Cellular and Molecular Mechanisms of SH2B1 Mutations That Cause Profound Childhood Obesity

摘要 肥胖是一个主要的健康问题，与代谢综合征、糖尿病和糖尿病的显著增加有关。 心脏病因此，肥胖症需要深入了解其在细胞，分子和 有机体水平。在一项令人兴奋的新进展中，鉴定了SH 2B 1中的人类突变， 与严重的儿童肥胖有关的支架蛋白SH 2B 1是体重的关键调节因子， 胰岛素敏感性和行为。我们对SH 2B 1如何调节神经元的理解存在根本性的差距。 维持能量稳态的回路以及SH 2B 1中的人类肥胖突变如何破坏该回路 导致肥胖。我们的长期目标是鉴定新的关键信号蛋白和/或基因调控蛋白， 由SH 2B 1调节的事件，对于建立和维持神经回路至关重要， 不良的进食行为和能量平衡，可以作为肥胖、胰岛素- 抵抗和/或适应不良行为。将使用原代神经元、新型小鼠模型和培养细胞 验证SH 2B 1对神经回路的建立和维持至关重要的中心假设 对正常进食行为和能量平衡很重要。从机制上讲，SH 2B 1作为一种支架， 一种增强质膜信号通路和细胞核循环的蛋白，两者都是必需的 用于调节基因转录和神经突生长。人类疾病突变损害了这些基因的一个子集， 海绵。具体目的是：1）确定SH 2B 1调控的神经营养配体信号通路， 2）确定神经突生长所需的细胞核SH 2B 1， 增强基因表达和人类突变损害增强;和3）定义的作用， SH 2B 1参与下丘脑神经元能量平衡的神经回路形成和转录这 研究是创新的，因为：1）SH 2B 1最近被认为是人类肥胖基因; 2）新发现的 固定的SH 2B 1突变为研究肥胖的原因提供了强有力的工具; 3）协调和治疗的概念， 通过支架蛋白在质膜之间的移动对神经营养配体的综合反应 和细胞核是新颖的;和4）许多提出的技术和小鼠模型是尖端的，包括- 使用CRISPR/Cas9技术删除或编辑Sh 2b 1，小鼠模型研究SH 2B 1对神经元的影响， 预测，研究下丘脑中LepRb神经元的小群体内的基因表达， 独特的Sh 2b 1 P322 S小鼠，能够在完整小鼠中研究人SH 2B 1突变的影响， 分离的神经元这项拟议中的研究意义重大，因为它将为细胞生物学提供重要的见解。 以及SH 2B 1和人类突变影响神经元功能的分子机制，包括 那些调节体重，以及如何复杂，多蛋白质为基础的信号是协调之间的plas， ma膜受体和细胞核。这一见解将促进我们对神经元功能的理解， 确定肥胖、胰岛素抵抗和/或适应不良行为的潜在新治疗靶点。

ABSTRACT Obesity is a major health problem associated with a significant increase in metabolic syndrome, diabetes, and heart disease. Thus, obesity demands an in-depth understanding of its causes at the cellular, molecular and organismal level. In an exciting new development, identification of human mutations in SH2B1 that associate with profound childhood obesity implicate the scaffold protein SH2B1 as a critical regulator of body weight, in- sulin sensitivity and behavior. There is a fundamental gap in our understanding of how SH2B1 regulates neural circuitry that maintains energy homeostasis and how human obesity mutations in SH2B1 disrupt that circuitry and contribute to obesity. Our long-term goal is to identify novel key signaling proteins and/or gene regulatory events that are regulated by SH2B1, critical for establishing and maintaining neural circuits important for nor- mal feeding behavior and energy balance, can be targeted for therapeutic intervention for obesity, insulin- resistance and/or maladaptive behavior. Primary neurons, novel mouse models and cultured cells will be used to test the central hypothesis that SH2B1 is crucial for the establishment and maintenance of neural circuits important for normal feeding behavior and energy balance. Mechanistically, SH2B1 serves as a scaffold pro- tein that enhances signaling pathways at the plasma membrane and cycles to the nucleus, both are required for regulating gene transcription and neurite outgrowth. Human disease mutations impair a subset of these re- sponses. The specific aims are: 1) Determine neurotrophic ligand signaling pathways regulated by SH2B1 and impaired by the human mutations; 2) Determine how nuclear SH2B1, which is required for neurite outgrowth, enhances gene expression and the human mutations impair that enhancement; and 3) Define the role for SH2B1 in neural circuit formation and transcription in hypothalamic neurons implicated in energy balance. This research is innovative because: 1) SH2B1 was recently implicated as a human obesity gene; 2) newly identi- fied SH2B1 mutations provide powerful tools to study the causes of obesity; 3) the concept of coordinating an integrated response to neurotrophic ligands by movement of scaffold proteins between the plasma membrane and the nucleus is novel; and 4) many of the proposed techniques and mouse models are cutting edge, includ- ing CRISPR/Cas9 technology to delete or edit Sh2b1, mouse models to study the effect of SH2B1 on neuronal projections, studying gene expression within the small population of LepRb neurons in the hypothalamus, and the unique Sh2b1P322S mouse that enables study of the impact of a human SH2B1 mutation in intact mice and isolated neurons. The proposed research is significant because it will provide critical insight into the cellular and molecular mechanisms by which SH2B1 and the human mutations affect the function of neurons, including those that regulate body weight, and how complex, multi-protein based signals are coordinated between plas- ma membrane receptors and the nucleus. This insight will advance our understanding of neuron function and identify potential new therapeutic targets for obesity, insulin resistance and/or maladaptive behavior.