Chloride Homeostasis in Lysosomal Function and Parkinson's Disease

溶酶体功能和帕金森病中的氯稳态

• 批准号: 10515961
• 负责人: ZIXU MAO
• 金额: $ 60.46万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10515961
• 关键词: Affect; Albers-Schonberg disease; Animal Model; Animals; Anions; Automobile Driving; Autophagocytosis; Autophagosome; Autopsy; Binding; Biochemical; Biological Markers; Biological Process; Biology; Brain; Brain Diseases; CLC Gene; Cathepsin C; Cell physiology; Cells; Chloride Channels; Chloride Ion; Chlorides; Complex; Data; Degenerative Disorder; Degradation Pathway; Disease; Environmental Risk Factor; Event; Family; Functional disorder; GTP-Binding Protein alpha Subunits, Gs; Genes; Genetic; Genetic Determinism; Genetic study; Health; Homeostasis; Human; Hydrolase; Impairment; Induced pluripotent stem cell derived neurons; Intracellular Space; LRRK2 gene; Link; Lysosomal Storage Diseases; Lysosomes; Modeling; Molecular; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organelles; Oxidative Stress; Parkinson Disease; Pathogenesis; Pathogenicity; Pathologic; Pathology; Pathway interactions; Patients; Play; Process; Proteins; Regulation; Reporting; Research; Rodent Model; Role; Signal Transduction; Site; Stress; Substantia nigra structure; Testing; base; design; detection of nutrient; dopaminergic neuron; experimental study; human model; induced pluripotent stem cell; innovation; member; mutant; nervous system disorder; new therapeutic target; pars compacta; response; targeted biomarker

Parkinson's disease (PD) is a progressive and degenerative disorder of the brain. It is pathologically characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). The key events driving the pathogenesis in PD are not completely understood. The long-term objective of my research is to understand the molecular and cellular processes by which neurons respond to stress and how dysfunction of these responsive mechanisms contributes to neurodegenerative process. I propose to investigate a new molecular regulatory process of lysosomal chloride and its role in PD pathogenesis. Chloride ion is the most abundant anion in both extra- and intracellular spaces of animal cells. No longer regarded as an inert anion, chloride is found to play discrete roles in cells and its homeostasis needs to be tightly regulated at a subcellular organelle level. Lysosomal chloride is important for the function of acidic hydrolases. In addition to this general role, lysosomal chloride itself also has specific roles affecting lysosomal functions such as binding directly to cathepsin C to regulate its activity. Lysosomal chloride is mainly regulated by chloride channel 7 (CLC-7) in complex with its beta subunit Ostm-1. Loss of either protein severely comprises lysosomal chloride homeostasis, reduces lysosomal degradation capacity, and causes accumulation of lysosomal storage materials and autophagosomes, leading to diseases in human and rodent models including neuronal damages and degeneration. How CLC-7 is regulated remains largely unknown and no studies have ever reported its involvement in PD pathogenesis. We discovered unexpectedly a regulatory link between leucine-rich repeat kinase 2 (LRRK2), one of the most common genetic determinants associated with PD, and CLC-7. Our new preliminary data show a direct interaction between LRRK2 and CLC-7. This interaction is pathogenically enhanced by LRRK2 G2019S mutation and by oxidative stress, leading to aberrantly high level of lysosomal chloride and reduced lysosomal activities. We hypothesize that LRRK2 interacts with CLC-7 to modulate lysosomal chloride homeostasis and pathogenic mutant LRRK2G2019S dysregulates this process and impairs lysosomal functions. We propose to assess the molecular effects of LRRK2 on CLC-7 function in DA neurons derived from iPSCs of PD patients (aim I), assess the cellular effects of aberrant LRRK2-CLCL-7 interaction under genetic and oxidative stress on lysosomal functions in DA neurons derived from control and PD patient iPSCs (aim II); assess the molecular and cellular effects of LRRK2 on lysosomal CLC-7, chloride, and functions in animal models of PD (aim III), and assess the lysosomal LRRK2, CLC-7, and chloride in postmortem brains of PD patients (aim IV). The study will identify the key process that controls lysosomal chloride, establish its role in PD cytopathogenesis, and possibly reveal new therapeutic targets and biomarkers for the disease.

帕金森病（PD）是一种进行性和退行性的大脑疾病。这是病理性的 其特征是黑质致密部 (SNc) 中多巴胺能神经元的丧失。关键 驱动帕金森病发病机制的事件尚不完全清楚。我研究的长期目标 是了解神经元对压力做出反应的分子和细胞过程以及功能障碍如何 这些反应机制有助于神经退行性过程。我建议研究一个新的 溶酶体氯化物的分子调控过程​​及其在PD发病机制中的作用。氯离子最多 动物细胞的细胞外和细胞内空间中含有丰富的阴离子。不再被视为惰性阴离子， 人们发现氯化物在细胞中发挥着离散的作用，其稳态需要在亚细胞中受到严格调节 细胞器水平。溶酶体氯化物对于酸性水解酶的功能很重要。除了这个一般 作用，溶酶体氯化物本身也具有影响溶酶体功能的特定作用，例如直接结合 组织蛋白酶 C 来调节其活性。溶酶体氯化物主要受氯离子通道 7 (CLC-7) 调节 与其β亚基Ostm-1形成复合物。任一蛋白质的严重损失均包括溶酶体氯化物 体内平衡，降低溶酶体降解能力，并导致溶酶体储存积累 材料和自噬体，导致人类和啮齿动物模型中的疾病，包括神经元损伤 和退化。 CLC-7 的监管方式仍然很大程度上未知，也没有研究报告过它的情况 参与 PD 发病机制。我们意外地发现富含亮氨酸重复序列之间存在调节联系 激酶 2 (LRRK2)（与 PD 相关的最常见遗传决定因素之一）和 CLC-7。我们的新 初步数据显示 LRRK2 和 CLC-7 之间存在直接相互作用。这种相互作用是致病的 LRRK2 G2019S 突变和氧化应激增强，导致溶酶体异常高水平 氯化物和溶酶体活性降低。我们假设 LRRK2 与 CLC-7 相互作用来调节 溶酶体氯化物稳态和致病突变体 LRRK2G2019S 失调该过程并损害 溶酶体功能。我们建议评估 LRRK2 对 DA 神经元 CLC-7 功能的分子影响 源自 PD 患者的 iPSC（目标 I），评估异常 LRRK2-CLCL-7 的细胞效应 遗传和氧化应激下的相互作用对 DA 神经元溶酶体功能的影响 对照和 PD 患者 iPSC（目标 II）；评估 LRRK2 对溶酶体的分子和细胞作用 CLC-7、氯和在 PD 动物模型中的功能（目标 III），并评估溶酶体 LRRK2、CLC-7 和 PD 患者死后大脑中的氯化物（目标 IV）。该研究将确定控制的关键过程 溶酶体氯化物，确定其在 PD 细胞病理发生中的作用，并可能揭示新的治疗靶点和 该疾病的生物标志物。