Examining the role of phosphatidylethanolamine and autophagic disruption in Lewy Body Dementias and Parkinson's disease

检查磷脂酰乙醇胺和自噬破坏在路易体痴呆和帕金森病中的作用

• 批准号: 10419671
• 负责人: Joseph R Mazzulli
• 金额: $ 64.24万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10419671
• 关键词: Affect; Alzheimer' s disease brain; Amino Acids; Autophagocytosis; Autophagosome; Autopsy; Basic Science; Binding; Biogenesis; Biological Assay; Brain; Brain Diseases; CDP ethanolamine; Carboxy-Lyases; Cell Line; Cells; Characteristics; Clinic; Communication; Complex; Data; Dementia; Dementia with Lewy Bodies; Disease; Electron Microscopy; Endoplasmic Reticulum; Endosomes; Enzymes; Ethanolamines; Functional disorder; GPI Membrane Anchors; Genes; Genetic; Goals; Human; Hydrolase; Induced pluripotent stem cell derived neurons; Knock-out; Knowledge; Lead; Lewy Body Dementia; Link; Lipids; Mediating; Membrane; Metabolic; Midbrain structure; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Molecular Conformation; Multiple System Atrophy; Natural Killer Cells; Neurons; Organelles; Parkinson Disease; Pathogenicity; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Phosphatidylethanolamine; Phosphatidylserines; Phospholipids; Play; Process; Protein Conformation; Proteins; Publishing; Research; Role; Set protein; Sorting - Cell Movement; Structure; Supplementation; Testing; Toxic effect; Transgenic Mice; Vacuole; Work; Yeasts; age related; alpha synuclein; cytotoxic; dopaminergic neuron; druggable target; exosome; experimental study; improved; induced pluripotent stem cell; inhibition of autophagy; insight; knockout gene; lipid transport; mitochondrial dysfunction; mitochondrial membrane; neuron loss; neurotoxicity; novel; novel therapeutics; overexpression; phosphoethanolamine; prevent; protein aggregation; protein degradation; proteostasis; synuclein; synucleinopathy; trafficking

Deficits in the phospholipid phosphatidylethanolamine (PE) and its metabolites, such as ethanolamine (ETA) and phosphoethanolamine, occur in the Parkinson’s disease (PD) and Alzheimer’s disease brain. We propose that these metabolic deficits likely also occur in diseases characterized by synuclein pathology such as dementia with Lewy bodies and multiple system atrophy. Whether these metabolic deficits are a cause or consequence of disease is not known. However, we do know that low levels of PE can lead to mitochondrial dysfunction, autophagy dysfunction, and the misprocessing of glycosylphosphatidylinositol-anchored proteins, and that the homeostasis of α-syn can certainly be impacted by decrements in these processes. In fact, data from yeast and worm models of synucleinopathies, have shown that the co-occurrence of low levels of PE (due to knocking out the mitochondrial enzyme phosphatidylserine decarboxylase, PISD) and α-syn are synthetically toxic. ETA rescues α-syn toxicity in PISD knockout cells, because ETA stimulates the synthesis of PE via the CDP-ethanolamine pathway, which resides in the endoplasmic reticulum. The long-term goal of this proposed research is to elucidate how α-syn modulates autophagy. To dissect the role of the PE-ETA axis in synucleinopathies and the role of α-syn in inhibiting autophagy, our specific aims are to: (1) determine the role of PE synthesis in autophagic-lysosomal function and clearance of α-syn in patient iPSC-neurons. We will determine if stimulating PE synthesis with ETA will rescue autophagic phenotypes in patient neurons and whether ETA decreases the accumulation of pathologic conformations of α-syn by increasing autophagic flux. (2) Determine the mechanism by which α-syn (and A53T) decreases the level of PISD in patient iPSC-midbrain and excitatory-cortical cells, and in SH-SY5Y cells. We propose that a deficit in PISD produces a deficit in PE with a parallel inhibition of autophagy. We will determine whether α-syn (and A53T) blocks the import of PISD into mitochondria by disrupting mitochondrial associated membranes (MAMs) that connect the ER with mitochondria. Alternatively, α-syn (and A53T) may trigger the release of PISD from cells via endosomes or promote the rapid degradation of the protein. These possibilities will be analyzed by electron microscopy, isolation of mitochondria with attending lipid analysis, analysis of autophagy flux, and exosome isolation. (3) Determine whether α-syn inhibits mitochondrion-vacuole and mitochondrion-ER contacts. We will knock out genes that regulate mitochondrion-ER contacts and separately knockout genes that regulate mitochondrion- vacuole contacts in cells with and without α-syn expression and then evaluate how disruptions in these mitochondrion-organelle contacts affect the autophagic flux of Atg8-GFP. We have already shown that α-syn inhibits the autophagic flux of Atg8-GFP, but these experiments dig deeper and will reveal whether the α-syn inhibits autophagy by disrupting molecular contacts between mitochondria and the ER or vacuole. The proposed experiments will give insight into why α-syn aggregates and how it can be prevented.

磷脂酰乙醇胺 (PE) 及其代谢物（例如乙醇胺）缺乏 (ETA) 和磷酸乙醇胺，存在于帕金森病 (PD) 和阿尔茨海默病的大脑中。我们 提出这些代谢缺陷也可能发生在以突触核蛋白病理学为特征的疾病中，例如 路易体痴呆和多系统萎缩。这些代谢缺陷是否是原因或 疾病的后果尚不清楚。然而，我们确实知道低水平的 PE 会导致线粒体 功能障碍、自噬功能障碍和糖基磷脂酰肌醇锚定蛋白的错误加工， 并且 α-syn 的稳态肯定会受到这些过程的减少的影响。事实上，数据 来自突触核蛋白病的酵母和蠕虫模型的研究表明，低水平 PE 的同时发生（由于 敲除线粒体酶磷脂酰丝氨酸脱羧酶 (PISD) 和 α-syn 是合成的 有毒的。 ETA 可以挽救 PISD 敲除细胞中的 α-syn 毒性，因为 ETA 通过 CDP-乙醇胺途径，存在于内质网中。本提议的长期目标 研究目的是阐明 α-syn 如何调节自噬。剖析 PE-ETA 轴在 突触核蛋白病和α-syn在抑制自噬中的作用，我们的具体目标是：（1）确定作用 PE 合成在自噬溶酶体功能中的作用以及患者 iPSC 神经元中 α-syn 的清除。我们将 确定用 ETA 刺激 PE 合成是否会挽救患者神经元的自噬表型，以及 ETA是否通过增加自噬通量来减少α-syn病理构象的积累。 (2) 确定 α-syn（和 A53T）降低患者 iPSC 中脑 PISD 水平的机制 和兴奋性皮质细胞以及 SH-SY5Y 细胞。我们认为 PISD 的赤字会导致 PE 的赤字 并同时抑制自噬。我们将确定 α-syn（和 A53T）是否阻止 PISD 的导入 通过破坏连接 ER 的线粒体相关膜 (MAM) 进入线粒体 线粒体。或者，α-syn（和 A53T）可能会通过内体或内体触发细胞释放 PISD 促进蛋白质的快速降解。这些可能性将通过电子显微镜进行分析， 线粒体的分离，包括脂质分析、自噬流分析和外泌体分离。 (3) 确定 α-syn 是否抑制线粒体-液泡和线粒体-ER 接触。我们将淘汰 调节线粒体-ER接触的基因和单独敲除调节线粒体-的基因 具有和不具有 α-syn 表达的细胞中的液泡接触，然后评估这些细胞中的破坏如何 线粒体-细胞器接触影响 Atg8-GFP 的自噬通量。我们已经证明 α-syn 抑制 Atg8-GFP 的自噬流，但这些实验进行了更深入的研究，并将揭示 α-syn 是否 通过破坏线粒体和内质网或液泡之间的分子接触来抑制自噬。拟议的 实验将深入了解 α-syn 聚集的原因以及如何预防它。