Dynamic regulation of Shank3 and ASD

描述(由申请人提供)：这是5RO1NS070301-02的竞争性续订，标题为“Shank3和自闭症谱系障碍的动态监管”，从2009年9月30日开始作为ARRA赠款授予。在人类遗传学研究中，SHANK3与自闭症谱系障碍(ASD)和精神分裂症都有关联，我们的广泛目标是定义与Shank3突变相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，突变缺失Shank3[Shank3(+/？exon21)]的C-末端，并发表了支持ASD(1)的新分子模型的发现。我们发现，Shank3(+/？外显子21)小鼠表达缺失C末端的Shank3突变蛋白(Shank3？C)，这是一种“功能获得”的方式，选择性地增加了WT Shank3和NMDA受体的NR1亚单位的泛素化，并减少了它们在突触的表达。SHANK3(+/？外显子21)小鼠表现出NMDA受体依赖的突触可塑性降低，以及mGluR依赖的长期抑郁增强。SHANK3(+/？外显子21)小鼠具有正常的记忆功能，但在相互的社会互动中表现出显著的行为缺陷，以及典型的与精神分裂症有关的表型。这些发现提供了重要的验证，即在小鼠模型中，Shank3突变导致的分子机制可能是与ASD相关的行为障碍的基础。这种竞争性延续的目的是；目标1将测试我们的假设，即Shank3和NR1泛素化的增加以及相关的行为缺陷依赖于(相反)由荷马(1)进行的Shank3的交联度。我们将使用删除Hmer 2(以减少Hmer交联度)或选择性地删除早期直接基因形式Hmer 1a(以增加交联度)的小鼠遗传模型，并确定这如何改变Shank3(+/？)外显子21小鼠的表型。由于Hmer 1a受行为经验的动态调节，这些研究将测试将行为经验与表型严重性联系起来的分子机制。目的2将检测Shank3的一个候选泛素连接酶，并确定其对Shank3(+/？外显子21)小鼠表型的贡献。目的3将评估在Shank3(+/？外显子21)小鼠中mTORC1(雷帕霉素复合体1的哺乳动物靶标)信号改变的观察结果，并检验假设的将蛋白质周转与mTORC1活性联系起来的一般机制。目的4将研究Shank3C的表达与增强的mGluR5信号之间的联系，并测试抑制mGluR5是否可以降低表型的严重性。目的5将利用Shank3小鼠的条件属性来研究Shank3？C产生的表型的发育和细胞基础。这些研究旨在解决NIH设定的重要目标，即建立行为疾病的动物模型，并为开发合理的治疗方法提供科学依据。

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.