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 (a-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 a-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 a-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. Overall design: 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.