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