Molecular Mechanisms of Purkinje Cell Degeneration in Ataxia-Telangiectasia

项目总结 共济失调-毛细血管扩张症(A-T)是一种常染色体隐性遗传的多系统疾病，由基因突变引起 普遍表达的共济失调-毛细血管扩张突变(ATM)基因影响约1：40,000-1：100,000 生育，这是无法治愈的。以进行性小脑神经变性为特征，没有 A-T的有效治疗，患者死于慢性鼻窦肺部感染或A-T相关癌症 到了生命的第三个十年。此外，小脑神经变性的原因，主要影响浦肯野 细胞(PC)，小脑的初级输出神经元，自从第一次描述A-A以来一直难以捉摸。 大约80年前，很大程度上是因为小鼠模型不能概括人类小脑的表型 个人电脑的死亡。因此，这项提议的关键目标是开发第一个人类A-T模型系统，该系统 总结了小脑的表型，并使用该系统来识别患者之间的分子差异 和未受影响的PC以及人和鼠标PC之间的差异。为此，我们将使用我们的 最近发表的方案(Buchholz等人，2020)，以产生诱导多能干细胞(IPSC)模型 系统，并使用该系统来研究A-T患者突变对人类PC发育的影响。 使用我们的方案，我们已经能够分化出与年轻成人相匹配的小脑浦肯野细胞。 在转录水平上的PC(Buchholz等人，2020)，并在与其共同培养时激发特定的钙电流 靶神经元、颗粒细胞(GC)。因此，我们在这项提案中的具体目标是使用该议定书来 区分来自A-T患者的IPSCs和来自家系的未受影响的对照IPSCs 成员，进入PC研究A-T PC表型，包括在共培养中生存和突触功能的缺陷 与GC一起。为了发现A-T突变的浦肯野细胞的分子变化，我们将研究全局基因 与对照组比较，表达和蛋白质磷酸化。重要的是，我们将使用CRISPR-Cas9 Prime 编辑以更正自动柜员机突变和救援测试，包括识别救援与救援中的关键更改 突变基因表达和蛋白质组学。综上所述，这些研究将确定A-T所涉及的途径 PC表型并将发现改变的通路，可能为治疗提供新的靶点。

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.