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

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.