Localizing endogenous synaptic proteins in vivo

体内定位内源性突触蛋白

• 批准号: 10308511
• 负责人: Megan Elise Williams
• 金额: $ 19.06万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308511
• 关键词: Adult; Age; Antibodies; Axon; Brain; Brain Diseases; CRISPR/Cas technology; Cadherins; Cells; Cellular biology; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; Data; Defect; Dendrites; Elements; Embryo; Engineering; Ensure; Enterobacteria phage P1 Cre recombinase; Epitopes; Functional disorder; Genes; Genetic; Goals; Guide RNA; Hippocampus (Brain); In Vitro; Infection; Intravenous; Intraventricular; Knockout Mice; Knowledge; Label; Mediating; Mental disorders; Methodology; Methods; Modification; Molecular; Monsters; Mus; N-Cadherin; Neonatal; Neurons; Neurosciences; Pattern; Positioning Attribute; Property; Protein Overexpression; Proteins; Reporter; Resolution; Route; Specificity; Synapses; System; Technology; Testing; Tissues; Transcript; Vertebral column; Viral; Visualization; Western Blotting; adeno-associated viral vector; arm; brain tissue; cadherin 10; cadherin-6; deep sequencing; experimental study; flexibility; fluorophore; gain of function; genome editing; genomic locus; high resolution imaging; in vivo; in vivo evaluation; in vivo imaging; innovation; insight; interest; microscopic imaging; neurotransmission; new technology; polarized cell; protein function; public health relevance; relating to nervous system; response; success; synaptic function; synaptogenesis; tool; trafficking; virtual

PROJECT SUMMARY Determining the subcellular localization of a protein under different cellular states is a critical aspect of assessing protein function and dysfunction. This is especially important in neuroscience as neurons are complex, polarized cells with distinct functional compartments, including synapses. Synapse dysfunction underlies many neural and psychiatric disorders. Interestingly, synapses connecting different neurons develop unique structural and functional properties that differentially modulate circuit function. This structural and functional diversity is mediated by molecular differences. However, our understanding of the proteins located at different types of synapses is very limited. Suitable antibodies are simply not available for many proteins and protein overexpression drives mis-localization. Therefore, methods to localize endogenous synaptic proteins in brain tissue are urgently needed. Here, we developed a CRISPR gene editing strategy that, in one seamless genetic modification, inserts an epitope tag onto a protein of interest and drives expression of a cell marker in postmitotic neurons using a single AAV. Our innovative new method is the first to provide an integrated means for selectively identifying only those neurons that correctly integrated the protein tag and provide a cell filling, structural reference necessary for determining the synapse-specific localization of a protein. Our method is highly flexible for a variety of proteins, tags, and cell markers. Preliminary data indicate that our method correctly tags the synaptic protein N-cadherin in cultured neurons but it requires further optimization and expansion in vitro (Aim 1) and in vivo (Aim 2). Successful completion of our proposal will yield new technologies that allow the study of endogenously expressed, synapse-specific proteins in the brain. As an example, we will test the hypothesis that different cadherins associated with distinct mental illnesses localize to different types of synapses in vivo. Taken together, our results are expected to result in a new technology that can be broadly applied to study synapse formation and function and provide new molecular insight to mechanisms underlying synapse diversity.

项目摘要 确定蛋白质在不同细胞状态下的亚细胞定位是蛋白质组学的一个关键方面。 评估蛋白质功能和功能障碍。这在神经科学中尤其重要，因为神经元 具有不同功能区室的复杂极化细胞，包括突触。突触功能障碍 是许多神经和精神疾病的基础有趣的是，连接不同神经元的突触 独特的结构和功能特性，差异调制电路功能。这种结构和 功能多样性由分子差异介导。然而，我们对位于 不同类型的突触是非常有限的合适的抗体对于许多蛋白质是根本不可用的， 蛋白质过表达导致错误定位。因此，定位内源性突触蛋白的方法， 急需脑组织在这里，我们开发了一种CRISPR基因编辑策略， 基因修饰，将表位标签插入到感兴趣的蛋白质上，并驱动细胞标志物在细胞中的表达。 有丝分裂后的神经元使用单一的AAV。我们创新的新方法是第一个提供综合手段 为了选择性地仅鉴定那些正确整合了蛋白质标签并提供细胞填充的神经元， 确定蛋白质的突触特异性定位所必需的结构参考。我们的方法是 对于各种蛋白质、标签和细胞标记物具有高度灵活性。初步数据表明，我们的方法 正确标记培养神经元中的突触蛋白N-钙粘蛋白，但需要进一步优化， 体外扩增（目的1）和体内扩增（目的2）。成功完成我们的提案将产生新的 这些技术允许研究大脑中内源性表达的突触特异性蛋白质。作为 例如，我们将检验与不同精神疾病相关的不同钙粘蛋白定位于 不同类型的突触。总之，我们的研究结果有望产生一种新技术， 可以广泛应用于研究突触的形成和功能，并为研究 突触多样性的潜在机制