Elucidating the Roles of SHANK3 and FXR in the Autism Interactome

描述(由申请人提供)：本提案响应RFA-MH-09-170，《恢复法限制竞争：解决自闭症谱系障碍异质性的研究》(R01)。正如RFA中指出的那样，自闭症谱系障碍(ASD)形成了一个非常不同的群体：超过30个不同的基因被确定为导致或促成自闭症的原因。为了更好地了解如此多样化的基因阵列如何导致相似的ASD表型，我们搜索了由自闭症相关基因编码的蛋白质的结合伙伴，并开发了一个自闭症蛋白质相互作用网络(“相互作用体”)，其中包含900多种蛋白质相互作用，其中许多是新的。这个相互作用组揭示的最令人兴奋的发现之一是发现有三个“中枢”蛋白质是相互作用的中心：FXR1、SHANK3和TSC1。此外，SHANK3与两类不同的蛋白质相互作用：一类涉及突触结构，另一类涉及RNA新陈代谢和翻译。后者将SHANK3与脆性X蛋白(FMRP)及其类似基因(FXR1和FX2)以及结节性硬化症复合蛋白(TSC1和TSC2)连接起来。部分基于这些发现，我们假设在各种ASD之间存在共同的致病机制，其中之一是突触的维持和可塑性。我们认为，SHANK3在突触中扮演着两个不同的角色，一个涉及突触的组织和维持，另一个涉及突触的局部RNA翻译。为了深入了解ASD发病的关键机制，我们将在体内识别和表征突触上的天然SHANK3复合体。为了实现这一点，我们将产生标记SHANK3蛋白的敲入小鼠，以允许在体内纯化SHANK3相关的复合体。我们将研究SHANK3天然复合体，以确定是否存在不同类别的SHANK3蛋白质复合体以及这些亚复合体中的蛋白质类型。我们将产生黑色或过度表达Shank3的小鼠，我们将描述这些Shank3小鼠模型的特征。此外，我们还将分析在缺乏FXR基因的小鼠中调节Shank3表达的行为、解剖学和生理学后果。详细的行为和生化研究将使我们能够在体内建立SHANK3和FXR蛋白之间的功能关系，并为详细的机制研究提供基础，这些研究将使广大ASD患者受益。 与公共卫生相关：自闭症是一种由不同基因突变引起的异质性疾病，被认为是一个主要的公共卫生问题，因为估计每150-200名儿童中就有一人患有自闭症。我们发现，一些导致自闭症的蛋白质与相同的伴侣相互作用，这表明有共同的途径导致自闭症。在这笔赠款中，我们将描述一些导致自闭症的常见途径，这将与公共卫生相关，因为我们的研究将帮助许多类型的自闭症，而不是一两个亚型。

DESCRIPTION (provided by applicant): This proposal is responsive to RFA-MH-09-170, the Recovery Act Limited Competition: Research to Address the Heterogeneity in Autism Spectrum Disorders (R01). As noted in the RFA, Autism spectrum disorders (ASD) form a very heterogeneous group: over 30 distinct genes have been identified as either causing or contributing to the cause of autism. To better understand how such a diverse array of genes can cause similar ASD phenotypes, we searched for the binding partners of proteins encoded by autism-associated genes and developed an autism protein interaction network (an "interactome") that contains over 900 protein interactions, many of which are novel. One of the most exciting discoveries unveiled by this interactome is the finding that there are three "hub" proteins that are centers for interaction: FXR1, SHANK3, and TSC1. Moreover, SHANK3 interacts with two distinct classes of proteins: one involved in synapse structure, the other involved in RNA metabolism and translation. This latter class connects SHANK3 to the Fragile X protein (FMRP) and its paralogs (FXR1 and 2) as well as the tuberous sclerosis complex proteins (TSC1 and TSC2). Based in part on these findings, we hypothesize that there are shared pathogenic mechanisms amongst various ASD, one of which centers on synapse maintenance and plasticity. We propose that SHANK3 plays two distinct roles at the synapse, one involving synapse organization and maintenance and the other involving local RNA translation at the synapse. To gain insight into key mechanisms underlying ASD pathogenesis, we will identify and characterize the native SHANK3 complexes at the synapse in vivo. To accomplish this, we will generate knock-in mice that tag the SHANK3 protein to permit in vivo purification of SHANK3-associated complexes. We will examine SHANK3 native complexes to determine whether there are distinct classes of SHANK3 protein complexes and the type of proteins in these sub-complexes. We will generate mice that either black or over-express Shank3 and we will characterize these Shank3 mouse models. In addition we will analyze behavioral, anatomical, and physiological consequences of modulating Shank3 expression in mice lacking FXR genes. Detailed behavioral and biochemical studies will allow us to establish a functional relationship between SHANK3 and FXR proteins in vivo and provide a foundation for detailed mechanistic studies that should benefit a broad population of ASD patients. PUBLIC HEALTH RELEVANCE: Autism is a heterogeneous disorder caused by mutations in different genes and is considered a major public health problem given the estimated prevalence of 1 in 150-200 children. We discovered that some autism- causing proteins interact with the same partners suggesting that there are shared pathways that lead to autism. In this grant, we will characterize some common pathways leading to autism, which would be of public health relevance because our studies will help many types of autism rather than one or two subtypes.