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、沟通缺陷以及重复或刻板行为。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，多达每 150 名儿童中就有 1 名被诊断为自闭症谱系障碍 (ASD) 之一，包括阿斯伯格综合症和 PDD-NOS（未另行指定的广泛性发育障碍） 。与 ASD 相关的经济成本估计为每年 350 亿美元，其中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与生产力损失和专门护理相关的成本。制定有效治疗 ASD 的治疗策略的关键是对 ASD 背后的细胞和分子机制的基本了解。该拨款申请的目标是设计一种基于成像的筛查方法，以评估一组与自闭症谱系障碍 (ASD) 相关的基因是否位于共同的信号通路中。我们的方法不仅将定义 ASD 涉及的信号通路的关键分子成分，还将创建一个筛选小分子库的平台，以确定 ASD 的潜在药物疗法。具体来说，我们将检验这样的假设：自闭症谱系障碍患者中的​​许多基因突变的功能是调节两种关键突触蛋白（跨突触细胞粘附分子 Neurexin 和 Neuroligin）之间复合物的形成，这反过来又介导突触蛋白的成熟、功能和可塑性。兴奋性谷氨酸突触。我们将首先建立一种基于成像的检测方法来检测和量化跨突触 Neurexin/Neuroligin (Nrx/Nlg) 复合物的水平。在这里，我们将结合通过 BirA/AP 生物素化的邻近标记技术（由我们的合作者麻省理工学院的 Alice Ting 开发）来标记突触 Nrx/Nlg 复合物、双顺反子载体，以同时将两种突触前或突触后蛋白质引入同一神经元，以及高分辨率定量成像监测 Nrx/Nlg 复合物的形成。接下来，我们将评估至少一部分 ASD 相关基因是否调节突触 Nrx/Nlg 复合体的形成。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，并进行中通量筛选以评估这些分子的下调是否影响 Nrx/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，从而能够识别基于 Nrx/Nlg 的信号通路的其他分子成分，以及使认知功能正常化的潜在药物靶点患有这些基因突变的 ASD 患者。 公共健康相关性：本次拨款申请的目标是设计一种基于成像的筛查方法，以评估与自闭症谱系障碍 (ASD) 相关的基因子集是否位于共同的信号通路中。具体来说，我们将测试以下假设：自闭症谱系障碍患者中的​​一组基因突变能够调节两种关键突触蛋白（细胞粘附分子 Neurexin 和 Neuroligin）之间跨突触复合物的形成，进而介导成熟、功能和可塑性兴奋性谷氨酸能突触。在目标 1 中，我们将建立一种基于成像的检测方法来检测和量化离体海马培养物中跨突触 Neurexin/Neuroligin (Nrx/Nlg) 复合物的水平。在这里，我们将结合通过 BirA/AP 生物素化的邻近标记技术（由我们的合作者麻省理工学院的 Alice Ting 开发）来标记突触 Nrx/Nlg 复合物、双顺反子载体，以同时将两种突触前或突触后蛋白质引入同一神经元，以及高分辨率定量成像监测 Nrx/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 Nrx/Nlg 复合体的形成。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，并进行中通量筛选以评估这些分子的下调是否影响 Nrx/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，从而能够识别基于 Nrx/Nlg 的信号通路的其他分子成分和潜在的药物靶标，以使认知功能正常化。 ASD 患者携带这些基因突变。