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

使用同基因人类多巴胺能神经元了解帕金森病的细胞类型脆弱性和氧化应激病理学

• 批准号: 10458745
• 负责人: Joel William Blanchard
• 金额: $ 39.83万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10458745
• 关键词: Affect; American; Autopsy; Biological Models; Brain; CRISPR-mediated transcriptional activation; Candidate Disease Gene; Caregivers; Cell Death; Cell Line; Cell model; Cell physiology; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Consequentialism; DNA Damage; Data; Data Set; Diagnosis; Disease; Disease model; Dopamine; Environment; Flow Cytometry; Fluorescent Dyes; Functional disorder; Gene Expression; Genes; Genetic; Genotype; Goals; Human; Hypothalamic structure; Image; Individual; Inherited; Knock-in; Knowledge; Lead; Lentivirus Vector; Lipids; Mendelian disorder; Metabolism; Midbrain structure; Mission; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Mutagenesis; Mutation; Nerve Degeneration; Neurons; Organoids; Outcome; Oxidative Stress; Oxidative Stress Pathway; PARK7 gene; PARK9 gene; Parkinson Disease; Parkinsonian Disorders; Pathologic; Pathology; Pathway Analysis; Pathway interactions; Patients; Pattern; Pharmacology; Phenotype; Population; Population Analysis; Prevention; Prevention strategy; Prosencephalon; Proteins; Proteomics; Public Health; Reactive Oxygen Species; Reporter; Research; Research Proposals; Risk; Scientist; Signal Transduction; Signaling Molecule; Source; System; Technology; Testing; Therapeutic; Time; Tissues; Tyrosine 3-Monooxygenase; United States National Institutes of Health; biological adaptation to stress; candidate validation; cell type; disease-causing mutation; disorder risk; dopaminergic neuron; early onset; gene network; human pluripotent stem cell; improved; innovation; insight; knock-down; loss of function; molecular subtypes; motor impairment; multiple omics; new therapeutic target; novel; novel strategies; patient stratification; protein aggregation; response; single-cell RNA sequencing; tool; trafficking; transcriptomics

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.

约有一百万美国人患有帕金森氏病（PD），其特征是逐渐丧失 ni中脑多巴胺能神经元（DNS）的亚群，导致运动障碍和其他 使人衰弱的条件。家族性PD基因在大脑中表现出广泛的表达和PD的神经退行性 可以普遍存在；但是，目前尚不清楚为什么Nigral DNS与 其他细胞类型，包括其他DN群体。验尸研究表明氧化应激（OS） 有助于家族性和零星PD。活性氧（ROS）是重要的信号分子 但是高水平的细胞内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ni骨DN的独特脆弱性，但与人相关 模型系统需要严格检验该假设。迫切需要发展 实验系统以更好地了解nigral DN脆弱性，确定新型疾病的信号传导 机制，改善分子亚型和患者分层。我们的长期目标是了解 通过遗传学的相互作用，nigral DNS的脆弱性，赋予细胞类型的特定功能 漏洞和可量化的表型，以识别新的治疗靶标。为了支持这个目标，我们有 开发的敲入人类多能干细胞（HPSC）报告基因识别和分离酪氨酸 来自大型类器官自旋培养物的羟化酶（Th）阳性DN。使用CRISPR诱变我们 创建的IS基因丧失功能模型的早期常染色体隐性PD（Parkin - / - ，DJ1 - / - 和） 在Th-Reporter细胞系中的ATP13A2 - / - ）。我们检测到线粒体蛋白的失调，显着 在中脑DNS中的等源性PD细胞系中的OS和细胞死亡增加，但在等源性WT-Control DNS中没有增加。到 了解nigral DN脆弱性我们建议使用我们的ISEOGENIC RETORTER PD模型和单细胞RNA 人中脑，下丘脑和前脑DNS的测序方法鉴定细胞种群 这表明对OS和细胞死亡的脆弱性增加，并确定受差异影响的DN群体。 表达和网络分析将确定赋予脆弱性的细胞功能。我们已经发展了 遗传工具将主要失调与新兴表型区分开以进一步发展机械 了解基因型 - 表型相互作用。使用创新的CRIS-PR-激活和抑制 技术我们将测试确定的候选基因，以减轻OS表型和细胞的潜力 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，将阐明常见和 独特的途径，这些途径赋予脆弱性DNS的脆弱性或保护，并提出新颖的策略 预防和治疗以改善患者及其护理人员的生活。