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

磷脂酰乙醇胺（PE）及其代谢产物（如乙醇胺）缺乏 (ETA)和磷酸乙醇胺，发生在帕金森病（PD）和阿尔茨海默病的大脑。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， 路易体痴呆和多系统萎缩。这些代谢缺陷是否是一个原因， 疾病的后果尚不清楚。然而，我们知道低水平的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， α-syn的体内平衡肯定会受到这些过程中的减少的影响。事实上，数据 从酵母和蠕虫模型的突触核蛋白病，已经表明，共同出现的低水平的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ETA在PISD敲除细胞中拯救α-syn毒性，因为ETA通过PISD基因刺激PE的合成。 CDP-乙醇胺途径，其存在于内质网中。这一长期目标的提出， 研究的目的是阐明α-syn如何调节自噬。为了剖析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 自噬-溶酶体功能中的PE合成和患者iPSC-神经元中α-syn的清除。我们将 确定用ETA刺激PE合成是否会拯救患者神经元中的自噬表型， ETA是否通过增加自噬流量来减少α-syn的病理构象的积累。 (2)确定α-syn（和A53 T）降低患者iPSC-中脑中PISD水平的机制 和兴奋皮质细胞，以及SH-SY 5 Y细胞。我们认为，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A53 T）是否阻止PISD的导入 通过破坏线粒体相关膜（MAMs）将ER与 线粒体或者，α-syn（和A53 T）可以通过核内体或核外体触发PISD从细胞释放。 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， 分离线粒体并伴随脂质分析、自噬通量分析和外泌体分离。（三） 确定α-syn是否抑制胞浆素-空泡和胞浆素-ER接触。我们将淘汰 调节Escherichia coli-ER接触的基因和分别敲除调节Escherichia coli-ER接触的基因， 空泡接触的细胞和无α-syn表达，然后评估如何破坏这些 细胞器-细胞器接触影响Atg 8-GFP的自噬通量。我们已经证明α-syn 抑制Atg 8-GFP的自噬通量，但这些实验深入研究并将揭示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 实验将深入了解为什么α-syn聚集以及如何防止它。

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.