Dynamic regulation of Shank3 and ASD

描述(由申请人提供)：这项题为“Shank3和ASD的动态调节”的提案研究了将Shank3突变与自闭症谱系障碍联系起来的分子机制。已经在多个ASD病例中发现了Shank3开放阅读框的突变，然而，这些突变是如何导致疾病的尚不清楚。SHANK3是一种支架蛋白，它与代谢型谷氨酸受体、AMPA型谷氨酸受体和NMDA受体相关蛋白相互作用，并在兴奋性突触中协调这些受体之间的生理作用。SHANK3还与荷马病毒相互作用，破坏荷马病毒结合的突变与最严重的ASD有关。为了探索ASD的分子基础，我们产生了Shank3的条件KI，它模仿人类ASD，因为突变蛋白缺乏C-末端和Hmer结合位点。Aim 1结合行为学、生化和生理学方法，将这只Shank3Ki小鼠作为ASD的模型进行研究。这些研究将探索这样一种假设，即突变的Shank3通过一种“功能获得”机制发挥作用，要么改变突触上的信号转导，要么加速包括Wt等位基因表达的Shank3在内的相关蛋白的降解。后一种假设是基于初步的发现，即Shank3的表达严重依赖于荷马结合。目的2研究Shank3降解的分子机制，探讨深橙(Dor)的作用。DOR是空泡蛋白分类(VPS)复合体的一员，它与蛋白酶体和溶酶体一起发挥作用，并以荷马抑制的方式与Shank3物理结合。研究将利用Dor的条件KO来测试Dor控制突触中Shank3水平的预测，反式杂合子分析将测试Dor的基因敲除是否可以修复由于Shank3突变造成的缺陷。这些研究检验了突触生物学中的新机制，并可能为Shank3突变如何导致ASD提供新的见解。 公共卫生相关性：这项建议涉及恢复法有限竞争：解决自闭症谱系障碍异质性的研究(R01)，目标是模型开发，以了解神经生物学机制和行为模型开发。研究将检查两个新的小鼠遗传模型，以评估Shank3的突变如何改变突触功能和行为。

DESCRIPTION (provided by applicant): This proposal entitled "Dynamic Regulation of Shank3 and ASD" examines molecular mechanisms that link mutations of Shank3 with Autism Spectrum Disorder. Mutations of the open reading frame of Shank3 have been identified in multiple cases of ASD, however, it is not yet known how these mutations produce disease. Shank3 is a scaffolding protein that interacts with metabotropic glutamate receptors, AMPA type glutamate receptors and the NMDA receptor associated proteins, and appears to play a role in coordinating physiological interactions between these receptors at excitatory synapses. Shank3 also interacts with Homer, and mutations that disrupt Homer binding are associated with the most severe forms of ASD. To explore the molecular basis of ASD, we have generated a conditional KI of Shank3 that mimics human ASD in that the mutant protein lacks the C-terminus and the Homer binding site. Aim 1 examines this Shank3 KI mouse as a model of ASD using a combination of behavioral, biochemical and physiological approaches. These studies will explore the hypothesis that mutant Shank3 acts by a "gain of function" mechanism, either to alter signaling at the synapse or to accelerate the degradation of associated proteins including Shank3 expressed by the Wt allele. The latter hypothesis is based on preliminary findings that the expression of Shank3 is critically dependent on Homer binding. Aim 2 will examine the molecular mechanism of Shank3 degradation and explore the role of Deep orange (Dor). Dor is a member of the Vacuolar protein sorting (VPS) complex that functions with both proteosomes and lysosomes, and physically associates with Shank3 in a manner that is inhibited by Homer. Studies will utilize a conditional KO of Dor to test the prediction that Dor controls the level of Shank3 at synapses and trans heterozygote analysis will test if genetic knock down of Dor can rescue deficits due to Shank3 mutation. These studies examine novel mechanisms in synaptic biology and may provide new insights into how mutation of Shank3 leads to ASD. PUBLIC HEALTH RELEVANCE: This proposal relates to Recovery Act Limited Competition: Research to Address the Heterogeneity in Autism Spectrum Disorders (R01) targeted to Model Development for Understanding Neurobiological Mechanisms and Behavioral Model Development. Studies will examine two new mouse genetic models to evaluate how mutations of Shank3 alter synaptic function and behavior.