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

• 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
• 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

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.

挑战与影响：亨廷顿氏病（HD）是一种致命的遗传神经退行性疾病，所有疾病 病例是由huntingtin基因中的CAG三核苷酸重复膨胀引起的。目前没有 HD治疗治疗疗法，因此对于开发新的治疗靶标有至关重要的需求。 纹状体培养基神经元（MSN）被认为是HD中受影响最大的神经元细胞类型，但它是 仍然尚不完全了解亨廷顿突变是如何导致神经元细胞死亡的一般或MSN的。 突变亨廷顿蛋白（MHTT）的表达相当普遍，表明MSN具有脆弱性 因素，否则缺乏保护因素。如果已知这些因素，它们将提高机械 理解并指出新的高清治疗靶标。一个主要的假设是亨廷顿突变 导致基因表达的功能功能功能失调。许多研究使用了基因表达 研究转录失调及其在HD中的机理基础的分析，但至今 受解剖细胞中混合的限制，因此未检测到全基因组MSN细胞类型 由于跨细胞类型的信号平均，基因表达的特定变化。但是，这样的数据是 完全了解转录失调是原因还是突变体的结果 Huntingtin（MHTT）毒性，以及MSN是否具有独特的脆弱因素或缺乏保护因素 本质上或响应MHTT。该提案中概述的研究将提高机理 理解并指出HD的新治疗靶标。 方法：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 核糖体亲和力纯化（TRAP）方法学对HD小鼠模型的研究。陷阱报告 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模型陷阱研究已经确定了大量先前未表征的变化 在早期症状前HD小鼠模型时点上的mRNA翻译，并指向早期失调 FORKHEAD BOX O1，FOXO1，响应MHTT的MSN中的转录因子活性。在AIM1中，我们将表演 细胞类型特异性陷阱分析以研究小鼠的症状前和症状后基因表达变化 高清模型检查MSN是否具有不同的HD脆弱性因素或缺乏HD保护因素 本质上或响应MHTT。在AIM 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，以及如何改变HD 模型小鼠。在AIM 3中，我们将测试WildType小鼠FOXO1丢失和过表达的表型效应 和高清模型小鼠。如果成功，此目标的结果将证明FOXO1在引起 增强了对MHTT的脆弱性，因此提供了指向新型治疗的原则数据证明 高清目标途径。