Division of Neurology, Department of Medicine, University of Hong Kong, Hong Kong SAR, China.
Institute of Metabolism and Systems Research, University of Birmingham, Birmingham B15 2TT, UK.
Int J Mol Sci. 2022 Mar 26;23(7):3646. doi: 10.3390/ijms23073646.
Synaptogyrin-3 (SYNGR3) is a synaptic vesicular membrane protein. Amongst four homologues (SYNGR1 to 4), SYNGR1 and 3 are especially abundant in the brain. SYNGR3 interacts with the dopamine transporter (DAT) to facilitate dopamine (DA) uptake and synaptic DA turnover in dopaminergic transmission. Perturbed SYNGR3 expression is observed in Parkinson's disease (PD). The regulatory elements which affect SYNGR3 expression are unknown. Nuclear-receptor-related-1 protein (NURR1) can regulate dopaminergic neuronal differentiation and maintenance via binding to NGFI-B response elements (NBRE). We explored whether NURR1 can regulate SYNGR3 expression using an in silico analysis of the 5'-flanking region of the human SYNGR3 gene, reporter gene activity and an electrophoretic mobility shift assay (EMSA) of potential -acting sites. In silico analysis of two genomic DNA segments (1870 bp 5'-flanking region and 1870 + 159 bp of first exon) revealed one X Core Promoter Element 1 (XCPE1), two SP1, and three potential non-canonical NBRE response elements (ncNBRE) but no CAAT or TATA box. The longer segment exhibited gene promoter activity in luciferase reporter assays. Site-directed mutagenesis of XCPE1 decreased promoter activity in human neuroblastoma SH-SY5Y (↓43.2%) and human embryonic kidney HEK293 cells (↓39.7%). EMSA demonstrated NURR1 binding to these three ncNBRE. Site-directed mutagenesis of these ncNBRE reduced promoter activity by 11-17% in SH-SY5Y (neuronal) but not in HEK293 (non-neuronal) cells. C-DIM12 (Nurr1 activator) increased SYNGR3 protein expression in SH-SY5Y cells and its promoter activity using a real-time luciferase assay. As perturbed vesicular function is a feature of major neurodegenerative diseases, inducing expression by NURR1 activators may be a potential therapeutic target to attenuate synaptic dysfunction in PD.
突触融合蛋白 3（SYNGR3）是一种突触囊泡膜蛋白。在四个同源物（SYNGR1 到 4）中，SYNGR1 和 3 在大脑中尤为丰富。SYNGR3 与多巴胺转运体（DAT）相互作用，促进多巴胺（DA）摄取和多巴胺能传递中的突触 DA 周转率。帕金森病（PD）中观察到 SYNGR3 表达失调。影响 SYNGR3 表达的调节元件尚不清楚。核受体相关蛋白 1（NURR1）可以通过与 NGFI-B 反应元件（NBRE）结合来调节多巴胺能神经元分化和维持。我们通过对人类 SYNGR3 基因 5'-侧翼区的计算机分析、报告基因活性和潜在作用位点的电泳迁移率变动分析（EMSA），探讨了 NURR1 是否可以调节 SYNGR3 的表达。计算机分析两个基因组 DNA 片段（1870 bp 5'-侧翼区和 1870 + 159 bp 的第一个外显子）揭示了一个 X 核心启动子元件 1（XCPE1）、两个 SP1 和三个潜在的非经典 NBRE 反应元件（ncNBRE），但没有 CAAT 或 TATA 盒。较长的片段在荧光素酶报告基因检测中显示出基因启动子活性。XCPE1 的定点突变降低了人神经母细胞瘤 SH-SY5Y（↓43.2%）和人胚肾 HEK293 细胞中的启动子活性（↓39.7%）。EMSA 表明 NURR1 与这三个 ncNBRE 结合。这些 ncNBRE 的定点突变使 SH-SY5Y（神经元）中的启动子活性降低了 11-17%，但在 HEK293（非神经元）细胞中没有降低。C-DIM12（Nurr1 激活剂）通过实时荧光素酶测定增加了 SH-SY5Y 细胞中 SYNGR3 蛋白的表达及其启动子活性。由于囊泡功能紊乱是主要神经退行性疾病的特征，因此 NURR1 激活剂诱导 表达可能是减轻 PD 中突触功能障碍的潜在治疗靶点。
