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，影响到110名儿童中的1名。在对ASD不同临床表现背后的细胞和分子机制的理解上存在着根本的差距。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，SHANK3基因与ASD有关。SHANK3是一种支架蛋白，它在突触后密度的兴奋性突触上组织一个信号复合体。一组SHANK3亚型是由6个不同的启动子和编码外显子的拼接产生的。SHANK3基因缺失是导致22q13.3缺失的费兰-麦克德米德综合征ASD特征的主要原因。在患有自闭症和智力障碍的患者中，已经发现了整个SHANK3基因的微缺失和SHANK3破坏特定亚型的点突变。我们和其他人报道了带有不同外显子缺失的Shank3亚型敲除小鼠。在这些小鼠中，发现海马区、纹状体和新皮质的兴奋性突触的突触后反应以及ASD样行为减少，但也有显著的表型差异。这种表型的异质性可以用每个突变对Shank3异构体特异性表达的不同影响来解释。然而，现有的突变小鼠并不是Shank3完全敲除的，并且在不同的大脑区域和使用不同的方案进行了分析，这使得对这些差异的解释变得复杂。因为人类中95%的SHANK3分子缺陷会删除整个SHANK3基因，而且这些患者的表型比那些SHANK3点突变的患者更严重，所以我们通过删除外显子4-22产生了Shank3完全基因敲除小鼠，也产生了带有外显子4-22的Shank3条件性基因敲除小鼠。这些模型在剖析SHANK3缺失引起的细胞机制方面比现有的同型基因敲除小鼠更有效。这项建议的目标是使用跨学科的方法分析Shank3完全基因敲除小鼠。中心假设是SHANK3分子缺陷导致人类ASD的主要途径是通过不同脑区谷氨酸受体介导的突触后反应减少的细胞和突触缺陷。有了这些突变小鼠，我们独特地定位于研究以下特定目标：1)在Shank3完全敲除小鼠中建立SHANK3缺失的主要临床特征的模型；2)。阐明Shank3缺乏的细胞和分子机制；3)分析ASD发病机制中Shank3缺乏的时间和空间要求。这项研究具有重要意义，因为对Shank3基因完全敲除小鼠的分析可能揭示由shank3缺失引起的ASD的细胞和电路机制。从研究Shank3获得的知识将提供可推广和适用于理解ASD其他原因的病理生理学的见解。

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.