8-12) and a marked reduction in myocardial fibrosis (2.92%±0.13 vs 6.42% ±0.39, p<0.01; n=3) compared with controls. Hydroxyproline levels within drug-treated DMD mice was decreased compared with controls (44.6±5.3 vs 64.3±6.9 nmol/100mg heart weight, p<0.05, n=6). RNA-Seq data revealed an upregulation of cAMP signaling with downregulation of NF-kB signaling in isolated cardiac myocytes from drug-treated DMD mice as compared to controls (n=3). Western blot analyses revealed increased phosphorylation of CREB (1.97±0.02 vs 1.00±0.06, p<0.05, n=5-9) with decreased phosphorylation of p65 in drug-treated DMD cardiac nuclei as compared to controls (0.62±0.06 vs 1.00±0.09, p<0.05, n=3). Collectively, the data revealed augmentation of VIP signaling prevents the development of DMD-associated cardiomyopathy in DMD mice. The molecular mechanism underlying the benefits of VIP signaling suggests an upregulation of cAMP-CREB signaling with downregulation of NF-kB signaling leading to inhibition of inflammation and fibrosis within drug-treated DMD hearts. VIP signaling in DMD may serve as a new therapeutic target for the treatment of DMD-associated cardiomyopathy.
Single-minded 1 (SIM1) mutations are one of the few known causes of nonsyndromic monogenic obesity in both humans and mice. Although the role of Sim1 in the formation of the hypothalamus has been ...described, its postdevelopmental, physiological functions have not been well established. Here we demonstrate that postnatal CNS deficiency of Sim1 is sufficient to cause hyperphagic obesity. We conditionally deleted Sim1 after birth using CaMKII-Cre (alpha-calcium/calmodulin-dependent protein kinase II-Cre) lines to recombine a floxed Sim1 allele. Conditional Sim1 heterozygotes phenocopied germ line Sim1 heterozygotes, displaying hyperphagic obesity and increased length. We also generated viable conditional Sim1 homozygotes, demonstrating that adult Sim1 expression is not essential for mouse or neuron survival and revealing a dosage-dependent effect of Sim1 on obesity. Using stereological cell counting, we showed that the phenotype of both germ line heterozygotes and conditional Sim1 homozygotes was not attributable to global hypocellularity of the paraventricular nucleus (PVN) of the hypothalamus. We also used retrograde tract tracing to demonstrate that the PVN of germ line heterozygous mice projects normally to the dorsal vagal complex and the median eminence. Finally, we showed that conditional Sim1 homozygotes and germ line Sim1 heterozygotes exhibit a remarkable decrease in hypothalamic oxytocin (Oxt) and PVN melanocortin 4 receptor (Mc4r) mRNA. These results demonstrate that the role of Sim1 in feeding regulation is not limited to formation of the PVN or its projections and that the hyperphagic obesity in Sim1-deficient mice may be attributable to changes in the leptin-melanocortin-oxytocin pathway.
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