BIN1-interactome in Alzheimer's disease pathophysiology

项目概要 阿尔茨海默病 (AD) 是一种毁灭性的神经退行性疾病，目前尚无明确的治疗方法可以逆转 疾病的病程，我们仍然缺乏对老年人如何患上 AD 的确切了解。桥接积分器1 (BIN1) 是晚发性 AD 的第二大遗传风险因素。至少12个不同的交替拼接的BIN1 同工型在大脑中表达，包括神经元特异性 BIN1 同工型 1 (BIN1iso1) 和普遍存在的 表达 BIN1 同工型 9 (BIN1iso9)。 AD患者的大脑灰质中，神经元数量减少 与健康对照相比，BIN1iso1 和 BIN1iso9 增加。迄今为止，有证据表明神经元 BIN1 亚型参与网格蛋白介导的内吞作用、内吞再循环和突触小泡释放和 检索。然而，BIN1 贡献这些功能的机制以及邻域 BIN1 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不清楚。因此，一个 该领域的根本差距是 BIN1iso1 相互作用蛋白的公正表征和 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，与 BIN1iso1 融合，将允许 识别 10 纳米半径内的所有蛋白质。基于 TurboID 的邻近标记加上最新的 先进的定量质谱和数据分析方法代表了一种强大的策略 发现研究。我在小鼠 N2a 神经母细胞瘤细胞中使用这种方法进行的初步体外研究 结果发现了 234 个蛋白质作为 BIN1 相关（近端）或相互作用蛋白质。这些 结果确定了几种已知的 BIN1 相互作用因子，例如 tau、dynamin、synaptojanin 以及许多以前的相互作用因子 未知的近端蛋白质。以下具体目标将在体内显着地转化这些发现 推进该领域。目标 1. 使用野生型小鼠体内鉴定神经元 BIN1iso1 相互作用蛋白 稳态条件。目标 2. 在 AD 小鼠模型（5XFAD 和 PS19) 病理发作之前和之后。该项目不仅将通过提供这些来推动该领域的发展 使用一系列 BIN1iso1 近端蛋白研究 AD 和 BIN1，以产生新的假设，但也将支持 申请人接受过 AD 病理生理学、体内 AAV 等先进方法的博士前研究培训 转导、AD 病理学背景下的基于邻近的标记、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。充满活力且高度协作的研究环境 南佛罗里达大学健康伯德阿尔茨海默病研究所将增加细胞生物学和分子生物学的学习机会 在一个由忠诚的导师领导的已建立的 AD 实验室内进行病理学研究。此外，研究培训 合作者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 申请人在与年龄相关的神经退行性疾病方面的跨学科研究培训。

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.