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 帕金森病（PD）是一种常见的年龄相关性神经退行性疾病，其特征是神经元的丢失。 中脑中的多巴胺能（DA）神经元。家族性和散发性PD的一个标志是Lewy 体内含物，由蛋白质α-突触核蛋白（a-syn）的聚集形式组成，其由 SNCA基因。我们实验室最近的工作支持了大量的文献，证明a-syn 在PD的神经元模型中，累积破坏蛋白质稳态。在诱导分化的神经元中， 我们从携带SNCA突变的患者的多能干细胞（iPSC）中观察到自噬受损， 错误折叠蛋白的清除、ER形态学变化和溶酶体水解酶追踪缺陷。这些 自噬-溶酶体途径（ALP）的缺陷是有趣的，因为GWAS研究已经确定， 编码ALP蛋白的几种基因的变异改变了发展PD的风险。例如， GBA 1基因编码溶酶体水解酶β-葡萄糖脑苷脂酶（GCase），是目前已知的最大的遗传缺陷型基因， 发生PD和路易体痴呆（DLB）的风险因素。我们实验室以前的工作表明， 野生型GCase在SNCA三联突变神经元的ER中积累，伴随着显著的ER 碎片化然而，尽管这些细胞中ER应激增加，但未折叠蛋白反应（UPR）未能 激活并重新折叠或降解积累的不稳定ER蛋白，包括不溶性GCase。有趣的是， 无偏质谱分析显示，参与选择性自噬降解的几种蛋白质， 相对于同基因对照，ER或ER-吞噬物在SNCA三倍体神经元中显著减少， 提示ER-吞噬可能在这些细胞中失调。由于ER-吞噬在ER蛋白中起着重要作用 内稳态和ER膜重塑，这一假设与ER的观察结果一致， 在SNCA三倍体神经元中观察到碎片和不溶性蛋白质积累。虽然有强大的 提示a-syn和ALP参与PD发病机制和a-syn诱导PD的遗传和病理学证据 由于ER稳态和自噬的破坏，对ER-吞噬在PD中的作用知之甚少。在这里， 我们打算解决一些关于ER-吞噬在PD发病机制中的潜在作用的关键问题。在目标1中， 我们将测量SNCA三倍体神经元和同基因对照中的ER-吞噬通量，以确定α-syn 蓄积损害ER-吞噬作用，并描述了这种损害可能发生的方式。在目标2中， 我们将研究野生型GCase在SNCA三联体神经元ER中积累的机制， 增强ER-吞噬功能是否能通过减轻GCase的蓄积而恢复ER的稳态。作为一个同步 在PD和DLB中常见的是ER的积累，并且在PD和DLB中通常观察到ER蛋白稳态缺陷。 神经退行性疾病，通过解决这些问题，我们希望揭示的见解， 治疗潜力