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， 例由亨廷顿基因中CAG三核苷酸重复扩增引起。目前没有 因此，迫切需要开发新的治疗靶点。 纹状体中棘神经元（MSNs）被认为是HD中受影响最严重的神经元细胞类型，但它是 仍然没有完全理解亨廷顿蛋白突变是如何导致神经元细胞死亡的，特别是MSN。 突变亨廷顿蛋白（mHTT）的表达相当普遍，这表明MSN具有脆弱性 或缺乏保护性因素。如果这些因素是已知的，他们将推进机械 了解并指出新的HD治疗靶点。一个突出的假设是亨廷顿突变 导致基因表达的毒性功能获得性失调。许多研究使用基因表达 分析，以研究转录失调及其机制基础，在HD，但迄今为止，这些研究 受到解剖学细胞混合的限制，因此没有检测到全基因组MSN细胞类型- 由于跨细胞类型的信号平均化而导致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突变的结果。 亨廷顿蛋白（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 无论是内在的还是对mHTT的反应。本提案中概述的研究将推动机械 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的基因表达研究，我们已经开始应用翻译， 核糖体亲和纯化（TRAP）方法用于HD小鼠模型的研究。TRAP报告 细胞类型特异性翻译组，通过允许细胞类型特异性翻译的mRNA免疫沉淀。我们 初步的HD模型TRAP研究已经确定了大量以前未表征的变化， 在早期、症状前HD小鼠模型时间点的mRNA翻译，并指出 叉头框O 1，Foxo 1，MSNs对mHTT反应的转录因子活性。在AIM 1中，我们将执行 细胞类型特异性TRAP分析，以研究小鼠中症状前和症状后的基因表达变化 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 无论是内在的还是对mHTT的反应。在目标2中，我们将确定细胞类型特异性磷酸化 MSN中Foxo 1的状态、亚细胞定位和转录靶点，以及这些在HD中的改变 模型小鼠。在目标3中，我们将测试野生型小鼠中Foxo 1缺失和过表达的表型效应 和HD模型小鼠。如果成功的话，这个目标的结果将证明Foxo 1在导致 增强了对mHTT的脆弱性，从而提供了指向一种新的治疗方法的原理验证数据。 HD的靶向途径。

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.