Understanding Cell-type Vulnerability and Oxidative Stress Pathology in Parkinson's Disease Using Isogenic Human Dopaminergic Neurons

项目摘要：了解细胞类型的脆弱性和操纵氧化应激病理学 在帕金森病中使用同基因人类多巴胺能神经元 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，PD患者的神经退行性变可能与PD的发病有关。 然而，目前还不清楚为什么黑质DN与其他细胞相比表现出如此精致的脆弱性， 包括其他DN人群。尸检研究表明，氧化应激（OS）有助于 家族性和散发性PD。活性氧（ROS）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能 解释增加的ROS，OS和黑质DN的独特脆弱性，但人类相关的模型系统是 需要严格检验这个假设。迫切需要开发实验系统，以更好地 了解黑质DN的脆弱性，确定新的疾病相关的信号传导机制，并提高分子水平， 亚型和患者分层。在本补充中，我们建议使用我们开发的最新模型 并将其应用于解剖病理细胞类型特异性机制。我们最近开发了一种3D文化 整合诱导多能干细胞（iPSC）衍生的单个细胞类型（神经元，星形胶质细胞， 少突胶质细胞、内皮细胞来制造脑组织芯片。这种方法使我们能够将细胞与 不同的基因背景。为此，我们的目的是调查细胞类型的具体影响，家族性PD 基因PARKIN和DJ1，以更好地了解每种脑细胞类型在PD病理学中的作用。 具体来说，我们的目标是确定PARKIN变异体在每种细胞类型对人类氧化应激的影响。 通过在不同的细胞类型中创建具有PARKIN功能缺失突变的脑组织芯片，我们将 监测多巴胺能神经元的氧化应激。此外，我们的目标是确定DJ 1风险的影响， 通过引入不同的细胞类型，每种细胞类型对蛋白质稳态和多巴胺能神经元存活的变化 与DJ1丧失功能的变体。我们将测量蛋白水解活性和神经元死亡作为DJ1损失的结果。 不同细胞类型的功能。将3D组织芯片系统应用于PD将使我们能够识别 每种细胞类型都对PD病理有贡献。

Project Summary: Understanding cell-type vulnerability and manipulating oxidative stress pathology in Parkinson’s Disease using isogenic human dopaminergic neurons 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. In this supplement we propose to use a recent model that we have developed and apply it to dissect pathological cell-type-specific mechanisms. We have recently developed a 3D culture model integrating induced pluripotent stem cells (iPSCs) derived individual cell types (neurons, astrocytes, oligodendrocytes, endothelial cells to create brain tissue chips. This approach allows us to integrate cells with different genetic backgrounds. To this end, we aim to investigate the cell type specific effects of familial PD genes PARKIN and DJ1 to better understand the role each brain cell type plays to contribute to PD pathology. Specifically, we aim to determine the impact of PARKIN variants in each cell type on oxidative stress in human brain tissue by creating brain tissue chips with PARKIN loss of function mutations in different cell types. We will monitor oxidative stress in the dopaminergic neurons. Additionally, we aim to determine the impact of DJ1 risk variants in each cell type on proteostasis and dopaminergic neuron survival by introducing different cell types with DJ1 loss of function variants. We will measure proteolytic activity and neuronal death as a result of DJ1 loss of function in different cell types. Applying the 3D tissue chip system to PD will allow us to identify the role of each cell type in contributing to PD pathology.