Determinants of cell type-specific vulnerability in Huntington's disease

挑战和影响：亨廷顿病(HD)是一种致命的遗传性神经退行性疾病，所有 这些病例是由亨廷顿蛋白基因中CAG三核苷酸重复扩增引起的。目前没有 HD的治疗疗法，因此迫切需要开发新的治疗靶点。 纹状体中棘神经元(MSN)被认为是HD中受影响最大的神经细胞类型，但它是 仍然没有完全了解亨廷顿蛋白突变是如何导致神经细胞死亡的，或者说是特定的MSN。 突变的Huntingtin蛋白(MHTT)在相当普遍的地方表达，这表明MSN具有脆弱性 因素或其他缺乏保护性因素。如果知道这些因素，它们将推动机械论的发展 了解并指出新的HD治疗目标。一个突出的假设是亨廷顿蛋白突变 导致基因表达的毒性功能获得失调。许多研究都使用了基因表达 侧写研究HD转录失调及其机制基础，但到目前为止，这些研究已经 受到解剖细胞混合的限制，因此没有检测到全基因组的MSN细胞类型- 由于细胞类型的信号平均而导致的基因表达的特定变化。然而，这些数据是 有必要充分了解转录失调是突变的原因还是后果 亨廷顿(MHTT)毒性，以及MSN是否具有明显的脆弱性因素或缺乏保护因素 本质上或者是对mHTT的回应。这项提案中概述的研究将推进机械论 了解并指出HD的新治疗靶点。 方法：为了进行细胞类型特异性基因表达的研究，我们已经开始应用翻译 核糖体亲和纯化(TRAP)方法学用于HD小鼠模型的研究。陷阱报告上的 细胞类型特异的翻译组，通过允许细胞类型特异的翻译的mRNA免疫沉淀。我们的 初步的HD模型陷阱研究已经发现了大量以前没有表征的变化 在早期、症状前的HD小鼠模型时间点的mRNA翻译，并指出早期的调节失调 叉头盒O1，Foxo1，MSN转录因子活性对mHTT的响应。在Aim1，我们将表演 细胞类型特异性TRAP分析研究小鼠症状前后基因表达的变化 HD模型，以检查MSN是否具有明显的HD脆弱性因素或缺乏HD保护因素 本质上或者是对mHTT的回应。在目标2中，我们将确定细胞类型特异性的磷酸化。 Foxo1在MSN中的状态、亚细胞定位和转录靶点，以及这些在HD中的变化 模型小鼠。在目标3中，我们将在野生型小鼠中测试Foxo1缺失和过表达的表型效应 和HD模型小鼠。如果成功，这一目标的结果将表明Foxo1在导致 增强了对mHTT的易感性，从而提供了指向一种新的治疗方法的原则证明数据 HD的靶向通路。

Challenge and Impact: Huntington's disease (HD) is a fatal inherited neurodegenerative disease, and all cases are caused by CAG trinucleotide repeat expansions in the huntingtin gene. There are currently no curative therapeutics for HD, and thus there is a critical need for the development of new therapeutic targets. Striatal medium spiny neurons (MSNs) are thought to be the most affected neuronal cell type in HD, but it is still not fully understood how huntingtin mutation leads to neuronal cell death in general or MSNs in particular. Mutant Huntingtin protein (mHTT) is expressed fairly ubiquitously, suggesting that MSNs possess vulnerability factors or else lack protective factors. If these factors were known, they would advance mechanistic understanding and point to new HD therapeutic targets. One prominent hypothesis is that huntingtin mutation leads to a toxic gain-of-function dysregulation of gene expression. Many studies have used gene expression profiling to study transcriptional dysregulation and its mechanistic basis in HD, but to date these studies have been limited by anatomical cellular intermixing and thus have not detected genome-wide MSN cell type- specific changes to gene expression due to signal averaging across cell types. However, such data are necessary to fully understand whether transcriptional dysregulation is causative or a consequence of mutant Huntingtin (mHTT) toxicity, and whether MSNs possess distinct vulnerability factors or lack protective factors either intrinsically or in response to mHTT. The studies outlined in this proposal will advance mechanistic understanding and point to new therapeutic targets for HD. Approach: To perform cell type-specific gene expression studies, we have begun to apply the translating ribosome affinity purification (TRAP) methodology to the study of mouse models of HD. TRAP reports on the cell type-specific translatome, by allowing cell type-specific translated mRNA immunoprecipitation. Our preliminary HD model TRAP studies have identified a large number of previously uncharacterized changes to mRNA translation at an early, pre-symptomatic HD mouse model timepoint, and point to early dysregulation of forkhead box O1, Foxo1, transcription factor activity in MSNs in response to mHTT. In Aim1, we will perform cell type-specific TRAP analyses to investigate pre- and post-symptomatic gene expression changes in mouse models of HD to examine whether MSNs possess distinct HD vulnerability factors or lack HD protective factors either intrinsically or in response to mHTT. In Aim 2 we will determine the cell type-specific phosphorylation status, subcellular localization, and transcriptional targets of Foxo1 in MSNs, and how these are altered in HD model mice. In Aim 3 we will test the phenotypic effects of Foxo1 loss and overexpression in wildtype mice and HD model mice. If successful, the results of this aim will demonstrate a role for Foxo1 in causing enhanced vulnerability to mHTT, and thus provide proof-of-principle data that points to a novel therapeutic target pathway for HD.