Role of Ataxin-1 in BACE1 Expression and Alzheimer's Disease

项目摘要 最近发现导致阿尔茨海默病(AD)的遗传因素的努力已经确定 几十个相关的基因座。其中，我们的研究小组之前报道了一例家族性AD患者的ATXN1 全基因组关联研究。ATXN1以其他方式被认为含有突变，导致 脊髓小脑性共济失调1型，一种主要损害协调运动的神经退行性疾病。在一个 基于细胞的研究，下调Aaxin-1基因(ATXN1)的表达增加了淀粉样变的过程 淀粉样前体蛋白(APP)和A的分泌，这是AD大脑中衰老斑块的主要成分。 在对Aaxin-1 KO小鼠的初步检查中，我们发现Aaxin-1调节BACE1的表达， 选择性地在易患阿尔茨海默病的脑区。在AD小鼠中，Aaxin-1的缺失增加了分泌酶的裂解 APP、A沉积与脑内胶质细胞增生症此外，Aaxin-1 KO损伤了海马区 神经发生和轴突靶向，由BACE1调控。为了验证和扩展这些内容 在此，我们建议1.阐明Aaxin-1调节BACE1的分子机制 在脑中的表达；2.评估Aaxin-1功能丧失在AD发病机制中的影响；以及3.确定 并从AD DNA样本集中鉴定与AD相关的新的Aaxin-1基因变体和/或突变。 具体地说，对于目标1，我们将首先确定Aaxin-1是否通过影响其BACE1的mRNA水平来调节BACE1的mRNA水平 稳定性或它的转录。为了测试这一点，我们将使用急性脑片培养的Aaxin-1 KO和WT ，并检测转录抑制物对BACE1mRNA稳态水平的不同影响 并检测培养切片中新合成的含有核苷酸类似物的BACE1mRNA。 然后我们将研究Aaxin-1相互作用的转录因子是否通过以下方式与BACE1启动子结合并调节 芯片分析。关于AIM 2，我们将检查A斑块负荷增加和胶质增生是否 在APP-PS1/ATXN1-KO小鼠中维持较大年龄(9月龄)。以确定受损的海马区 神经发生和轴突靶向是由BACE1增加引起的，我们将产生ATXN1−/−：BACE1/− 并与atxn1−/−小鼠相比，检查这两个缺陷是否得到挽救。最后，在目标3中，我们将 通过分析确定增加AD风险或提供保护的ATXN1突变/变异 NIMH、NIA和ADNI AD DNA样本集的WGS/WES数据。对于最相关的突变/变异，我们 将通过将它们整合到人的基因组中来研究它们对BACE1表达和APP处理的影响 通过CRISPR/Cas9技术获得人神经细胞。在完成建议的研究后，我们相信我们 将提供急需的数据，说明Aaxin-1在调节BACE1表达中的作用，同时还 促进针对BACE1的新疗法，以预防和治疗阿尔茨海默病。

Project Summary Recent efforts to uncover genetic factors responsible for Alzheimer's disease (AD) have identified dozens of associated loci. Among these, our research group previously reported ATXN1 in a family-based AD genome-wide association study. ATXN1 has been otherwise known for to harbor mutations causing spinocerebellar ataxia type 1, a neurodegenerative disease that primarily impairs coordinated movement. In a cell-based study, knockdown of Ataxin-1 gene (ATXN1) expression increased amyloidogenic processing of the amyloid precursor protein (APP) and the secretion of A, the main component of senile plaques in AD brains. In the preliminary examination of Ataxin-1 KO mice, we found Ataxin-1 regulates BACE1 expression, selectively in AD-vulnerable brain regions. In AD mice, depletion of Ataxin-1 increased -secretase-cleavage of APP, A deposition and gliosis in the cerebrum. Furthermore, Ataxin-1 KO impaired hippocampal neurogenesis and axonal targeting, which are regulated by BACE1. To validate and expand upon these findings, here, we propose to 1. Elucidate the molecular mechanism by which Ataxin-1 regulates BACE1 expression in the brain; 2. Assess the impact of Ataxin-1 loss of function on AD pathogenesis; and 3. Identify and characterize novel AD-associated Ataxin-1 gene variants and/or mutations from AD DNA sample sets. Specifically, for Aim 1, we will first determine whether Ataxin-1 regulates BACE1 mRNA level by affecting its stability or by its transcription. To test this, we will employ acute brain slice cultures of Ataxin-1 KO and WT mice, and examine the differential effects of transcriptional inhibitors on the steady-state level of BACE1 mRNA and also measure newly synthesized BACE1 mRNA incorporating nucleotide analogs in the cultured slices. We will then examine if Ataxin-1-interacting transcriptional factors bind to and regulate BACE1 promoter by ChIP analysis. With regard to Aim 2, we will examine if the increased A plaque load and gliosis are maintained in APP-PS1/ATXN1-KO mice at older age (9 month). To determine if impaired hippocampal neurogenesis and axonal targeting are caused by increased BACE1, we will generate ATXN1 −/−:BACE1 +/− mice and examine if the two deficits are rescued, as compared to ATXN1 −/− mice. Finally, in Aim 3, we will identify ATXN1 mutations/variants that either increase risk or confer protection for AD by analysis of WGS/WES data of NIMH, NIA and ADNI AD DNA sample sets. For the most associated mutations/variants, we will examine their effects on BACE1 expression and APP processing by incorporating them into the genome of human neuronal cells via CRISPR/Cas9 technology. At the completion of the proposed study, we believe we will provide critically needed data addressing the role of Ataxin-1 in regulating BACE1 expression while also facilitating novel therapies aimed at targeting BACE1 to prevent and treat AD.