Understanding cell-type vulnerability and oxidative stress pathology in Parkinson's Disease using isogenic human dopaminergic neurons

大约有100万美国人患有帕金森氏病(PD)，其特征是进行性丧失 黑质中脑多巴胺能神经元亚群，导致运动障碍和其他 令人衰弱的条件。家族性帕金森病基因在脑内广泛表达，帕金森病患者神经变性 可以广泛传播；然而，尚不清楚为什么黑猩猩的dns与 其他细胞类型，包括其他糖尿病肾病人群。尸检研究表明，氧化应激(OS) 导致家族性和散发性帕金森病。活性氧(ROS)是重要的信号分子 但细胞内高水平的ROS会损害DNA、脂质和蛋白质。高能量需求和多巴胺 新陈代谢可以解释ROS、OS的增加和黑质DNS独特的脆弱性，但与人类有关 需要模型系统来严格检验这一假设。迫切需要发展 实验系统以更好地了解黑质糖尿病肾病的脆弱性，识别与疾病相关的新信号 机制，并改进分子亚型和患者分层。我们的长期目标是了解 黑质肾病的脆弱性通过遗传学、细胞类型特定功能的相互作用而产生 脆弱性和可量化的表型，以确定新的治疗靶点。为了支持这一目标，我们有 建立敲入人多能干细胞(HPSC)报告系以鉴定和分离酪氨酸 来自大规模有机体旋转培养的羟基酶(TH)阳性的DNS。使用CRISPR突变我们 早发性常染色体隐性帕金森病(Parkin-/-、DJ1-/-和DJ1-/-)等基因功能丧失模型的建立 ATP13A2-/-)。我们检测到线粒体蛋白质的失调，显著地 在同基因的PD细胞系中，中脑糖尿病患者的OS和细胞死亡增加，但在同基因的WT对照糖尿病患者中并不增加。至 了解黑质糖尿病肾病漏洞我们建议使用我们的同源报告PD模型和单细胞RNA 人中脑、下丘脑和前脑dns的测序方法识别细胞群体 这表明对OS和细胞死亡的易感性增加，并确定受不同影响的糖尿病肾病人群。 表达和网络分析将识别导致漏洞的蜂窝功能。我们已经开发出 基因工具用于区分初级调控失调和新出现的表型以进一步阐明发病机制 了解基因型与表型的相互作用。使用创新的CRISPR-激活和抑制 我们将测试已确定的候选基因是否有可能改善OS表型和细胞 在我们的PD模型中死亡。我们的模型在概念和技术上都是创新的，并将照亮共同和 在黑质肾病中提供脆弱性或保护的独特途径，并提出新的策略 预防和治疗，以改善患者及其照顾者的生活。

About one million Americans live with Parkinson's Disease (PD) which is characterized by progressive loss of subpopulations of nigral midbrain dopaminergic neurons (DNs), leading to motor impairment and other debilitating conditions. Familial PD genes show broad expression in the brain and neurodegeneration in PD can be widespread; however, it is unclear why nigral DNs show such exquisite vulnerability compared to other cell types, including other DN populations. Post-mortem studies suggest that oxidative stress (OS) contributes to familial and sporadic PD. Reactive oxygen species (ROS) are important signaling molecules but high levels of intracellular ROS will damage DNA, lipids and proteins. High energy needs and dopamine metabolism may explain increased ROS, OS and the unique vulnerability of nigral DNs but human-relevant model systems are required to rigorously test this hypothesis. There is an urgent need to develop experimental systems to better understand nigral DN vulnerability, identify novel disease-relevant signaling mechanisms, and improve molecular subtyping and patient stratification. Our long-term goal is to understand the vulnerability of nigral DNs through the interplay of genetics, cell type specific functions that confer vulnerability and quantifiable phenotypes to identify new therapeutic targets. In support of this goal, we have developed knock-in human pluripotent stem cell (hPSC) reporter lines to identify and isolate tyrosine hydroxylase (TH)-positive DNs from large-scale organoid spin cultures. Using CRISPR mutagenesis we created isogenic loss-of-function models of early-onset autosomal recessive PD (PARKIN-/-, DJ1-/- and ATP13A2-/-) in TH-reporter cell lines. We detected dysregulation of mitochondrial proteins, significantly increased OS and cell death in isogenic PD cell lines in midbrain DNs, but not in isogenic WT-control DNs. To understand nigral DN vulnerability we propose to use our isogenic reporter PD model and single-cell RNA sequencing approaches of human midbrain, hypothalamic and forebrain DNs to identify populations of cells that show increased vulnerability to OS and cell death and identify differentially affected DN populations. Expression and network analysis will identify cellular functions that confer vulnerability. We have developed genetic tools to distinguish primary dysregulation from emerging phenotypes to further the mechanistic understanding of genotype-phenotype interactions. Using innovative CRISPR-activation and inhibition technologies we will test identified candidate genes for their potential to ameliorate OS phenotypes and cell death in our PD model. Our model is conceptually and technically innovative and will illuminate common and unique pathways that confer vulnerability or protection in nigral DNs and propose novel strategies for prevention and treatment to improve the lives of patients and their caregivers.