Analysis of Shank3 Complete and Temporal and Spatial Specific Knockout Mice

描述（由申请人提供）：自闭症谱系障碍（ASD）是一个主要的公共卫生问题，影响110名儿童中的1名。在理解ASD的不同临床表现的细胞和分子机制方面存在根本性的差距。了解这些机制对于开发新的治疗方法至关重要。人类研究固有的局限性使得研究细胞和分子机制变得困难，这为动物模型的研究提供了理论基础。最近的遗传学证据表明SHANK 3基因与ASD有关。SHANK 3是在兴奋性突触的突触后密度处组织信号复合物的支架蛋白。SHANK 3同种型的阵列由6个选择性启动子和编码外显子的剪接产生。SHANK 3基因的缺失是22q13.3缺失型Escheran-McDermid综合征ASD特征的主要原因。整个SHANK 3基因的微缺失和SHANK 3破坏特定亚型的点突变已在ASD和智力残疾患者中被鉴定。我们和其他人报道了具有不同外显子缺失的Shank 3亚型敲除小鼠。在这些小鼠中发现海马、纹状体和新皮质中兴奋性突触的突触后反应减少以及ASD样行为，但也存在显著的表型差异。这种表型异质性可以通过每个突变对Shank 3亚型特异性表达的不同影响来解释。然而，这些差异的解释是复杂的，因为现有的突变小鼠不是Shank 3完全敲除的，并且在不同的脑区域和使用不同的方案进行了分析。因为人类中>95%的SHANK 3分子缺陷缺失整个SHANK 3基因，并且这些患者具有比具有SHANK 3点突变的那些患者更严重的表型，所以我们通过缺失外显子4-22产生了Shank 3完全敲除小鼠，并且还产生了具有floxed外显子4-22的Shank 3条件性敲除小鼠。这些模型对于剖析SHANK 3缺失引起的细胞机制比现有的同种型敲除小鼠更有效。本提案的目的是使用跨学科方法分析Shank 3完全敲除小鼠。中心假设是SHANK 3分子缺陷导致人类ASD的主要途径是通过不同脑区中减少的突触能受体介导的突触后反应的细胞和突触缺陷。利用这些突变小鼠，我们独特地定位于研究以下特定目的：1）在Shank 3完全敲除小鼠中模拟SHANK 3缺失的主要临床特征; 2）。阐明Shank 3缺陷的细胞和分子机制; 3）分析ASD发病机制中Shank 3缺陷的时间和空间要求。这些研究具有重要意义，因为对Shank 3基因完全敲除小鼠的分析可能揭示SHANK 3基因缺失导致ASD的细胞和电路机制。从研究Shank 3中获得的知识将提供可推广的见解，并适用于理解ASD其他原因的病理生理学。 公共卫生相关性：拟议的项目是通过分析Shank 3完全敲除小鼠来了解自闭症谱系障碍的细胞和突触机制。该项目将导致更好地了解ASD的发病机制和开发治疗方法。

DESCRIPTION (provided by applicant): Autism spectrum disorder (ASD) is a major public health problem affecting 1 out 110 children. There is a fundamental gap in understanding the cellular and molecular mechanisms underlying the diverse clinical presentation of ASD. Understanding these mechanisms is critical for developing novel therapeutic approaches. The limitations inherent in human studies make it difficult to study cellular and molecular mechanisms, providing a rationale for the study of animal models. Recent genetic evidence implicates the SHANK3 gene in ASD. SHANK3 is a scaffolding protein that organizes a signaling complex at postsynaptic density of excitatory synapses. An array of SHANK3 isoforms result from 6 alternative promoters and splicing of coding exons. Deletion of the SHANK3 gene is a major contributor to ASD features in the 22q13.3 deletion Phelan-McDermid syndrome. Microdeletions of the entire SHANK3 gene and point mutations of SHANK3 disrupting specific isoforms have been identified in patients with ASD and intellectual disability. We and others reported Shank3 isoform-knockout mice with different exonic deletions. Reduced postsynaptic response of excitatory synapses in hippocampus, striatum, and neocortex as well as ASD-like behaviors is found in these mice, but there were also notable phenotypic differences. This phenotypic heterogeneity could be explained by the different impact that each mutation has on isoform-specific expression of Shank3. However, interpretation of these differences is complicated by the fact that the existing mutant mice are not Shank3 complete knockout and were analyzed in different brain regions and using different protocols. Because >95% of SHANK3 molecular defects in humans delete the entire SHANK3 gene, and these patients have more severe phenotypes than those with point mutations of SHANK3, we have generated Shank3 complete knockout mice by deleting exons 4-22, and also Shank3 conditional knockout mice, with floxed exons 4-22. These models are more valid for dissecting the cellular mechanism arising from SHANK3 deletion than existing isoform-knockout mice. The objective of this proposal is to analyze Shank3 complete knockout mice using an interdisciplinary approach. The central hypothesis is that the primary path by which SHANK3 molecular defects lead to ASD in humans is via cellular and synaptic defects of reduced glutamatergic receptor-mediated postsynaptic responses in different brain regions. With these mutant mice, we are uniquely positioned to study the following Specific Aims: 1) To model major clinical features of SHANK3 deletion in Shank3 complete knockout mice; 2). To delineate the cellular and molecular mechanism underlying Shank3 deficiency; 3) To analyze the temporal- and spatial-requirements of SHANK3 deficiency underlying the pathogenesis of ASD. The proposed studies are significant because analysis of Shank3 complete knockout mice may uncover the cellular and circuit mechanism of ASD caused by SHANK3 deletion. The knowledge gained from studying Shank3 will provide insights that may be generalizable and applicable to understanding the pathophysiology of other causes of ASD. PUBLIC HEALTH RELEVANCE: The proposed project is to understand the cellular and synaptic mechanism underlying autism spectrum disorder by analyzing Shank3 complete knockout mice. This project will lead to better understanding the pathogenesis and developing treatment for ASD.