Neuronal control of body weight and size by the Gbeta5-R7 signaling complex

摘要 肥胖是糖尿病、癌症、心血管疾病、神经疾病和其他疾病的主要共同发病因素 疾病。最近与人类肥胖有关的一组基因是编码R7家族的基因 G蛋白信号蛋白(RGS6、7、9和11)和编码G蛋白β的Gnb5的调节因子 亚基G5。G5和R7蛋白形成必需的二聚体，因此Gnb5基因敲除导致完全 整个G5-R7复合体的降解。这项建议研究了Gnb5和R7基因的缺陷如何 可能导致肥胖；最终目标是找到与以下疾病相关的有效治疗方法 受这些基因调控的神经内分泌途径。 拟议的研究计划建立在早期发现的基础上，这些发现共同构成了一个强有力的前提 为了这项研究。去除一个Gnb5等位基因会导致小鼠肥胖和代谢综合征；这一发现是 后来被人类遗传学证实。随后在这个实验室中进行的机械研究确定了一个新的角色 G5-R7复合体位于胰岛β细胞中，G5-R7强烈促进胰岛素分泌。大脑是最重要的 主要器官调节体重，G5-R7的表达水平远高于胰腺或 任何其他外周组织。这些考虑导致了G5-RGS7调节身体的假设 体重通过其在中枢神经系统中的功能来实现，在中枢神经系统中，与分泌胰岛素的β细胞一样，G5-R7可以调节激素 和/或神经元的神经递质分泌。为了支持这一假设，实验室中最近的研究 证明了使用loxP/Cre技术在下丘脑局部敲除Gnb5引起了戏剧性的 成年小鼠体重增加和肥胖。拟议中的研究将发展这一发现。 具体目标1将分析下丘脑特定核团和神经元类型对GNB5消融的影响 体重和各种激素水平。利用病毒介导的基因转移过表达Gnb5将 以拯救肥胖症。目标2将确定哪些激素和信号蛋白(即GPCRs)是 与Gnb5在下丘脑神经元共表达。组织和急性培养的神经元将被检查 分泌特定的荷尔蒙和信号。特定目标3将研究G5-R7影响的分子事件 通过其基因敲除(CRISPR/Cas9)，在内源性表达M3R的细胞系中进行敲除和过表达 和G5-R7。实验将集中在G5-R7的信号、结构和功能分析以及信号刺激 使用药理制剂以及生化和生物物理分析的分泌物。通过解开一个 调节神经元功能的新机制，这个项目可能会对理解产生强烈的影响 肥胖和其他神经内分泌疾病的病因学。

Abstract Obesity is a major co-morbidity factor in diabetes, cancer, cardiovascular, neurological and other diseases. One group of genes recently linked to obesity in humans are genes encoding the R7 family of regulator of G protein signaling proteins (RGS6, 7, 9 and 11) and Gnb5, which encodes the G protein beta subunit G5. G5 and R7 proteins form obligatory dimers, so that the Gnb5 knockout causes complete degradation of the entire G5-R7 complex. This proposal studies how a deficiency in the Gnb5 and R7 genes can lead to obesity; the ultimate goal is to discover effective treatments of disorders associated with neuroendocrine pathways regulated by these genes. The proposed research plan builds upon earlier discoveries that together constitute a strong premise for this study. Ablation of one Gnb5 allele leads to obesity and metabolic syndrome in mice; this finding was later confirmed by human genetics. Subsequent mechanistic investigations in this lab identified a novel role for the G5-R7 complex in pancreatic beta cells where G5-R7 strongly promoted insulin secretion. Brain is the main organ regulating body weight, and the expression level of G5-R7 is much higher than in the pancreas or any other peripheral tissues. These considerations lead to the hypothesis that G5-RGS7 regulates body weight via its function in the CNS, where as in the insulin-secreting beta cells, G5-R7 can regulate hormone and/or neurotransmitter secretion by neurons. In support of this hypothesis, recent studies in the lab demonstrated that local knockout of Gnb5 in the hypothalamus using LoxP/Cre technology caused a dramatic increase of body weight and adiposity in adult mice. The proposed research will develop this discovery. Specific Aim 1 will analyze the effects of Gnb5 ablation in specific hypothalamic nuclei and types of neurons on body weight and levels of various hormones. Overexpression of Gnb5 using viral-mediated gene transfer will be performed to rescue obesity. Aim 2 will determine which hormones and signaling proteins (i.e., GPCRs) are co-expressed with Gnb5 in hypothalamic neurons. Tissue and acutely cultured neurons will be examined for secretion of specific hormones and signaling. Specific Aim 3 will study molecular events affected by G5-R7 via its knockout (CRISPR/Cas9), knockdown and overexpression in cell lines that endogenously express M3R and G5-R7. Experiments will focus on signaling, structure-function analysis of G5-R7 and signal-stimulated secretion utilizing pharmacological agents and biochemical and biophysical assays. Through unraveling a novel mechanism that regulates neuronal functions, this project may have a strong impact on understanding etiology of obesity and other neuroendocrine disorders.