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

大约有一百万美国人患有帕金森病（PD），其特征是进行性丧失。 黑质中脑多巴胺能神经元（DN）的亚群，导致运动障碍和其他 使人衰弱的状况。家族性PD基因在脑内广泛表达，并可导致PD的神经退行性变 可能是广泛的;然而，目前还不清楚为什么黑猩猩DN显示出如此精致的脆弱性相比， 其他细胞类型，包括其他DN群体。死后研究表明，氧化应激（OS） 导致家族性和散发性PD。活性氧是重要的信号分子 但高水平的细胞内ROS会破坏DNA、脂质和蛋白质。高能量需求和多巴胺 代谢可以解释增加的ROS，OS和黑质DNs的独特脆弱性，但人类相关的 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DN的脆弱性，确定新的疾病相关的信号 机制，并改善分子亚型和患者分层。我们的长期目标是了解 黑质DNA的脆弱性，通过遗传学的相互作用，细胞类型的特定功能，赋予 脆弱性和可量化的表型，以确定新的治疗靶点。为了实现这一目标，我们 开发了敲入人多能干细胞（hPSC）报告细胞系，以鉴定和分离酪氨酸 来自大规模类器官旋转培养物的羟化酶（TH）阳性DN。使用CRISPR诱变， 创建了早发性常染色体隐性PD的同基因功能丧失模型（PARKIN-/-，DJ 1-/-和 ATP13A2-/-）。我们检测到线粒体蛋白的失调， 增加OS和细胞死亡的同基因PD细胞系中脑DN，但不是在同基因WT对照DN。到 了解黑质DN的脆弱性，我们建议使用我们的同基因报告PD模型和单细胞RNA 人类中脑、下丘脑和前脑DNA的测序方法以鉴定细胞群 显示出对OS和细胞死亡的增加的脆弱性，并鉴定出不同影响的DN群体。 表达和网络分析将确定赋予脆弱性的细胞功能。我们已经开发 遗传工具，以区分原发性失调与新出现的表型， 了解基因型-表型相互作用。使用创新的CRISPR激活和抑制 技术，我们将测试确定的候选基因，以了解它们改善OS表型和细胞增殖的潜力。 我们的PD模型中的死亡我们的模型在概念上和技术上都是创新的， 独特的途径，赋予黑质DNs的脆弱性或保护，并提出新的策略， 预防和治疗，以改善患者及其护理人员的生活。

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.