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

鉴定调节突触 Nrx/Nlg 信号复合物的自闭症基因

• 批准号: 7978923
• 负责人: Craig C Garner
• 金额: $ 20万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-08 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7978923
• 关键词: Adhesions; Adult; Affect; Applications Grants; Asperger Syndrome; Autistic Disorder; Behavioral; Biological Assay; Biotinylation; Brain region; Cadherins; Caring; Cell Adhesion Molecules; Characteristics; Child; Communication; Complex; Coupled; DLG4 gene; Data; Defect; Deletion Mutation; Diagnosis; Down-Regulation; Drug Delivery Systems; Equilibrium; Etiology; Excitatory Synapse; Genes; Genetic; Glutamates; Goals; Heterogeneity; Hippocampus (Brain); Image; Individual; Inhibitory Synapse; Label; Lead; Link; Literature; Mediating; Molecular; Monitor; Mus; Mutate; Mutation; N-Methyl-D-Aspartate Receptors; Neurodevelopmental Disorder; Neurons; Patients; Pervasive Development Disorder; Pharmacological Treatment; Pharmacotherapy; Phenotype; Plastics; Population; Prevalence; Probability; Productivity; Property; Proteins; Publishing; RNA Interference; Resolution; Screening procedure; Services; Signal Pathway; Signal Transduction; Small Interfering RNA; Social Behavior; Special Education; Specific qualifier value; Stereotyped Behavior; Symptoms; Synapses; Synapsins; Synaptic Vesicles; Synaptic plasticity; System; Technology; Testing; Therapeutic; autism spectrum disorder; base; cognitive function; contactin; cost; design; economic cost; experience; expression vector; gene function; high throughput screening; loss of function; mouse model; postsynaptic; presynaptic; public health relevance; small molecule libraries; synaptic function; vector; vocalization

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.

描述（由申请人提供）：自闭症是一种神经发育障碍，其特征是异常的社会行为，沟通障碍，重复或刻板的行为。累积患病率文献表明，大约每1000名儿童中就有1名被诊断为自闭症，每150名儿童中就有1名被诊断为自闭症谱系障碍（ASD）之一，包括Alfreger综合征和PDD-NOS（未另行说明的广泛性发育障碍）。与ASD相关的经济成本估计为350亿美元/年，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与自闭症患者成年后的生产力损失和专业护理相关的成本。开发有效治疗ASD的治疗策略的关键是对其背后的细胞和分子机制的基本理解。这项资助申请的目标是设计一种基于成像的筛选试验，以评估一组与自闭症谱系障碍（ASD）相关的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中涉及的信号通路的关键分子组分，而且还将创建筛选小分子文库的平台，以确定ASD的潜在药物疗法。具体来说，我们将测试的假设，许多基因突变的ASD患者的功能，以调节两个关键的突触蛋白，跨突触细胞粘附分子Neurexin和Neuroligin，这反过来又介导的成熟，功能和可塑性的兴奋性神经元突触之间的复合物的形成。我们将首先建立一种基于成像的测定方法来检测和定量跨突触的Neurexin/Neuroligin（Nlg/Nlg）复合物的水平。在这里，我们将结合联合收割机技术，通过BirA/AP生物素化（由我们的合作者Alice Ting在麻省理工学院开发）来标记突触Nlg/Nlg复合物，双顺反子载体同时将两种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Nlg/Nlg复合物的形成。接下来，我们将评估是否至少有一个ASD相关基因的子集调节突触神经元/Nlg复合物的形成。具体来说，我们将针对已知的ASD相关基因产物创建短干扰（si）RNA，并进行中等通量筛选以评估这些分子的下调是否影响Nlg/Nlg复合物的形成。一旦到位，该测定将适用于siRNA和小分子文库的自动化，高通量筛选，从而能够鉴定基于Nlg/Nlg的信号传导途径的其他分子组分，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。 公共卫生关系：这项资助申请的目标是设计一种基于成像的筛选试验，以评估与自闭症谱系障碍（ASD）相关的基因子集是否存在于共同的信号通路中。具体来说，我们将测试的假设，一组基因突变的ASD患者的功能，以调节两个关键的突触蛋白，细胞粘附分子Neurexin和Neuroligin，这反过来介导的成熟，功能和可塑性的兴奋性突触之间的跨突触复合物的形成。在目的1中，我们将建立一个基于成像的测定，以检测和定量水平的跨突触的Neurexin/Neuroligin（Nlg/Nlg）复合物在分离的海马培养。在这里，我们将结合联合收割机技术，通过BirA/AP生物素化（由我们的合作者Alice Ting在麻省理工学院开发）来标记突触Nlg/Nlg复合物，双顺反子载体同时将两种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Nlg/Nlg复合物的形成。在目标2中，我们将评估ASD相关基因是否调节突触Nlg/Nlg复合物的形成。具体来说，我们将为已知的ASD相关基因产物创建短干扰（si）RNA，并进行中等通量筛选，以评估这些分子的下调是否影响Nlg/Nlg复合物的形成。一旦到位，该测定将适用于siRNA和小分子文库的自动化高通量筛选，从而能够鉴定基于Nlg/Nlg的信号传导途径的其他分子组分和潜在的药物靶点，以使携带这些基因突变的ASD患者的认知功能正常化。