Dynamic regulation of Shank3 and ASD

描述（由申请人提供）：这是一个5 RO 1 NS 070301 -02的竞争性更新，题为“小腿3和自闭症谱系障碍的动态调节”，这是作为ARRA赠款开始09/30/2009。Shank 3在人类遗传学研究中与自闭症谱系障碍（ASD）和精神分裂症有关，我们的广泛目标是定义与Shank 3突变有关的行为疾病的分子基础，并确定适用于ASD其他遗传原因的统一概念。我们创建了一个小鼠模型，模拟人类受试者的突变，删除了Shank 3的C-末端[Shank 3（+/<$exon21）]，并发表了支持ASD新分子模型的研究结果（1）。我们发现Shank 3（+/<$exon21）小鼠表达缺少C末端的Shank 3突变蛋白（Shank 3 <$C），这以“功能获得”的方式选择性地增加WT Shank 3和NMDA受体NR 1亚基的泛素化，并减少它们在突触处的表达。Shank 3（+/<$exon 21）小鼠表现出NMDA受体依赖性突触可塑性降低，以及mGluR依赖性长期抑制增强。柄3（+/â外显子21）小鼠具有正常的记忆功能，但在相互社会互动中显示出显著的行为缺陷，以及与精神分裂症经典相关的表型。这些发现提供了重要的验证，即Shank 3突变的分子机制可以成为小鼠模型中与ASD相关的行为功能障碍的基础。这种竞争性延续的目的是：目的1将检验我们的假设，即Shank 3和NR 1的泛素化增加以及相关的行为缺陷取决于Homer的Shank 3交联程度（反向）（1）。我们将使用删除Homer 2（以减少Homer交联）或选择性删除立即早期基因形式Homer 1a（以增加交联）的小鼠遗传模型，并确定这如何改变Shank 3（+/<$exon21）小鼠的表型。由于Homer 1a受行为经验的动态调节，这些研究将测试连接行为经验和表型严重程度的分子机制。目的2将检测Shank 3的候选泛素连接酶，并确定其对Shank 3（+/<$exon21）小鼠表型的贡献。目的3将评估在Shank 3（+/<$exon21）小鼠中mTORC 1（雷帕霉素复合物1的哺乳动物靶蛋白）信号传导改变的观察结果，并检测将蛋白质周转与mTORC 1活性联系起来的假设一般机制。目的4将检查Shank 3 C表达和增强的mGluR 5信号传导之间的联系，并测试mGluR 5的抑制是否可以降低表型的严重程度。目的5将利用Shank 3小鼠的条件特性来检查Shank 3 C产生的表型的发育和细胞基础。这些研究解决了NIH设定的建立行为疾病动物模型的重要目标，并为开发合理的治疗方法提供科学依据。 公共卫生相关性：为了发现导致自闭症谱系障碍（ASD）的分子机制，我们创建了一种新型小鼠模型，该模型模拟了与ASD相关的人类Shank 3突变，并报告了模仿人类ASD和精神分裂症的行为缺陷。最重要的是，我们发现了一种分子机制可以使细胞和行为表型合理化。在这项更新提案中，我们将更深入地了解分子机制，并评估可能导致ASD多种遗传原因的趋同机制，并合理化新的治疗方向。

DESCRIPTION (provided by applicant): This is a competitive renewal of 5RO1NS070301-02, entitled "Dynamic Regulation of Shank3 and Autism Spectrum Disorders", which was awarded as an ARRA grant beginning 09/30/2009. Shank3 is linked in human genetic studies to both autism spectrum disorders (ASD) and schizophrenia, and our broad goals are to define the molecular basis for behavioral diseases linked to mutations of Shank3, and to identify unifying concepts that are applicable to other genetic causes of ASD. We created a mouse model that mimics human subjects with mutations that delete the C-terminus of Shank3 [Shank3(+/ ¿exon21)], and have published findings that support a novel molecular model for ASD(1). We discovered that Shank3 (+/ ¿exon21) mice express Shank3 mutant protein lacking the C-terminus (Shank3¿C), and this acts in a "gain-of-function" manner to selectively increase the ubiquitination of WT Shank3 and the NR1 subunit of the NMDA receptor, and reduce their expression at synapses. Shank3(+/¿exon 21) mice show reduced NMDA receptor-dependent synaptic plasticity, as well as enhanced mGluR-dependent long-term depression. Shank3(+/¿exon21) mice have normal memory function but display prominent behavioral deficits in reciprocal social interaction, together with phenotypes classically associated with schizophrenia. These findings provide important validation that molecular mechanisms consequent to mutation of Shank3 can underlie behavioral dysfunction relevant to ASD in a mouse model. Aims of this competitive continuation are; Aim 1 will test our hypothesis that increased ubiquitination of Shank3 and NR1, and associated behavioral deficits, are dependent (inversely) on the degree of Shank3 cross-linking by Homer(1). We will use mouse genetic models that delete Homer2 (to reduce Homer crosslinking), or selectively delete the immediate early gene form Homer1a (to increase crosslinking), and determine how this modifies phenotypes in Shank3 (+/ ¿exon21) mice. Since Homer1a is dynamically regulated by behavioral experience, these studies will test a molecular mechanism linking behavioral experience and severity of phenotypes. Aim 2 will examine a candidate ubiquitin ligase for Shank3 and determine its contribution to phenotypes in Shank3 (+/ ¿exon21) mice.Aim 3 will evaluate the observation that mTORC1 (mammalian target of rapamycin complex 1) signaling is altered in Shank3 (+/¿exon21) mice, and examine a hypothesized general mechanism that links protein turnover with mTORC1 activity. Aim 4 will examine the link between Shank3¿C expression and enhanced mGluR5 signaling, and test if inhibition of mGluR5 can reduce the severity of phenotypes. Aim 5 will utilize the conditional property of the Shank3 mouse to examine the developmental, and cellular bases of phenotypes produced by Shank3 ¿C. These studies address important goals set by the NIH to establish animal models of behavioral disease, and to provide a scientific basis for development of rational therapies. PUBLIC HEALTH RELEVANCE: With a goal to discover molecular mechanisms that contribute to autism spectrum disorders (ASD), we have created a novel mouse model that mimics human mutations of Shank3 linked to ASD and have reported behavioral deficits that mimic aspects of human ASD and schizophrenia. Most importantly, we have discovered a molecular mechanism can rationalize cellular and behavioral phenotypes. In this renewal proposal, we will gain a deeper understanding of molecular mechanisms and evaluate convergent mechanisms that could underlie multiple genetic causes of ASD, and rationalize new directions for therapy.