Molecular Mechanisms of Purkinje Cell Degeneration in Ataxia-Telangiectasia

共济失调毛细血管扩张症浦肯野细胞变性的分子机制

基本信息

批准号：
10193587
负责人：
Mary Elizabeth Hatten
金额：
$ 46.61万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2023-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10193587
关键词：
ATM Gene Mutation ATM Signaling Pathway ATM gene Affect Ataxia Telangiectasia Ataxia Telangiectasia Patients Biological Assay Biological Models Birth CRISPR/Cas technology Calcium Cell Death Cell Survival Cells Cellular Assay Cerebellar degeneration Cerebellum Cessation of life Chronic Coculture Techniques Control Groups DNA Damage Data Data Set Defect Development Disease Etiology Family member Fibroblasts Fire - disasters Functional disorder Gene Expression Gene Proteins Genes Genetic Diseases Human Image Immunologic Deficiency Syndromes Infection Life Live Birth Lymphocyte Malignant Neoplasms Messenger RNA Methods Mitochondria Modeling Molecular Morphology Mus Mutation Nerve Degeneration Neurologic Neurons Output Oxidative Stress Pathway interactions Patients Phenotype Phosphorylation Predisposition Proteins Proteomics Protocols documentation Publishing Purkinje Cells Research Role Specificity System Techniques Telangiectasis Testing Tuberous Sclerosis ataxia telangiectasia mutated protein cell type comparative effective therapy experimental study granule cell human disease human embryonic stem cell induced pluripotent stem cell insight mouse model mutant mutation correction new therapeutic target phosphoproteomics response single-cell RNA sequencing stem cell differentiation stem cell model synaptic function synaptogenesis transcriptome transcriptomics young adult

项目摘要

PROJECT SUMMARY Ataxia-telangiectasia (A-T) is an autosomal recessive, multi-system, disorder caused by mutations in the universally expressed ataxia-telangiectasia, mutated (ATM) gene affecting approximately 1:40,000-1:100,000 births, for which there is no cure. Characterized by progressive cerebellar neurodegeneration, there are no effective treatments for A-T, with patients succumbing to chronic sinopulmonary infections or A-T related cancer by the third decade of life. Furthermore, the cause of cerebellar neurodegeneration, chiefly affecting Purkinje cells (PCs), the primary output neuron of the cerebellum, has remained elusive since the first descriptions of A- T nearly 80 years ago, largely because mouse models do not recapitulate the human cerebellar phenotype of PC death. Thus, the critical objectives of this proposal are to develop the first human A-T model system that recapitulates the cerebellar phenotype and to use that system to identify molecular differences between patient and unaffected PCs as well as differences between human and mouse PCs. Toward that end, we will use our recently published protocol (Buchholz et al, 2020) to generate an induced pluripotent stem cell (iPSC) model system and use that system to study the effects of A-T patient mutations on developing human PCs. Using our protocol, we have been able to differentiate cerebellar Purkinje cells that match young adult PCs on a transcriptomic level (Buchholz et al., 2020) and fire specific calcium currents in co-culture with their target neurons, granule cells (GCs). Our specific aims in this proposal are therefore to use this protocol to differentiate iPSCs derived from patients with A-T, as well as unaffected control iPSCs derived from family members, into PCs to study A-T PC phenotypes, including defects in survival and synaptic function in co-culture with GCs. To discover molecular changes in Purkinje cells with the A-T mutation, we will study global gene expression and protein phosphorylation compared to controls. Critically, we will then use CRISPR-Cas9 prime editing to correct the ATM mutation and test for rescue, including identifying key changes in rescued versus mutant gene expression and proteomics. Taken together, these studies will identify pathways involved in A-T PC phenotypes and will discover altered pathways that could provide novel targets for therapy.

项目概要共济失调毛细血管扩张症 (A-T) 是一种常染色体隐性遗传、多系统疾病，由基因突变引起普遍表达的共济失调毛细血管扩张症突变 (ATM) 基因影响约 1:40,000-1:100,000 分娩，目前尚无治愈方法。以进行性小脑神经变性为特征，没有 A-T 的有效治疗方法，患者死于慢性鼻窦肺部感染或 A-T 相关癌症到生命的第三个十年。此外，小脑神经变性的原因主要影响浦肯野病细胞（PC）是小脑的主要输出神经元，自从首次描述 A- 近 80 年前，主要是因为小鼠模型没有重现人类小脑表型电脑死亡。因此，该提案的关键目标是开发第一个人类 A-T 模型系统概括小脑表型并使用该系统来识别患者之间的分子差异和不受影响的 PC 以及人类和鼠标 PC 之间的差异。为此，我们将利用我们的最近发布的生成诱导多能干细胞 (iPSC) 模型的方案（Buchholz 等人，2020）系统并使用该系统研究 A-T 患者突变对人类 PC 发育的影响。使用我们的方案，我们已经能够区分与年轻人匹配的小脑浦肯野细胞 PC 在转录组水平上（Buchholz 等人，2020）并在与其共培养时发射特定的钙电流目标神经元、颗粒细胞（GC）。因此，我们在本提案中的具体目标是使用该协议来区分来自 A-T 患者的 iPSC 以及来自家庭的未受影响的对照 iPSC 成员进入PC来研究A-T PC表型，包括共培养中的存活和突触功能缺陷与 GC。为了发现浦肯野细胞中 A-T 突变的分子变化，我们将研究全局基因与对照相比，表达和蛋白质磷酸化。至关重要的是，我们将使用 CRISPR-Cas9 prime 编辑以纠正 ATM 突变并测试救援，包括识别救援与救援的关键变化突变基因表达和蛋白质组学。总而言之，这些研究将确定 A-T 涉及的途径 PC表型并将发现改变的途径，为治疗提供新的靶点。