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(如乙醇胺)缺乏 (ETA)和磷乙醇胺，存在于帕金森氏病(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为特征的疾病中，如 伴有路易体和多系统萎缩的痴呆。无论这些新陈代谢缺陷是原因还是 疾病的后果不得而知。然而，我们确实知道，低水平的PE可以导致线粒体 糖基磷脂酰肌醇锚定蛋白的功能障碍、自噬功能障碍和错误加工， α-SYN的动态平衡肯定会受到这些过程中的减量的影响。事实上，数据 从酵母和蠕虫的联核症模型中，已经表明低水平的PE(由于 为了敲除线粒体酶磷脂酰丝氨酸脱羧酶(PISD)和α-SYN 有毒的。Eta挽救了PISD基因敲除细胞中的α-SYN毒性，因为Eta通过 CDP-乙醇胺途径，存在于内质网中。这一提议的长期目标是 研究旨在阐明α-SYN是如何调节自噬的。剖析PE-ETA轴在脑出血中的作用 α-SYN在抑制自噬中的作用，我们的具体目的是：(1)确定其作用 自噬-溶酶体功能中PE合成的变化和患者IPSC神经元中α-SYN的清除。我们会 确定ETA刺激PE合成是否会挽救患者神经元的自噬表型和 Eta是否通过增加自噬通量减少α-SYN的病理构象积累。 (2)确定α-SYN(和A53T)降低IPSC-中脑PISD水平的机制 和兴奋性皮质细胞，以及SH-SY5Y细胞。我们提出，PISD的赤字会导致PE的赤字 同时抑制自噬。我们将确定α-SYN(和A53T)是否阻止PISD的进口 通过破坏连接内质网和内质网的线粒体相关膜(MAM)进入线粒体 线粒体。或者，α-SYN(和A53T)可能通过内体或 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， 分离线粒体，同时进行脂质分析、自噬通量分析和外切体分离。(3) 确定α-SYN是否抑制线粒体-空泡和线粒体-内质网的接触。我们将击倒对手 调控线粒体-内质网接触的基因和分别敲除调控线粒体的基因- 有和没有α-SYN表达的细胞中的空泡接触，然后评估这些细胞中的破坏 线粒体-细胞器接触影响Atg8-GFP的自噬通量。我们已经证明了α-syn 抑制atg8-gfp的自噬通量，但这些实验挖掘得更深，并将揭示α-syn 通过破坏线粒体和内质网或液泡之间的分子联系来抑制自噬。建议数 实验将深入了解α-SYN聚集的原因以及如何防止它。