The role of ER-phagy in maintaining protein homeostasis in PD patient derived neurons.

ER 吞噬在维持 PD 患者衍生神经元蛋白质稳态中的作用。

• 批准号: 10607434
• 负责人: Annie Zalon
• 金额: $ 4.77万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607434
• 关键词: Autophagocytosis; Biochemical; Cells; Co-Immunoprecipitations; Defect; Dementia with Lewy Bodies; Disease; Dose; Electron Microscopy; Endoplasmic Reticulum; Failure; Functional disorder; Genes; Genetic; Genetic study; Genomic Segment; Golgi Apparatus; Homeostasis; Hydrolase; Impairment; In Vitro; Inclusion Bodies; Laboratory Finding; Lewy Bodies; Link; Literature; Mass Spectrum Analysis; Measures; Mediating; Membrane; Midbrain structure; Modeling; Morphology; Movement Disorders; Mutation; Nature; Neurodegenerative Disorders; Neuronal Differentiation; Neurons; Parkinson Disease; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Patients; Persons; Phenotype; Play; Process; Proteins; Resistance; Risk; Role; SNCA gene; Severities; Stress; Structure; Symptoms; Techniques; Testing; Therapeutic; Toxic effect; Variant; Vesicle; Western Blotting; Work; age related; age related neurodegeneration; alpha synuclein; dopaminergic neuron; endoplasmic reticulum stress; functional improvement; functional restoration; genetic risk factor; genome wide association study; glucosylceramidase; immunocytochemistry; improved; in vivo; induced pluripotent stem cell; insight; loss of function mutation; misfolded protein; neuronal survival; novel therapeutic intervention; protein aggregation; proteostasis; receptor; response; risk variant; sporadic Parkinson' s Disease; trafficking

Project Summary Parkinson’s disease (PD) is a common age-related neurodegenerative disorder characterized by loss of dopaminergic (DA) neurons in the midbrain. A hallmark of both familial and sporadic PD is the presence of Lewy body inclusions consisting of aggregated forms of the protein alpha-synuclein (a-syn), which is encoded by the SNCA gene. Recent work by our lab has supported a large body of literature demonstrating that a-syn accumulation disrupts protein homeostasis in neuronal models of PD. In neurons differentiated from induced pluripotent stem cells (iPSCs) from patients harboring mutations in SNCA, we observed impaired autophagic clearance of misfolded proteins, ER morphology changes, and deficits in lysosomal hydrolase tracking. These deficits in the autophagy-lysosomal pathway (ALP) are intriguing given that GWAS studies have identified variants in several genes encoding for ALP proteins that modify risk for developing PD. For instance, variants in the gene GBA1, which encodes the lysosomal hydrolase β-glucocerebrosidase (GCase), are the greatest genetic risk factor for developing PD and Dementia with Lewy Bodies (DLB). Previous work from our lab has shown that wild-type GCase accumulates in the ER of SNCA triplication mutation neurons, accompanied by dramatic ER fragmentation. Yet, despite an increase in ER stress in these cells, the unfolded protein response (UPR) fails to activate and either refold or degrade accumulating unstable ER proteins, including insoluble GCase. Intriguingly, unbiased mass spectrometry revealed that several proteins involved in the selective autophagic degradation of the ER, or ER-phagy, are significantly depleted in SNCA triplication neurons relative to isogenic controls, suggesting that ER-phagy may be dysregulated in these cells. As ER-phagy plays an important role in ER protein homeostasis and ER membrane remodeling, this hypothesis is consistent with the observations of ER- fragmentation and insoluble protein accumulation observed in SNCA triplication neurons. While there is strong genetic and pathological evidence implicating a-syn and the ALP in PD pathogenesis and a-syn-induced disruptions to ER homeostasis and autophagy, relatively little is known about the role of ER-phagy in PD. Here, we intend to resolve some key questions regarding a potential role of ER-phagy in PD pathogenesis. In Aim 1, we will measure ER-phagy flux in SNCA triplication neurons and isogenic controls to determine whether a-syn accumulation impairs ER-phagy and delineate the means through which this impairment may occur. In aim 2, we will investigate the mechanism of wild-type GCase accumulation in the ER of SNCA triplication neurons, and if enhancement of ER-phagy can restore ER homeostasis through alleviating accumulated GCase. As a-syn accumulation is common across PD and DLB, and ER proteostasis defects are commonly observed across neurodegenerative diseases, by resolving these questions, we hope to uncover insights that hold broad therapeutic potential.

项目摘要 帕金森氏病（PD）是一种常见的与年龄相关的神经退行性疾病，其特征是失去 中脑的多巴胺能（DA）神经元。家族性和零星PD的标志是Lewy的存在 由蛋白质α-突触核蛋白（A-Syn）组成的体内夹杂物，由蛋白 SNCA基因。我们的实验室最近的工作支持了大量文献，证明了A-syn 积累会破坏PD神经元模型中的蛋白质稳态。在与诱导的神经元中 来自SNCA突变的患者的多能干细胞（IPSC），我们观察到自噬受损 清除错误折叠的蛋白质，ER形态的变化，并在溶酶体水解酶跟踪中定义。这些 鉴于GWAS研究已经确定 编码ALP蛋白的几种基因中的变体，可改变开发PD的风险。例如，变体 编码溶酶体水解酶β-葡萄糖酶（GCASE）的基因GBA1是最大的通用性 使用路易体（DLB）发展PD和痴呆症的危险因素。我们实验室的先前工作表明 野生型gcase积聚在SNCA三重突变神经元的ER中，由戏剧性的ER完成 分散。然而，目的地增加了这些细胞中的ER应力，展开的蛋白质反应（UPR）未能 激活并降解或降解累积不稳定的ER蛋白，包括不溶性GCASE。有趣的是， 公正的质谱法表明，与选择性自噬降解有关的几种蛋白质 ER或ER-Phagy在SNCA三重元中显着耗尽相对于等源性对照， 表明在这些细胞中ER-PHAGY可能失调。由于ER-Phagy在ER蛋白中起重要作用 稳态和ER膜重塑，该假设与ER-的观察结果一致 在SNCA三分之一神经元中观察到的碎裂和不溶性蛋白积累。虽然有强大 在PD发病机理中牵涉A-Syn和ALP的遗传和病理证据以及A-SYN诱导的 ER稳态和自噬的中断，关于ER-Phagy在PD中的作用相对较少。这里， 我们打算解决有关ER-Phagy在PD发病机理中潜在作用的一些关键问题。在AIM 1中， 我们将测量SNCA三重元神经元和等源性控制中的ER-PHAGY通量，以确定A-Syn是否是否 积累会损害ER-PHAGY，并描述可能发生这种障碍的手段。在AIM 2中， 我们将研究SNCA三重神经元ER中野生型GCASE积累的机理，以及 如果增强ER-Phagy可以通过减轻累积的GCASE来恢复ER稳态。作为a-syn 在PD和DLB中的积累很常见，ER蛋白毒素缺陷通常在整个环境中观察到 神经退行性疾病，通过解决这些问题，我们希望揭示具有广泛的见解 治疗潜力。