Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process whereby cellular cytoplasmic contents are sequestered in double membraned vesicles and delivered to lysosomes for proteolytic degradation. Dysfunction in the autophagic machinery or in the lysosomes is a hallmark of numerous neurodegenerative diseases, including Parkinson’s disease (PD). Deficient clearance of damaged cellular components, misfolded proteins, and aggregates leads to the accumulation of neurotoxic proteins, and an increase in cellular stressors such as reactive oxygen species – eventually causing neuronal cell death. In the case of PD, neuronal inclusions are composed primarily of the small, disordered protein alpha synuclein (α-Syn), which can be an autophagy substrate.

To assist in the identification genes that modify the accumulation of α-Syn and may represent potential therapeutic targets, we conduct an unbiased whole-genome CRISPR/Cas9 knockout screen to identify modulators of α-Syn levels in human neuroblastoma cells. We identify both positive and negative regulators of α-Syn accumulation, and, with reference to the literature, select a group of hits for further investigation. To determine whether the effects of these genes on α-Syn are primarily autophagy dependent, we conduct follow up CRISPR/Cas9 knockout screens in comparable autophagy-competent and autophagy-null HeLa cell lines. We identify a small group of genes that appear to impact the autophagic clearance of α-Syn, which we hope may provide leads for future work.

RNA binding proteins stand out as enriched amongst genes that modify α-Syn levels in our screens – particularly core protein components of stress granules (SGs). SGs are autophagy substrates, and have received much attention as a result of newly discovered links to protein aggregation in neurodegenerative diseases. We demonstrate that α-Syn can localise to SGs under various cellular stresses, and that the C-terminal of α-Syn is dispensable for this association, as is the presence of a canonical SG-nucleating protein. We also provide initial data indicating that α-Syn may develop secondary structure and fibrillise within SGs.

A SG-associated RNA binding protein is identified as strong regulator of a-Syn levels in our whole genome CRISPR screen. We demonstrate that in basal conditions (where no SGs are present) depletion of this protein blocks both the synthesis and the autophagic degradation of α-Syn. The blockage in α-Syn degradation is correlated with widespread vesicle trafficking defects in cells depleted of our target protein. We demonstrate that this phenotype is characterised by a blockage of autophagic flux and accumulation of autophagy substrates; perinuclear lysosomal clustering; defective trafficking of lysosomal hydrolases and lysosomal deacidification; defective endocytosis; and a block in constitutive secretion. Cells deficient in our target protein also exhibit profoundly abnormal architecture of the Golgi complex, defective fission of the recycling endosome, and perturbations in the microtubule cytoskeleton. While future research is required to uncover the precise mechanism by which this protein facilitates vesicle trafficking and maintains the architecture of the Golgi apparatus, the discovery of a new role for a much-studied RNA binding protein has implications for a number of neurodegenerative diseases to which the protein has been linked, and may help to shed light on pathogenic mechanisms.