BIN1-interactome in Alzheimer's disease pathophysiology

项目总结 阿尔茨海默病(AD)是一种毁灭性的神经退行性疾病，没有明确的治疗方法可以逆转 我们仍然缺乏对老年人如何发展为阿尔茨海默病的确切把握。桥接积分器1 BIN1基因是晚发性AD的第二大遗传风险因素。至少12个不同的可选拼接BIN1 异构体在大脑中表达，包括神经元特异性的BIN1异构体1(BIN1so1)和普遍存在的 表达BIN1亚型9(BIN1so9)。阿尔茨海默病患者脑灰质神经元减少 与健康对照组相比，BIN1so1和BIN1so9的表达增加。到目前为止，证据表明神经元 BIN1亚型参与网状蛋白介导的内吞作用、内吞循环和突触小泡释放 取回。然而，BIN1对这些功能和邻域的贡献机制 BIN1与哪些蛋白质相互作用以完成这些细胞任务在很大程度上仍然是未知的。因此，a 该领域的基本差距是BIN1so1相互作用蛋白和BIN1so1相互作用蛋白的无偏见特征 近邻。缩小这一差距将有助于确定BIN1的S在健康和疾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BIN1so1，将允许 识别半径为10纳米的所有蛋白质。基于TurboID的邻近标记与最新的 先进的定量质谱仪和数据分析方法代表了一种强大的战略 发现性研究。我在小鼠N2a神经母细胞瘤细胞中用这种方法进行的初步体外研究 结果发现了234种蛋白质作为BIN1相关(近端)或相互作用的蛋白质。这些 结果确定了几个已知的BIN1相互作用因子，如tau、Dynamin、synaptojanin以及许多以前 未知的近端蛋白质。以下具体目标将在体内和戏剧性地转化这些发现 把场地向前推进。目的1.利用野生型小鼠在体内鉴定神经元BIN1so1相互作用蛋白 动态平衡状态。目的2.建立阿尔茨海默病(AD)模型小鼠神经元BIN1iso相互作用体 PS19)发病前后。该项目不仅将通过提供 用BIN1so1近端蛋白清单研究AD和BIN1，以产生新的假说，但也将支持 申请者在AD病理生理学、体内AAV等先进方法方面的博士前研究培训 转导，AD病理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、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细胞生物学和分子方面的学习机会 由一位忠诚的导师领导的已建立的AD实验室内的病理学。此外，研究性培训在 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.