Analysis of Shank3 Complete and Temporal and Spatial Specific Knockout Mice

Shank3 完全敲除小鼠和时空特异性敲除小鼠的分析

• 批准号: 8477306
• 负责人: YONG-HUI JIANG
• 金额: $ 40.82万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8477306
• 关键词: 22q13.3; Affect; Age; Animal Model; Behavior; Behavior assessment; Behavioral; Biochemical; Brain region; Characteristics; Child; Clinical; Code; Complex; Corpus striatum structure; Defect; Development; Disease; Etiology; Excitatory Synapse; Exons; Functional disorder; Gene Mutation; Genes; Genetic; Glutamate Receptor; Glutamates; Heterogeneity; Hippocampus (Brain); Human; Impairment; Intellectual functioning disability; Knock-out; Knockout Mice; Knowledge; Language Delays; Lead; Mediating; Methods; Modeling; Molecular; Molecular Genetics; Mus; Mutant Strains Mice; Mutation; Neocortex; Neurobiology; Neurologic; Pathogenesis; Patients; Phenotype; Point Mutation; Positioning Attribute; Protein Isoforms; Protocols documentation; Public Health; RNA Splicing; Reporting; Scaffolding Protein; Signal Transduction; Staging; Synapses; Syndrome; Testing; Tissues; Triad Acrylic Resin; Work; autism spectrum disorder; base; clinical Diagnosis; density; design; gain of function; insight; interdisciplinary approach; loss of function; microdeletion; mouse model; novel therapeutic intervention; postsynaptic; promoter; receptor; response; social communication; tool

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.

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，每110名儿童中就有1名受到影响。在理解ASD不同临床表现背后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依据。最近的遗传证据表明SHANK3基因与ASD有关。SHANK3是一种支架蛋白，在兴奋性突触的突触后密度组织信号复合物。一系列SHANK3异构体是由6个不同的启动子和编码外显子的剪接产生的。SHANK3基因的缺失是22q13.3缺失费伦-麦克德米综合征中ASD特征的主要因素。在ASD和智力残疾患者中发现了整个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Shank3异构体敲除小鼠具有不同的外显子缺失。这些小鼠海马、纹状体和新皮层的兴奋性突触突触后反应和asd样行为均有所减少，但也存在显著的表型差异。这种表型异质性可以用每种突变对Shank3亚型特异性表达的不同影响来解释。然而，对这些差异的解释是复杂的，因为现有的突变小鼠没有完全敲除Shank3，并且在不同的大脑区域和使用不同的方案进行了分析。由于人类中>95%的SHANK3分子缺陷缺失了整个SHANK3基因，并且这些患者的表型比SHANK3点突变的患者更严重，因此我们通过删除4-22外显子产生了SHANK3完全敲除小鼠，以及4-22外显子被封住的SHANK3条件敲除小鼠。这些模型在剖析SHANK3缺失引起的细胞机制方面比现有的同种异构体敲除小鼠更有效。本提案的目的是使用跨学科方法分析Shank3完全敲除小鼠。核心假设是SHANK3分子缺陷导致人类ASD的主要途径是通过不同脑区减少谷氨酸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，我们具有独特的优势来研究以下具体目标：1)在SHANK3完全敲除小鼠中模拟SHANK3缺失的主要临床特征；2）. 阐明Shank3缺乏的细胞和分子机制；3)分析SHANK3缺失在ASD发病机制中的时空要求。这些研究具有重要意义，因为对Shank3完全敲除小鼠的分析可能揭示Shank3缺失引起的ASD的细胞和电路机制。从研究Shank3中获得的知识将为理解ASD的其他原因的病理生理提供可推广和适用的见解。