DRP1表現變異和帕金森症：啟動子多型性和轉錄調控研究

Abstract

在帕金森症(PD)中，粒線體的代謝被破壞，而抑制電子傳遞鏈複合物I的神經毒素在人類和動物模型中會產生類似於帕金森氏症的症狀。遺傳性帕金森氏症的連鎖分析也顯示粒線體功能障礙與帕金森症的發病相關。粒線體在細胞中以動態管狀網絡存在，它通過裂變和融合不斷改變形狀，來適應局部的環境改變。Dynamin-related protein 1 (DRP1)是控制粒線體分裂的關鍵蛋白，它會經過轉譯後修飾和移位至粒線體外膜，來調節粒線體分裂。先前60個帕金森症患者週邊血單核細胞的研究顯示，DRP1表現水平顯著較53個正常對照組上升。DRP1啟動子的遺傳變異可能調節DRP1表達。通過直接定序，發現在DRP1啟動子區域中有-556 G/A (rs565216693)、-318 -/AAT、-315 A/T (rs201231372)和-311 A/T (rs11423175)等核苷酸多型性。接著藉PCR-RFLP技術，對533個帕金森症患者和544個正常對照組的病例對照研究，確認了-556 G/A多型性與帕金森症相關。-311 A/T與連鎖的-318 -/AAT及-315 A/T多型性與帕金森症相關性，DRP1啟動子直接定序檢測了各63個帕金森症患者和正常人的樣品，但因樣品數不夠大，差異未達顯著性。經由電腦模擬搜尋上述多型性鄰近區域序列，發現-556位點的變異會影響轉錄因子TFAP2A (Transcription factor AP-2 alpha)、CEBPB (CCAAT enhancer binding protein beta)的結合，而-318、-311位點的變異會影響轉錄因子FOXA1 (Forkhead box A1)的結合。TFAP2A、CEBPB、FOXA1在人的神經組織中皆有表現。-556 G/A可能因影響TFAP2A的結合而影響DRP1表現，-318 -/AAT、-311 A/T則可能因移動FOXA1結合位置改變染色質重建(Chromatin remodeling)，來影響DRP1表現。過度表現FOXA1或CEBPB於SHSY-5Y細胞，可增強內生性DRP1表現。共轉染對照組DRP1-GFP報導質體和FOXA1/CEBPB到SHSY-5Y細胞，GFP的轉錄活性增加。本研究表明了-556 G/A基因型的人患上帕金森症的風險較大，且在SHSY-5Y細胞中，轉錄因子FOXA1或CEBPB會増加DRP1的轉錄活性。

In Parkinson’s disease (PD), mitochondrial metabolism is disrupted and neurotoxins that inhibit complex I of the electron transport chain produce PD-like symptoms in humans and animal models. Linkage analysis of hereditary PD has also suggested that mitochondrial dysfunction contributes to the pathogenesis of PD. Mitochondria in cells exist as a dynamic tubular network shaped continuously by fission and fusion events in response to local environmental changes. Dynamin-related protein 1 (DRP1), a key protein controlling fission, undergoes posttranslational modifications and translocation to the mitochondrial outer membrane to regulate mitochondrial fission. Previously DRP1 expression level was significantly increased in peripheral blood mononuclear cells from 60 PD patients compared with 53 normal controls. Genetic variations in the DRP1 promoter may modulate DRP1 expression. Through directing sequencing, polymorphisms -556 G/A (rs565216693), -318 -/AAT, -315 A/T (rs201231372), and -311 A/T (rs11423175) were found in the DRP1 promoter region. A case-control study (n=533 for PD and n=544 for controls) confirmed the association between -556 G/A and PD. The potential associations of -311 A/T and the linked -318 -/AAT, -315 A/T and PD were tested by direct sequencing. Due to small sample size (n=63 for both PD and normal controls), the difference was not significant. In silico searches of the regions flanking these variations revealed TFAP2A (transcription factor AP-2 alpha) and CEBPB (CCAAT enhancer binding protein beta) binding in -556 site and FOXA1 (forkhead box A1) binding in -318 and -311 sites. TFAP2A, CEBPB and FOXA1 are expressed in human nervous tissues. -556 G/A may sway TFAP2A binding and -318 -/AAT, -311 A/T may influence chromatin remodeling by shifting FOXA1 binding position to affect DRP1 expression. Overexpression of FOXA1 and CEBPB up-regulated DRP1 expression on SH-SY5Y cells. Co-transfection control DRP1-GFP reporter plasmid with FOXA1 or CEBPB could increase transcription activity of GFP. This study indicate that people with -556 G/A genotype have a higher risk of developing Parkinson’s disease. In SH-SY5Y cells FOXA1 or CEBPB could increase transcription activity of DRP1.