Generation Of Alpha Synuclein (SNCA) Cell Models For High-Throughput Drug Screening Using Zinc-Finger Nuclease Technology (P1.044)

Objectives/Background: Elevated levels of misfolded α−synuclein cause dopaminergic neuronal loss in Parkinson Disease. Treatment with HADC inhibitor was neuroprotective to dopaminergic neurons in PD animal and cell models suggesting that manipulating transcriptional gene expression and histone deacytylation could be used to prevent dopaminergic neuronal death in PD patients. However, there is no cell line model that contains all SNCA regulatory elements in the proper chromatin context for identifying specific HADC inhibitors and compounds that act on the SNCA regulatory elements. We aimed to produce cell lines that express knock-in α−synuclein-luciferase or α−synuclein-GFP for high-throughput drug screens. Methods: We used a Zinc-Finger Nuclease based approach to knock-in luciferase or GFP directly downstream of the most 3’-SCNA exon such that the reporters were in-frame with SCNA. Proper integration was confirmed by RT-PCR and sequence analysis as well as western blot analysis. Bafilomycin A1 treatment was used as a positive control. Results: Two SH-SY5Y cell lines, SH-SY5Y_[SNCA-GFP] and SH-SY5Y_[SNCA-Luc,] were generated. RT-PCR confirmed that the marker gene was located at the desired locus. Western blots using anti-GFP, anti-luciferase, and anti-alpha synuclein antibodies confirmed that both the SH-SY5Y_[SNCA-GFP] and SH-SY5Y_[SNCA-Luc] lines produced α−synuclein-GFP and α−synuclein-luciferase fusion protein, respectively. No evidence of nonspecific donor DNA integration was observed. Treatment with bafilomycin A1, a lysosomal inhibitor, significantly increased the luciferase level in the SH-SY5YSNCA-Luc cell line. Conclusions: Using the ZFN method, we have generated two cell lines that will be useful in high-throughput drug screenings to identify compounds that can regulate the expression of α−synuclein. These cell lines provide unique tools for drug screens as they include human SCNA regulatory control regions in promoters, introns, and even distant sites that potentially may interact through chromatin loops. Disclosure: Dr. Dansithong has nothing to disclose. Dr. Thai has nothing to disclose. Dr. Paul has nothing to disclose. Dr. Pulst has received personal compensation for activities with Athena Diagnostics. Dr. Pulst has received license or royalty payments from Cedars-Sinai Medical Center. Dr. Huynh has received personal compensation in an editorial capacity for the Hanoi Medical Journal.