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

项目摘要 最近的努力，以揭示遗传因素负责阿尔茨海默氏病（AD）已确定 几十个相关的基因座其中，我们的研究小组先前报道了ATXN 1在一个以家庭为基础的AD中的表达， 全基因组关联研究ATXN 1已经被认为具有突变， 脊髓小脑共济失调1型，一种主要损害协调运动的神经退行性疾病。中 一项基于细胞的研究表明，Ataxin-1基因（ATXN 1）表达的敲低增加了 淀粉样前体蛋白（APP）与AD脑内老年斑的主要成分Aβ的分泌有关。 在对Ataxin-1基因敲除小鼠的初步研究中，我们发现Ataxin-1调节BACE 1的表达， 选择性地在易患AD的大脑区域。在AD小鼠中，Ataxin-1的缺失增加了β-分泌酶对AD小鼠的β-分泌酶切割。 脑内APP、Aβ沉积及胶质增生。此外，Ataxin-1 KO损害海马 神经发生和轴突靶向，这是由BACE 1调节。为了验证和扩展这些 调查结果，在这里，我们建议1。阐明Ataxin-1调节BACE 1的分子机制 在大脑中的表达; 2。评估共济失调蛋白-1功能丧失对AD发病机制的影响;以及3.识别 并表征来自AD DNA样品集的新的AD相关共济失调蛋白-1基因变体和/或突变。 具体而言，对于目标1，我们将首先确定Ataxin-1是否通过影响其表达来调节BACE 1 mRNA水平。 稳定性或转录。为了测试这一点，我们将使用共济失调蛋白-1 KO和WT的急性脑切片培养物， 小鼠，并检查转录抑制剂对BACE 1 mRNA稳态水平的差异作用 并且还测量在培养的切片中掺入核苷酸类似物的新合成的BACE 1 mRNA。 然后我们将检查共济失调蛋白-1相互作用转录因子是否通过以下方式结合并调节BACE 1启动子 ChIP分析。关于目标2，我们将检查Aβ斑块负荷增加和神经胶质增生是否是 在APP-PS1/ATXN 1-KO小鼠中维持较年长（9个月）。为了确定受损的海马 神经发生和轴突靶向是由增加的BACE 1引起的，我们将产生ATXN 1 −/−：BACE 1 +/− 小鼠，并检查与ATXN 1 −/−小鼠相比，这两种缺陷是否得到挽救。在目标3中，我们将 通过分析识别增加AD风险或提供AD保护的ATXN 1突变/变体 NIMH、NIA和ADNI AD DNA样品集的WGS/WES数据。对于最相关的突变/变体，我们 将通过将它们整合到BACE 1基因组中来研究它们对BACE 1表达和APP加工的影响。 通过CRISPR/Cas9技术获得人类神经元细胞。在完成拟议的研究后，我们认为， 将为阐明Ataxin-1在调节BACE 1表达中的作用提供急需的数据， 促进旨在靶向BACE 1的新疗法以预防和治疗AD。

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 Aβ, 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 β-secretase-cleavage of APP, Aβ 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 Aβ 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.