ABCA1介导ANXA1膜/核竞争性转运在视网膜神经节细胞凋亡中的机制研究

Glaucoma is a common cause of irreversible blindness, but the mechanism has not been fully clarified yet. Annexin A1 (ANXA1) has been identified as a bifunctional protein on cell destiny. ANXA1 membrane mirgation protects neural cells, however, the nuclear translocation induced cell apoptosis. Our previous study has indicated that increased intraocular pressure (IOP) promotes nuclear translocation of ANXA1 in retinal ganglion cells (RGCs), thus leading to RGC loss. ABCA1 is a membrane transport protein and has been reported to colocalize with ANXA1 in pituitary tumor cell. Our preliminary data shown that increased IOP downregulated ABCA1 expression and membrane translocation of ANXA1 in RGCs. Therefore, we hypothesized that increased IOP reduces ABCA1 expression, leading to downregulation of membrane translocation and upregulation of nuclear translocation of ANXA1, which eventually trigger RGCs apoptosis. Based on our hypothesis, we aim to ① figure out the influences of ABCA1 downregulation induced by high IOP on membrane and nuclear translocation of ANXA1; ② clarify mechanisms by which ABCA1 induces membrane and nuclear translocation of ANXA1; ③ synthesize small peptide of ABCA1 to maintain its function of ANXA1 membrane translocation and protect high IOP induced RGC damage. Our study offers new strategies and targets to reduce RGC loss caused by glaucoma.

青光眼是一类机制尚未完全阐明的严重致盲性眼病。Annexin A1（ANXA1）蛋白核转位促凋亡、膜转位神经保护，是决定细胞命运的双刃剑。申请人发现，高眼压引起视网膜神经节细胞ANXA1核转位增多，促进凋亡。前人发现，在垂体瘤细胞中膜转运蛋白ABCA1与ANXA1存在共定位，而在预实验中发现，高眼压引起神经节细胞中ABCA1蛋白表达下降，ANXA1膜转位减少。据此推测，高眼压引起ABCA1表达减少会导致ANXA1膜转运减少、核转位增多，触发神经节细胞凋亡。在此假说之上，本课题拟利用分子生物学的研究手段，①明确高眼压引起ABCA1表达下调对ANXA1蛋白核、膜转位的影响，②阐明ABCA1影响ANXA1核、膜转运的分子机制，③制备ABCA1小分子多肽维持其膜转运ANXA1功能，探讨其对高眼压引起神经节细胞损伤的保护效应。本研究将为青光眼引起的视网膜神经节细胞凋亡机制研究提供新的思路与靶点。

神经系统中，Annexin A1（ANXA1）蛋白核转位促进细胞凋亡、膜转位促进神经保护，是决定神经细胞命运的双刃剑。在本项目研究中，我们重点关注了ABCA1蛋白介导的ANXA1核/膜竞争性转位的分子效应与机制。我们发现，缺血再灌注损伤模型可以引起视网膜神经节细胞（RGCs）中ABCA1蛋白表达下调；与此同时，给予30μM的ABCA1激动剂TO901317治疗，可以有效缓解RGCs凋亡；进一步我们发现，TO901317治疗可以促进ANXA1蛋白的膜定位，TO901317可以促进ABCA1蛋白与ANXA1蛋白结合；机制上，我们构建了ANXA1不同结构域质粒，发现ANXA1蛋白196-274氨基酸序列是与ABCA1结合的关键结构域；同时，我们构建了不同的ABCA1蛋白胞内结构域质粒，发现ABCA1蛋白903-1344氨基酸序列是与ANXA1结合的关键结构域；进而我们发现，促进ABCA1蛋白可以抑制ANXA1蛋白与核转运蛋白importinβ结合，反之，抑制了ABCA1蛋白表达可以促进ANXA1-importinβ结合；我们还意外的发现，TBK1蛋白介导的泛素化降解，可能是造成ABCA1蛋白表达减少的关键；治疗上，我们利用慢病毒载体装载了ABCA1全长结构域，利用腺相关病毒载体装载了ABCA1 903-1344氨基酸结构域，我们发现，无论是ABCA1全长结构域治疗，还是ABCA1-ANXA1结合关键结构域治疗，都可以有效改善缺血再灌注损伤引起的RGCs凋亡。该研究为阐明缺血再灌注损伤引起的RGCs凋亡，提供了新的分子机制，为进一步的分子治疗提供了新的研究策略及干预靶点。

内容获取失败，请点击重试