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

亨廷顿病细胞类型特异性脆弱性的决定因素

基本信息

批准号：
9285159
负责人：
Myriam Heiman
金额：
$ 40.03万
依托单位：
BROAD INSTITUTE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9285159
关键词：
Affect Affinity Chromatography Alpha Cell Anatomy Antibodies Bioinformatics Brain-Derived Neurotrophic Factor CAG repeat Cell Nucleus Cells Cellular Stress Cessation of life ChIP-seq Corpus striatum structure Data Data Analyses Dependence Development Disease Disease model Dominant-Negative Mutation FOXO1A gene Flow Cytometry Fluorescence Gene Expression Gene Expression Profiling Genes Genetic Transcription Genetic Translation Goals Huntington Disease Huntington gene Immunoprecipitation Indirect Immunofluorescence Inherited Lead Mediating Messenger RNA Methodology Molecular Mus Mutation Neurodegenerative Disorders Neurons Nuclear Pathway interactions Phenotype Phosphorylation Process RNA Regulation Regulator Genes Reporting Resolution Ribosomes Role Sensorimotor functions Signal Transduction Testing Thalamic structure Tissues Toxic effect Translating base cell type curative treatments gain of function genome-wide knock-down mouse model mutant neuron loss new therapeutic target overexpression response therapeutic target transcription factor transcriptome sequencing

项目摘要

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.

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，所有病例是由亨廷顿基因中的 CAG 三核苷酸重复扩增引起的。目前没有 HD 的治疗方法，因此迫切需要开发新的治疗靶点。纹状体中型多棘神经元 (MSN) 被认为是 HD 中受影响最严重的神经元细胞类型，但它仍然不完全了解亨廷顿突变如何导致神经元细胞死亡，特别是 MSN 死亡。突变亨廷顿蛋白 (mHTT) 表达相当普遍，表明 MSN 具有脆弱性因素或缺乏保护因素。如果这些因素是已知的，它们将促进机械化了解并指出新的 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失调。许多研究都使用基因表达分析来研究 HD 中的转录失调及其机制基础，但迄今为止，这些研究已经受到解剖细胞混合的限制，因此尚未检测到全基因组 MSN 细胞类型由于跨细胞类型的信号平均而导致的基因表达的特定变化。然而，这样的数据是有必要充分了解转录失调是突变的原因还是结果亨廷顿蛋白 (mHTT) 毒性，以及 MSN 是否具有明显的脆弱性因素或缺乏保护性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进机制了解并指出 HD 的新治疗靶点。方法：为了进行细胞类型特异性基因表达研究，我们已经开始应用翻译核糖体亲和纯化 (TRAP) 方法用于研究 HD 小鼠模型。 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。我们的初步的 HD 模型 TRAP 研究已发现大量以前未表征的变化早期、症状前 HD 小鼠模型时间点的 mRNA 翻译，表明早期失调叉头框 O1、Foxo1、MSN 中响应 mHTT 的转录因子活性。在Aim1中，我们将执行细胞类型特异性 TRAP 分析可研究小鼠症状前和症状后基因表达变化 HD 模型来检查 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 Foxo1 的状态、亚细胞定位和转录靶标，以及这些在 HD 中如何改变模型小鼠。在目标 3 中，我们将测试 Foxo1 缺失和过度表达对野生型小鼠的表型影响和HD模型小鼠。如果成功，这一目标的结果将证明 Foxo1 在引起增强了 mHTT 的脆弱性，从而提供了指向新型治疗方法的原理验证数据 HD 的目标途径。