The Parkinson's Disease Variant rs356182 Regulates Neuronal Differentiation Independently from Alpha-Synuclein

One of the most significant risk variants for Parkinson's disease (PD), rs356182, is located at the PD-associated locus near the alpha-synuclein (α-syn) encoding gene, SNCA. SNCA-proximal variants, including rs356182, are thought to function in PD risk through enhancers via allele-specific regulatory effects on SNCA expression. However, this interpretation discounts the complex activity of genetic enhancers and possible nonconical functions of α-syn. Here we investigated a novel risk mechanism for rs356182. We use CRISPR-Cas9 in LUHMES cells, a model for dopaminergic midbrain neurons, to generate precise hemizygous lesions at rs356182. The PD-protective (A/-), PD-risk (G/-), and wildtype (A/G) clones were neuronally differentiated and then compared transcriptionally and morphologically. Among the affected genes was SNCA, whose expression was promoted by the PD-protective allele (A) and repressed in its absence. In addition to SNCA, hundreds of genes were differentially expressed and associated with neurogenesis and axonogenesis- an effect not typically ascribed to α-syn. We also found that the transcription factor FOXO3 specifically binds to the rs356182 A-allele in differentiated LUHMES cells. Finally, we compared the results from the rs356182-edited cells to our previously published knockouts of SNCA and found only minimal overlap between the sets of significant differentially expressed genes. Together, the data implicate a risk mechanism for rs356182 in which the risk-allele (G) is associated with abnormal neuron development, independent of SNCA expression. We speculate that these pathological effects manifest as a diminished population of dopaminergic neurons during development leading to the predisposition for PD later in life. To determine the role of rs356182 in dopaminergic neurons, we generated three biological replicate hemizygous clones for the Parkinson's disease protective allele (A/-) and PD risk allele (G/-), as well as 3 sorted and cloned WT controls using the LUHMES cell line. The clones were expanded and differentiated to day 6 before being submitted for RNA-sequencing.