The Role of IRX3 Neurons in the Regulation of Body Weight Homeostasis

项目摘要/摘要 肥胖是由环境和遗传因素共同驱动的能量失衡造成的。而当 许多基因组座位与肥胖相关，其中一些非编码单核苷酸多态(SNPs)与肥胖有关 脂肪质量和肥胖(FTO)基因的第一个内含子与人类肥胖有最强的关联。 包含这些SNPs的肥胖相关区域在成人中形成了功能性的长距离连接 带有邻近基因易洛魁同源框蛋白3(IRX3)启动子的小鼠大脑，但不是FTO， 提示IRX3可能是体重增加的调节因子。有趣的是，与肥胖相关的SNP， Rs1421085与人脑样本中IRX3表达的增加高度相关，并已被提出 通过干扰转录抑制因子的结合位点直接增加IRX3的表达 IRX3.然而，IRX3功能的机制及相关脑区(S)尚未见报道。因此， 迫切需要更好地了解导致肥胖的潜在潜在机制。 流行病。这项提案的总体目标是研究中枢回路和机制，通过这些回路和机制 IRX3以及与肥胖相关的SNPs会影响体重的动态平衡。为了实现这一目标，我们 建立了一种携带人类rs1421085单核苷酸多态(OB-SNPrs142/Rs142)的新型小鼠模型。重要的是，OB- SNPrs142/Rs142小鼠在人源化中温和高脂饮食条件下的表型概括 与拥有rs1421085 SNP的人类相关，包括脂肪质量百分比增加和 体重增加约5%。我们的初步数据还显示，OB-SNPrs142/Rs142小鼠表现出 以剂量依赖的方式增加大脑中IRX3mRNA的表达，类似于在 携带风险等位基因的人类大脑样本。利用一种新的IRX3-Cre小鼠与tdTomato报告基因杂交 ，我们已经确定IRX3在下丘脑的表达主要表达在 乳头体前腹核(VPM)。这一数据和其他初步数据使我们假设 下丘脑VPM中IRX3的表达增加改变体重稳态并增加身体 体重增加。这一假设将通过以下目标进行评估：在目标1中，我们将评估 VPM IRX3通过利用病毒载体增加VPM中IRX3水平对代谢表型的贡献 然后监测能量参数。此外，通过对VPM中IRX3(+)神经元的化学激活， 我们将探索IRX3神经元在调节新陈代谢中的作用。在目标2中，我们将调查潜力 VPM中IRX3表达上调的电生理和转录机制 IRX3(+)神经元通过膜片钳记录、单细胞RNA测序和染色质影响新陈代谢 免疫沉淀。这一提议意义重大，因为它是第一个询问 作为肥胖相关SNPs代谢效应的驱动力，VPM中IRX3的增加。

PROJECT SUMMARY / ABSTRACT Obesity results from an imbalance of energy that is driven by both environmental and genetic factors. While many genomic loci are associated with obesity, several non-coding single nucleotide polymorphisms (SNPs) in the first intron of the fat mass and obesity (FTO) gene have the strongest association with obesity in humans. The obesity-associated region containing these SNPs forms functional long-distance connections in the adult mouse brain with the promoter of a neighboring gene iroquois homeobox protein 3 (IRX3), but NOT FTO, suggesting that IRX3 may be a regulator of body weight gain. Interestingly, the obesity-associated SNP, rs1421085, is highly associated with increased IRX3 expression in human brain samples and has been proposed to increase IRX3 expression directly through interfering with the binding site of a transcriptional repressor of IRX3. However, the mechanism and relevant brain region(s) of IRX3 function have never been reported. Thus, there is a critical need to better understand the potential underlying mechanisms contributing to the obesity epidemic. The overall objective of this proposal is to investigate the central circuits and mechanisms by which IRX3, and by extension obesity-associated SNPs, influences body weight homeostasis. To accomplish this, we have developed a novel mouse model harboring the human rs1421085 SNP (OB-SNPrs142/rs142). Importantly, OB- SNPrs142/rs142 mice under humanized thermoneutral and high-fat diet conditions recapitulate phenotypes associated with humans possessing the rs1421085 SNP including increased fat mass percentage and an approximately 5% increase in bodyweight. Our preliminary data also reveals that OB-SNPrs142/rs142 mice exhibit increased Irx3 mRNA expression in the brain in a dose-dependent manner, similar to what was reported in the brain samples of humans harboring the risk-allele. Using a novel Irx3-CRE mouse crossed to a tdTomato reporter mouse, we have determined that IRX3 expression in the hypothalamus is primarily expressed in neurons in the ventral premammillary nucleus (vPM). This and additional preliminary data has led us to hypothesize that increased IRX3 expression in the vPM of the hypothalamus alters body weight homeostasis and increases body weight gain. This hypothesis will be evaluated through the following aims: In Aim 1, we will evaluate the contribution of vPM IRX3 to metabolic phenotypes, by utilizing a viral vector to increase IRX3 levels in the vPM and then monitor energy parameters. In addition, using chemogenetic activation of IRX3(+) neurons in the vPM, we will explore the role of IRX3 neurons to regulate metabolism. In Aim 2, we will investigate potential electrophysiological and transcriptional mechanisms through which increased expression of IRX3 in vPM IRX3(+) neurons impacts metabolism using patch-clamp recording, single cell RNA sequencing, and chromatin immunoprecipitation. This proposal is significant because it is the first to interrogate the physiological impact of increased IRX3 in the vPM as the driver of the metabolic effects of the obesity-associated SNPs.