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

项目摘要 帕金森病(PD)是一种常见的与年龄相关的神经退行性疾病，其特征是 中脑的多巴胺能(DA)神经元。家族性和散发性帕金森病的一个特征是存在路易 体包涵体由聚集态的α-突触核蛋白(a-syn)组成，由 SNCA基因。我们实验室最近的工作支持了大量文献，证明a-syn 在帕金森病神经元模型中，蓄积破坏蛋白质的动态平衡。在诱导分化的神经元中 SNCA基因突变患者的多能干细胞(IPSCs)，我们观察到自噬功能受损 错误折叠的蛋白质清除，内质网形态变化，以及溶酶体水解酶跟踪缺陷。这些 自噬-溶酶体途径(ALP)的缺陷是耐人寻味的，因为GWAS的研究已经发现 几个编码碱性磷酸酶蛋白的基因的变异改变了发展为帕金森病的风险。例如，中的变体 编码溶酶体水解酶β-葡萄糖脑苷酶(GCase)的基因gba1是最大的遗传基因。 发生帕金森病和路易体痴呆的危险因素(DLB)。我们实验室之前的研究表明， SNCA三倍体突变神经元的内质网中有野生型GCase积聚，并伴有明显的内质网 碎片化。然而，尽管这些细胞的内质网应激增加，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的内质网蛋白，包括不溶性GCase。有趣的是， 无偏质谱显示，几种蛋白质参与了选择性自噬降解 与同基因对照相比，SNCA三倍体神经元中的ER，或ER吞噬显著减少， 提示内质网吞噬功能在这些细胞中可能处于失调状态。内质网吞噬功能在内质网蛋白中起重要作用 动态平衡和内质网重构，这一假说与内质网的观察结果一致。 观察到SNCA三倍体神经元的碎裂和不溶性蛋白积聚。当有强大的 α-SYN和ALP参与帕金森病发病机制及α-SYN诱导的遗传学和病理学证据 对于内质网稳态和自噬的破坏，人们对内质网吞噬在帕金森病中的作用知之甚少。这里, 我们打算解决一些关于内质网吞噬在帕金森病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的ER吞噬通量，以确定a-syn 蓄积损害了内质网吞噬功能，并描述了这种损害可能发生的途径。在目标2中， 我们将研究野生型GCase在SNCA三倍体神经元内质网中积累的机制，以及 增强内质网吞噬功能是否能通过减轻GCase积聚来恢复内质网内稳态。作为a-syn 在PD和DLB中积聚是常见的，ER蛋白平衡缺陷在 神经退行性疾病，通过解决这些问题，我们希望揭示出具有广泛意义的见解。 治疗潜力。

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.