Cell autonomous and non-cell autonomous roles of the GWAS risk factor BIN1 in Alzheimer's disease neuropathology

这项建议侧重于BIN1，这是最近在晚发性阿尔茨海默病(LOAD)主要易感基因(仅次于APOE)的主要易感基因中发现的共同风险基因之一。BIN1(桥接整合子-1)是适配器蛋白家族中的一员，在内吞和膜重塑的背景下调节膜的动力学。BIN1基因20个外显子的交替剪接产生了普遍存在的和组织特异性的亚型，这些亚型在组织分布、亚细胞定位和功能上都不同。这些功能包括细胞周期进程、细胞凋亡、细胞骨架组织和DNA修复。据报道，BIN1的表达增加和BIN1的交替剪接在负荷个体的大脑中被报道，但这些观察如何转化为AD风险的增加完全不清楚。在初步研究中，我们在啮齿动物和人脑的灰质和白质的成熟少突胶质细胞中发现了显著的BIN1表达。通过建立少突胶质细胞特异性BIN1条件性基因敲除(CKO)小鼠，我们证实BIN1主要在成熟少突胶质细胞中表达。有趣的是，我们观察到BIN1在人类老年斑附近的异常表达，以及在AD转基因小鼠模型的淀粉样蛋白沉积中BIN1的增加。基于这些新的发现，我们假设BIN1在成熟的少突胶质细胞中发挥作用，并且BIN1表达和/或功能的改变在AD相关的发病过程和神经退行性变中以非细胞自主的方式发挥作用。我们提出以下具体目的来检验与BIN1在AD中的作用相关的新假说。该方案的具体目的是：目的1：验证BIN1细胞在AD脑内表达和定位改变的假说。我们将研究BIN1在正常和疾病人脑中的表达和交替剪接。我们将通过分离和共聚焦显微镜确定BIN1的亚细胞定位，并利用免疫电子显微镜阐明BIN1的超微结构定位。目的：验证BIN1表达下调将减轻转基因小鼠淀粉样蛋白和tau病理的假说。我们将确定神经元或成熟少突胶质细胞中BIN1表达的细胞类型特异性缺失是否会减轻转基因AD小鼠模型中与AD相关的神经病理和行为缺陷。目的：验证BIN1在体内参与少突胶质细胞分化、存活和成熟以及髓鞘形成的假说。我们将利用BIN1 CKO小鼠模型和培养的少突胶质细胞来验证这一假说。这一建议是及时的、独特的、极具创新性的。我们相信，我们的研究将揭示BIN1在大脑中的S功能，表征AD领域感兴趣的新的BIN1CKO小鼠模型，确定BIN1表达的减弱是否为疾病干预提供了新的策略，并为鉴定与AD相关的BIN1功能变异奠定基础，并指导这一主要负载风险基因调控的生物通路和致病机制的未来功能表征。

This proposal focuses on BIN1, one of the recently identified common risk genes within the major susceptibility loci for late-onset Alzheimer's disease (LOAD) (second only to APOE). BIN1 (Bridging INtegrator-1) is a member of a family of adaptor proteins that regulate membrane dynamics in the context of endocytosis and membrane remodeling. Alternate splicing of the 20 exons in BIN1 gene generates ubiquitous and tissue- specific isoforms, which differ in their tissue distribution, subcellular localization, and function. These functions include cell cycle progression, apoptosis, cytoskeletal organization, and DNA repair. An increase of BIN1 expression and alternate splicing of BIN1 have been reported in the brains of individuals with LOAD but how these observations translate to increased risk for AD is entirely not clear. In preliminary studies, we have characterized prominent BIN1 expression in mature oligodendrocytes in the gray and white matter in rodent and the human brain. By generating oligodendrocyte-specific Bin1 conditional knock out (cKO) mice, we confirmed that BIN1 is mainly expressed in mature oligodendrocytes. Interestingly, we observe aberrant BIN1 expression near human senile plaques and BIN1 accrual in amyloid deposits of AD transgenic mouse models. Based on these novel findings, we hypothesize that BIN1 functions in mature oligodendrocytes, and that alteration in BIN1 expression and/or function in oligodendrocytes plays a role in AD-related pathogenic processes and neurodegeneration in a non-cell autonomous manner. We propose the following specific aims to test novel hypothesis related to the role of BIN1 in AD. The Specific Aims of this proposal are: Aim 1: To test the hypothesis that BIN1 cellular expression and localization are altered in AD brain. We will investigate the BIN1 expression and alternate splicing in normal and diseased human brain. We will determine the subcellular localization of BIN1 by fractionation and confocal microscopy, and clarify the ultrastructural BIN1 localization using immunoelectron microscopy. Aim 2: To test the hypothesis that downregulation of BIN1 expression will attenuate amyloid and tau pathology in transgenic mice. We will ascertain whether cell-type specific loss of BIN1 expression in neurons or mature oligodendrocytes will attenuate AD-related neuropathology and behavior deficits in transgenic AD mouse models. Aim 3: To test the hypothesis that BIN1 plays a role in oligodendrocyte differentiation, survival, and maturation, as well as myelination in vivo. We will utilize Bin1 cKO mouse models and cultured oligodendrocytes to test this hypothesis. This proposal is timely, unique, and highly innovative. We believe that our investigation will uncover significant insights on BIN1's function in the brain, characterize novel Bin1 cKO mouse models of interest to the AD field, establish whether attenuation of BIN1 expression provides a novel strategy for disease intervention, and lay the foundation for characterization of BIN1 functional variants linked to AD, and guide future functional characterization of biological pathways and pathogenic mechanisms regulated by this major LOAD risk gene.