Dynamic regulation of Shank3 and ASD

描述（由申请人提供）：这项题为“Shank 3和ASD的动态调节”的提案研究了将Shank 3突变与自闭症谱系障碍联系起来的分子机制。Shank 3开放阅读框的突变已在多个ASD病例中被鉴定，然而，尚不清楚这些突变如何产生疾病。Shank 3是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的支架蛋白，并且似乎在协调兴奋性突触处这些受体之间的生理相互作用中起作用。Shank 3还与Homer相互作用，破坏Homer结合的突变与最严重形式的ASD相关。为了探索ASD的分子基础，我们已经产生了模拟人ASD的Shank 3的条件性KI，因为突变蛋白缺乏C-末端和Homer结合位点。目的1使用行为、生物化学和生理学方法的组合来检查作为ASD模型的Shank 3 KI小鼠。这些研究将探索突变体Shank 3通过“功能获得”机制起作用的假设，要么改变突触处的信号传导，要么加速相关蛋白质的降解，包括Wt等位基因表达的Shank 3。后一种假设基于初步发现，即Shank 3的表达严重依赖于Homer结合。目的2将研究Shank 3降解的分子机制，并探索深橙子（Dor）的作用。Dor是与蛋白体和溶酶体一起起作用的蛋白质分选（VPS）复合物的成员，并且以被Homer抑制的方式与Shank 3物理缔合。研究将利用Dor的条件性KO来测试Dor控制突触处的Shank 3水平的预测，并且反式杂合子分析将测试Dor的遗传敲低是否可以挽救由于Shank 3突变引起的缺陷。这些研究探讨了突触生物学中的新机制，并可能为Shank 3突变如何导致ASD提供新的见解。 公共卫生相关性：该提案涉及恢复法有限竞争：研究，以解决自闭症谱系障碍（R 01）的异质性，目标是模型开发，以了解神经生物学机制和行为模型开发。研究将检查两种新的小鼠遗传模型，以评估Shank 3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.