VPS35 D620N Inhibits Autophagy and Causes Cell Death by Regulating RNA-binding Protein Activity

Parkinson’s disease (PD) is the most common motor disease in the USA. The primary clinical motor symptoms of PD result from loss of dopaminergic (DA) neurons in the substantia nigra with autophagy dysfunction being closely linked to this disease. Recently, a PD-causing mutation in VPS35 (D620N) was reported to block autophagy. However, preliminary investigation by other groups into a causal mechanism was limited to canonical VPS35 protein interactors in HeLa cells. To overcome these limitations we have performed an unbiased screen using mass spectrometry and RNA sequencing (RNA seq) to identify key protein interactors and pathways in a widely-used cellular model of PD. We have discovered that VPS35 protein interactors show a high enrichment for RNA-binding proteins including several known or suspected to be causal for ALS. Additionally, the D620N mutation resulted in a dramatic decrease in RNA-binding protein interaction. From our screen, Fused in Sarcoma (FUS) and Ewing sarcoma breakpoint region 1 (EWSR1) have emerged as lead candidates for mediating VPS35 D620N autophagy dysfunction. Based upon RNA-binding protein interaction, we examined the transcriptome of VPS35 WT and D620N cells and found changes indicative of alterations in RNA metabolism and autophagy. We hypothesize that VPS35 D620N inhibits autophagy and causes cell death by regulating RNA-binding protein activity.