BIN1-interactome in Alzheimer's disease pathophysiology

BIN1-相互作用组在阿尔茨海默病病理生理学中的作用

• 批准号: 10677190
• 负责人: Joseph McMillan
• 金额: $ 3.79万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-14 至 2025-08-13
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677190
• 关键词: Address; Adult; Affect; Age; Alternative Splicing; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease pathology; Alzheimer' s disease patient; Alzheimer' s disease risk; American; Amyloidosis; Bioinformatics; Biological Process; Biotin; Biotinylation; Brain; Brain Diseases; Cellular biology; Clathrin; Cluster Analysis; Complement; Core Facility; Coupled; Data Analyses; Development; Disease; Dynamin; Elderly; Endocytosis; Environment; Enzymes; Experimental Designs; Functional disorder; Future; Genes; Genetic; Gliosis; Health; Human; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Interdisciplinary Study; Investigation; Kinetics; Knowledge; Label; Laboratories; Late Onset Alzheimer Disease; Learning; Ligase; Link; Mass Spectrum Analysis; Mediating; Mentors; Methods; Mus; Muscle Development; Nature; Neighborhoods; Neuroblastoma; Neurodegenerative Disorders; Neurons; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Protein Isoforms; Proteins; Proteome; Proteomics; Radial; Recycling; Research; Research Training; Retrieval; Risk; Risk Factors; SYNJ1 gene; Severities; Skeletal Muscle; Synapses; Synapsins; Synaptic Vesicles; System; Therapeutic; Translating; Translations; Tumor Suppressor Proteins; Wild Type Mouse; Work; age related neurodegeneration; anticancer research; apolipoprotein E-4; brain tissue; detection method; experimental study; genetic risk factor; genome wide association study; grasp; gray matter; human old age (65+); in vivo; innovation; interest; lifestyle factors; molecular pathology; mouse model; neuroblastoma cell; neuron loss; novel; pre-doctoral; promoter; protein protein interaction; protein purification; superresolution microscopy; tau Proteins; tool; vesicular release

PROJECT SUMMARY Alzheimer’s disease (AD) is a devastating neurodegenerative disorder with no definitive treatment that reverses the course of the disease, and we still lack a firm grasp on how older people develop AD. Bridging Integrator 1 (BIN1) is the second-largest genetic risk factor for late-onset AD. At least 12 different alternatively spliced BIN1 isoforms are expressed in the brain, including the neuron-specific BIN1 isoform 1 (BIN1iso1) and the ubiquitously expressed BIN1 isoform 9 (BIN1iso9). In the brain gray matter of patients with AD, there is a decrease in neuronal BIN1iso1 and an increase in BIN1iso9 compared to healthy controls. Thus far, evidence has shown that neuronal BIN1 isoforms participate in clathrin-mediated endocytosis, endocytic recycling, and synaptic vesicle release and retrieval. However, the mechanisms by which BIN1 contributes to these functions and the neighborhood of proteins that BIN1 interacts with to accomplish these cellular tasks remain largely undefined. Therefore, a fundamental gap in the field is an unbiased characterization of BIN1iso1 interacting proteins and proximal neighbors. Closing this gap will help define BIN1’s biological functions in healthy and diseased brain neurons. Utilizing the highly innovative proximity biotin ligase, TurboID, fused to BIN1iso1, will allow the identification of all proteins within a 10-nm radius. TurboID-based proximity labeling coupled with the most recent advanced quantitative mass spectrometry and data analysis methods represents a powerful strategy for discovery research. My preliminary in vitro studies using this approach in mouse N2a neuroblastoma cells resulted in the discovery of 234 proteins as BIN1-associated (proximal) or interacting proteins. These results identified several known BIN1 interactors such as tau, dynamin, synaptojanin, and many previously unknown proximal proteins. The following Specific Aims will translate these findings in vivo and dramatically advance the field. Aim 1. Identify neuronal BIN1iso1 interacting proteins in vivo using wild-type mice under homeostatic conditions. Aim 2. Establish neuronal BIN1iso interactome in mouse models of AD (5XFAD and PS19) before and after the onset of pathology. This project will not only advance the field by providing those studying AD and BIN1 with a list of BIN1iso1 proximal proteins to generate novel hypotheses but will also support the applicant’s pre-doctoral research training in AD pathophysiology, advanced methods such as in vivo AAV transduction, proximity-based labeling in the context of AD pathology, large-scale proteomics data analysis, and bioinformatics analysis of BIN1 functional pathways. The dynamic and highly collaborative research environment at the USF Health Byrd Alzheimer’s Institute will enhance the learning opportunities in cell biology and molecular pathology within an established AD laboratory led by a committed mentor. Furthermore, research training in proteomics approaches offered by the collaborator’s lab and the Proteomics Core facilities will complement the applicant’s interdisciplinary research training in age-related neurodegenerative disease.

项目摘要 阿尔茨海默病（AD）是一种破坏性的神经退行性疾病，没有明确的治疗方法可以逆转 疾病的过程，我们仍然缺乏对老年人如何发展AD的坚定把握。桥接集成器1 BIN1是晚发性AD的第二大遗传风险因素。至少12种不同的选择性剪接的BIN1 同种型在脑中表达，包括神经元特异性BIN1同种型1（BIN1iso1）和普遍存在的 表达BIN1同种型9（BIN1iso9）。在AD患者的脑灰质中，神经元的数量减少， 与健康对照相比，BIN1iso1和BIN1iso9增加。到目前为止，有证据表明，神经元 BIN1亚型参与网格蛋白介导的内吞作用、内吞再循环和突触囊泡释放， 检索然而，BIN1对这些功能的贡献机制以及BIN1的邻近区域是不确定的。 与BIN1相互作用以完成这些细胞任务的蛋白质在很大程度上仍然不确定。因此 该领域的一个基本空白是BIN1iso1相互作用蛋白质的无偏表征， 近邻缩小这一差距将有助于确定BIN1在健康和患病大脑中的生物学功能 神经元利用高度创新的邻近生物素连接酶，TurboID，融合到BIN1iso1，将允许 识别10 nm半径内的所有蛋白质。基于TurboID的邻近标记加上最近的 先进的定量质谱和数据分析方法代表了一种强有力的策略， 发现研究我的初步体外研究使用这种方法在小鼠N2a神经母细胞瘤细胞 结果发现了234种BIN1相关蛋白或相互作用蛋白。这些 结果鉴定了几种已知的BIN1相互作用物，如tau、发动蛋白、突触蛋白和许多先前的 未知近端蛋白质以下具体目标将在体内和戏剧性地转化这些发现 推进领域。目标1.使用野生型小鼠在体内鉴定神经元BIN1iso1相互作用蛋白， 稳态条件目标2.在AD小鼠模型中建立神经元BIN1iso相互作用组（5XFAD和5XFAD）。 PS19）在病理发生之前和之后。该项目不仅将通过提供这些 研究AD和BIN1与BIN1iso1近端蛋白的列表，以产生新的假设，但也将支持 申请人在AD病理生理学，先进的方法，如体内AAV的博士前研究培训 转导，AD病理学背景下基于邻近的标记，大规模蛋白质组学数据分析，以及 BIN1功能通路的生物信息学分析。动态和高度协作的研究环境 在USF健康伯德阿尔茨海默氏症研究所将增加学习机会，在细胞生物学和分子 病理学在一个既定的AD实验室领导下的承诺导师。此外，研究培训 由合作者的实验室和蛋白质组学核心设施提供的蛋白质组学方法将补充 申请人在年龄相关神经退行性疾病方面的跨学科研究培训。