Discovery of new E3 ligases for alpha-synuclein clearance

Parkinson’s disease (PD) is one of the most common motor-related neurodegenerative diseases and thus far they are untreatable. Genetic alterations in SNCA gene have been shown to be linked to PD and accumulation of aberrant fibrillary forms of alpha-synuclein constituting major part of Lewy bodies that are key hallmark of PD. Accumulation of fibrillary forms of alpha-synuclein is suggested to be contributed to neuronal cell death. Therefore, removal of pathogenic forms of alpha-synuclein offers a therapeutic potential. Cells have two major systems for removal of proteins: autophagy and ubiquitin-proteasome system. Both systems utilize polyubiquitylation as a degradation signal, which are conjugated to the target proteins by the E3 ubiquitin ligases and removed by deubiquitylating enzymes (DUBs) reversibly. In this study, we performed a siRNA-based high-throughput screen to identify E3s and DUBs that regulate alpha-synuclein turnover. The detection of alpha-synuclein in cell extracts was done by a sensitive ELISA assay. From the screen, several E3s previously unlinked to PD pathogenesis or alpha-synuclein turnover were found to significantly alter the abundance of endogenous alpha-synuclein levels in cells. We discovered that one of our key hits directly modifies alphasynuclein with ubiquitin on several lysine residues. Knockdown and knockout tests further validated key regulatory role of the E3 on alpha-synuclein stability. We are now also validating whether other E3 ligases also target alpha-synuclein in a similar manner. Uncovering the roles of these will potentially allow opportunities for a better understanding of the PD pathogenesis and new therapeutic developments against PD.