Dansithong Warunee, Paul Sharan, Scoles Daniel R, Pulst Stefan M, Huynh Duong P
Department of Neurology, University of Utah, 175 North Medical Center Drive East, 5th Floor, Salt Lake City, Utah, 84132, United States of America.
PLoS One. 2015 Aug 28;10(8):e0136930. doi: 10.1371/journal.pone.0136930. eCollection 2015.
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.
帕金森病（PD）是一种由黑质多巴胺能神经元丧失引起的进行性神经退行性疾病。PD的标志是出现称为路易小体和路易神经突的神经元蛋白聚集体，其中α-突触核蛋白是主要成分。家族性PD很少见，与SNCA基因的错义突变或基因拷贝数增加导致SNCA过表达有关。这表明降低SNCA表达可能对PD具有治疗作用。支持这一假设的是，在细胞系和啮齿动物PD模型中，SNCA的减少具有神经保护作用。我们开发了表达与报告基因荧光素酶（luc）或绿色荧光蛋白（GFP）融合的SNCA的新型细胞系，目的是能够对可降低SNCA表达的小分子进行高通量化合物筛选（HTS）。由于SNCA的表达可能受远上游元件（包括位于转录位点上游8852 bp处的NACP-REP1）调控，我们采用锌指核酸酶（ZFN）基因组编辑技术将报告基因框内插入SNCA基因下游，以保留天然SNCA表达控制。这确保了完全保留控制SNCA表达的已知和未知的上游和下游遗传元件。用组蛋白去乙酰化酶抑制剂丙戊酸（VPA）处理细胞导致SNCA-luc和SNCA-GFP表达显著增加，这支持了使用我们的细胞系来鉴定改变复杂表达控制模式的小分子。用荧光素酶抑制剂或SNCA siRNA处理表达SNCA-luc的细胞，Z'值≥0.75，表明这些细胞系适用于HTS。本研究提出了基因组编辑的一种新用途，即创建完全在天然表达控制下表达α-突触核蛋白融合构建体的细胞系。这些细胞系非常适合用于高通量筛选直接降低SNCA表达或通过作用于SNCA启动子和5'-UTR的远距离位点来降低SNCA表达的化合物。
