Cellular mechanism of action of SH2B1 isoforms implicated in human obesity

 描述（由申请人提供）：肥胖是一个主要的健康问题，导致糖尿病、代谢综合征和心脏病的大幅增加，以及医疗保健系统的严重压力。减少肥胖需要在细胞、分子和生物体水平上深入了解其原因。在一个令人兴奋的新发展中，已经确定了支架蛋白SH 2B 1中罕见的人类变体，这些变体与严重的儿童肥胖，胰岛素不敏感以及某些个体的适应不良行为和言语和语言延迟有关。这些突变中的一些是特定的SH 2B 1亚型所特有的。在体外，SH 2B 1的亚型具有明显不同的亚细胞定位和增强神经突起的能力， 生长和基因表达。我们对SH 2B 1如何调节神经元回路的理解存在根本性的差距，这些神经元回路调节体重，胰岛素敏感性，行为和学习。我们的长期目标是确定由SH 2B 1亚型调节的通路，这些通路对于建立和/或维持对正常进食行为和能量平衡至关重要的神经回路至关重要。新的小鼠模型，原代培养的神经元和良好表征的培养细胞将用于测试中心假设，即每种SH 2B 1亚型对正常摄食行为和能量平衡重要的神经回路的建立和维持做出独特而关键的贡献。该假设将通过追求3个具体目标来检验：1）使用新的体内小鼠模型来确定SH 2B 1同种型的独特C-末端尾部如何影响下丘脑中表达LepRb的神经元的功能; 2）在细胞和分子水平上确定SH 2B 1同种型的C-末端尾部如何调节神经元中SH 2B 1的功能，特别关注脑特异性SH 2B 1 δ;和3）确定SH 2B 1亚型在调节体重和胰岛素敏感性中的作用。这项研究是创新的，因为：1）SH 2B 1最近才被认为是人类肥胖基因; 2）在一个独特的肥胖个体队列中新发现的SH 2B 1突变为研究肥胖的原因提供了有用的工具; 3）通过质膜，细胞质，细胞核和核仁之间的支架蛋白的运动来协调对神经营养因子的综合反应的概念是新颖的; 4）目前的知识和工具现在使得有可能研究各种SH 2B 1亚型的神经元功能的机制基础，它们在体内调节能量稳态的能力，以及它们对LepRb神经元的转录组和神经元回路的调节;和5）许多提出的技术和小鼠模型是尖端的。这些包括编辑Sh 2b 1的CRISPR/Cas9方法;研究SH 2B 1对与摄食行为和能量消耗相关的LepRb神经元的神经元投射和基因表达的影响的新型小鼠模型;以及缺乏特定SH 2B 1亚型的新型小鼠模型，以研究特定SH 2B 1亚型在完整小鼠和分离神经元中的影响。

 DESCRIPTION (provided by applicant): Obesity is a major health problem, resulting in a substantial increase in diabetes, metabolic syndrome, and heart disease and severe strains on healthcare systems. Reducing obesity demands an in-depth understanding of its causes at the cellular, molecular and organismal level. In an exciting new development, rare human variants in the scaffold protein SH2B1 have been identified that associate with profound childhood obesity, insulin insensitivity, and in some individuals, maladaptive behavior and speech and language delay. Some of these mutations are unique to specific SH2B1 isoforms. In vitro, isoforms of SH2B1 have distinctly different subcellular localization and ability to enhance neurite outgrowth and gene expression. There is a fundamental gap in our understanding of how SH2B1 regulates the neuronal circuitry that regulates, body weight, insulin sensitivity, behavior and learning. Our long-term goal is to identify pathways regulated by the SH2B1 isoforms that are critical for the establishment and/or maintenance of neural circuits important for normal feeding behavior and energy balance. Novel mouse models, primary cultured neurons and well characterized cultured cells will be used to test the central hypothesis that each SH2B1 isoform makes a unique and crucial contribution to the establishment and maintenance of neural circuits important for normal feeding behavior and energy balance. This hypothesis will be tested by pursuing 3 specific aims: 1) Use novel in vivo mouse models to determine how the unique C-terminal tails of SH2B1 isoforms impact the function of LepRb-expressing neurons in the hypothalamus; 2) Determine at the cellular and molecular level how the C-terminal tails of SH2B1 isoforms regulate the function of SH2B1 in neurons with a particular focus on brain-specific SH2B1δ; and 3) Define the role for SH2B1 isoforms in the regulation of body weight and insulin sensitivity. This research is innovative because: 1) SH2B1 was only recently implicated as a human obesity gene; 2) newly identified SH2B1 mutations in a unique cohort of obese individuals provide useful tools for studying causes of obesity; 3) the concept of coordinating an integrated response to neurotrophic factors by movement of scaffold proteins between the plasma membrane, cytoplasm, nucleus and nucleoli is novel; 4) present knowledge and tools now make it possible to study the mechanistic basis for the neuronal function of the various SH2B1 isoforms, their ability to regulate energy homeostasis in vivo, and their regulation of the transcriptome and neuronal circuitry of LepRb neurons; and 5) many of the proposed techniques and mouse models are cutting edge. These include CRISPR/Cas9 methodology to edit Sh2b1; novel mouse models to study the effect of SH2B1 on neuronal projections and gene expression of LepRb neurons relevant to feeding behavior and energy expenditure; and novel mouse models lacking specific isoforms of SH2B1 to study the impact of the specific SH2B1 isoforms in intact mice and isolated neurons.