title: Generation of SNCA Cell Models Using Zinc Finger Nuclease (ZFN) Technology for Efficient High-Throughput Drug Screening.
abstract: Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by loss of dopaminergic neurons of the substantia nigra. The hallmark of PD is the appearance of neuronal protein aggregations known as Lewy bodies and Lewy neurites, of which α-synuclein forms a major component. Familial PD is rare and is associated with missense mutations of the SNCA gene or increases in gene copy number resulting in SNCA overexpression. This suggests that lowering SNCA expression could be therapeutic for PD. Supporting this hypothesis, SNCA reduction was neuroprotective in cell line and rodent PD models. We developed novel cell lines expressing SNCA fused to the reporter genes luciferase (luc) or GFP with the objective to enable high-throughput compound screening (HTS) for small molecules that can lower SNCA expression. Because SNCA expression is likely regulated by far-upstream elements (including the NACP-REP1 located at 8852 bp upstream of the transcription site), we employed zinc finger nuclease (ZFN) genome editing to insert reporter genes in-frame downstream of the SNCA gene in order to retain native SNCA expression control. This ensured full retention of known and unknown up- and downstream genetic elements controlling SNCA expression. Treatment of cells with the histone deacetylase inhibitor valproic acid (VPA) resulted in significantly increased SNCA-luc and SNCA-GFP expression supporting the use of our cell lines for identifying small molecules altering complex modes of expression control. Cells expressing SNCA-luc treated with a luciferase inhibitor or SNCA siRNA resulted in Z'-scores ≥ 0.75, suggesting the suitability of these cell lines for use in HTS. This study presents a novel use of genome editing for the creation of cell lines expressing α-synuclein fusion constructs entirely under native expression control. These cell lines are well suited for HTS for compounds that lower SNCA expression directly or by acting at long-range sites to the SNCA promoter and 5'-UTR.
authors: Department of Neurology, University of Utah, 175 North Medical Center Drive East, 5th Floor, Salt Lake City, Utah, 84132, United States of America.
authors: Warunee
authors: W
authors: Dansithong
authors: Department of Neurology, University of Utah, 175 North Medical Center Drive East, 5th Floor, Salt Lake City, Utah, 84132, United States of America.
authors: Sharan
authors: S
authors: Paul
authors: Department of Neurology, University of Utah, 175 North Medical Center Drive East, 5th Floor, Salt Lake City, Utah, 84132, United States of America.
authors: Daniel R
authors: DR
authors: Scoles
authors: Department of Neurology, University of Utah, 175 North Medical Center Drive East, 5th Floor, Salt Lake City, Utah, 84132, United States of America.
authors: Stefan M
authors: SM
authors: Pulst
authors: Department of Neurology, University of Utah, 175 North Medical Center Drive East, 5th Floor, Salt Lake City, Utah, 84132, United States of America.
authors: Duong P
authors: DP
authors: Huynh
mesh_terms: D002460:Cell Line; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008954:Models, Biological; D010300:Parkinson Disease / Q000188:drug therapy / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects*; D016335:Zinc Fingers; D051844:alpha-Synuclein / Q000235:genetics* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; C497604:SNCA protein, human; D054852:Small Molecule Libraries; D051844:alpha-Synuclein; D003851:Deoxyribonucleases