Identification of Autism genes that regulate synaptic Nrx/Nlg signaling complexes

描述(申请人提供)：自闭症是一种神经发育障碍，特征是不正常的社交行为，沟通障碍，以及重复或刻板印象的行为。累积患病率文献表明，大约每1000名儿童中就有1人被诊断为自闭症，多达150人中有1人被诊断为自闭症谱系障碍(ASD)之一，包括阿斯伯格综合症和PDD-NOS(普适性发育障碍，未另行规定)。与自闭症相关的经济成本估计为每年350亿美元，包括特殊教育服务和减轻症状的治疗。这些估计甚至没有考虑到自闭症患者成年后失去生产力和专门护理的成本。制定有效治疗ASD的治疗策略的关键是对其背后的细胞和分子机制有基本的了解。这项拨款申请的目标是设计一种基于成像的筛查试验，以评估与自闭症谱系障碍(ASD)相关的一组基因是否存在于共同的信号通路中。我们的方法不仅将定义ASD中涉及的信号通路(S)的关键分子组件，而且还将创建一个筛选小分子文库的平台，以确定ASD的潜在药物治疗。具体地说，我们将测试这一假设，即ASD患者中的许多基因突变的功能是调节两种关键突触蛋白-跨突触细胞黏附分子Neuresin和Neuroigin之间复合体的形成，这反过来又介导兴奋性谷氨酸能突触的成熟、功能和可塑性。我们将首先建立一种基于成像的分析方法来检测和量化跨突触神经尿素素/神经连接蛋白(NRX/NLG)复合体的水平。在这里，我们将结合通过Bira/AP生物素化的邻近标记技术(由我们的合作者MIT的Alice Ting开发)来标记突触NRX/NLG复合体，双顺反子载体同时将两个突触前或突触后蛋白引入同一神经元，以及高分辨率定量成像来监测NRX/NLG复合体的形成。接下来，我们将评估是否至少有一部分ASD相关基因调控突触NRX/NLG复合体的形成。具体地说，我们将针对已知的ASD相关基因产物创建短干扰(Si)RNA，并进行中通量筛选，以评估这些分子的下调是否影响NRX/NLG复合体的形成。一旦到位，这种检测方法将适用于siRNA和小分子文库的自动化、高通量筛选，从而能够识别基于NRX/NLG的信号通路的其他分子成分，以及用于使携带这些基因突变的ASD患者的认知功能正常化的潜在药物靶点。 公共卫生相关性：这项拨款申请的目标是设计一种基于成像的筛查试验，以评估与自闭症谱系障碍(ASD)相关的基因亚集是否存在于共同的信号通路中。具体地说，我们将测试一组在ASD患者中突变的基因的功能，以调节两种关键突触蛋白-细胞黏附分子Neuresin和Neuroigin之间跨突触复合体的形成，这反过来又介导兴奋性谷氨酸能突触的成熟、功能和可塑性。在目标1中，我们将建立一种基于成像的分析方法来检测和定量分离的海马区培养物中跨突触神经尿素素/神经连接蛋白(NRX/NLG)复合体的水平。在这里，我们将结合通过Bira/AP生物素化的邻近标记技术(由我们的合作者MIT的Alice Ting开发)来标记突触NRX/NLG复合体，双顺反子载体同时将两个突触前或突触后蛋白引入同一神经元，以及高分辨率定量成像来监测NRX/NLG复合体的形成。在目标2中，我们将评估ASD相关基因是否调节突触NRX/NLG复合体的形成。具体地说，我们将为已知的ASD相关基因产物创建短干扰(Si)RNA，并进行中通量筛选，以评估这些分子的下调是否影响NRX/NLG复合体的形成。一旦到位，这种分析将适用于siRNA和小分子文库的自动化、高通量筛选，从而能够识别基于NRX/NLG的信号通路的其他分子成分，以及潜在的药物靶点，以使携带这些基因突变的ASD患者的认知功能正常化。

DESCRIPTION (provided by applicant): Autism is a neurodevelopmental disorder characterized by abnormal social behavior, communication deficits, and repetitive or stereotyped behaviors. Cumulative prevalence literature suggests that approximately 1 in 1000 children are diagnosed with Autism, and as many as 1 in 150 are diagnosed with one of the Autism Spectrum Disorders (ASDs), including Asperger's Syndrome and PDD-NOS (pervasive developmental disorder not otherwise specified). Economic costs associated with ASDs are estimated at $35 billion/year, including special education services and treatments to reduce symptoms. These estimates do not even factor in the costs associated with lost productivity and specialized care for Autistic individuals once they reach adulthood. A key to developing therapeutic strategies to effectively treat ASDs is a fundamental understanding of the cellular and molecular mechanisms that underlie them. The goal of this grant application is to design an imaging-based screening assay in order to assess whether a group of genes associated with Autism Spectrum Disorders (ASDs) lie in a common signaling pathway. Our approach will not only define key molecular components of the signaling pathway(s) involved in ASDs, but also create a platform for screening small molecule libraries in order to identify potential pharmacotherapies for ASDs. Specifically, we will test the hypothesis that many of the genes mutated in ASD patients function to regulate the formation of complexes between two key synaptic proteins, the transsynaptic cell adhesion molecules Neurexin and Neuroligin, which in turn mediate the maturation, function, and plasticity of excitatory glutamatergic synapses. We will first establish an imaging-based assay to detect and quantify levels of transsynaptic Neurexin/Neuroligin (Nrx/Nlg) complexes. Here, we will combine the technologies of proximity labeling via BirA/AP biotinylation (developed by our collaborator Alice Ting at MIT) to label synaptic Nrx/Nlg complexes, bicistronic vectors to simultaneously introduce two pre- or postsynaptic proteins into the same neuron, and high-resolution quantitative imaging to monitor Nrx/Nlg complex formation. Next, we will evaluate whether at least a subset of ASD-associated genes regulate the formation of synaptic Nrx/Nlg complexes. Specifically, we will create short interfering (si) RNAs against known ASD-associated gene products, and perform a medium-throughput screen to assess whether downregulation of these molecules affects Nrx/Nlg complex formation. Once in place, this assay will be adaptable for automated, higher-throughput screens of siRNA and small molecule libraries, thus enabling the identification of other molecular components of the Nrx/Nlg-based signaling pathway, and of potential drug targets to normalize cognitive function in ASD patients carrying mutations in these genes. PUBLIC HEALTH RELEVANCE: The goal of this grant application is to design an imaging-based screening assay in order to assess whether subsets of genes associated with Autism Spectrum Disorders (ASDs) lie in a common signaling pathway. Specifically, we will test the hypothesis that a set of the genes mutated in ASD patients function to regulate the formation of transsynaptic complexes between two key synaptic proteins, the cell adhesion molecules Neurexin and Neuroligin, which in turn mediate the maturation, function, and plasticity of excitatory glutamatergic synapses. In Aim 1, we will establish an imaging-based assay to detect and quantify levels of transsynaptic Neurexin/Neuroligin (Nrx/Nlg) complexes in dissociated hippocampal cultures. Here, we will combine the technologies of proximity labeling via BirA/AP biotinylation (developed by our collaborator Alice Ting at MIT) to label synaptic Nrx/Nlg complexes, bicistronic vectors to simultaneously introduce two pre- or postsynaptic proteins into the same neuron, and high-resolution quantitative imaging to monitor Nrx/Nlg complex formation. In Aim 2, we will evaluate whether ASD-associated genes regulates the formation of synaptic Nrx/Nlg complexes. Specifically, we will create short interfering (si) RNAs for the known ASD-associated gene products, and perform a medium-throughput screen to assess whether downregulation of these molecules affects Nrx/Nlg complex formation. Once in place, this assay will be adaptable for automated, high-throughput screens of siRNA and small molecule libraries, thus enabling the identification of other molecular components of the Nrx/Nlg-based signaling pathway and of potential drug targets to normalize cognitive function in ASD patients carrying mutations in these genes.