Ishikawa Makoto, Takaseki Sanae, Yoshitomi Takeshi, Covey Douglas F, Zorumski Charles F, Izumi Yukitoshi
Department of Ophthalmology, Akita University Graduate School of Medicine, Akita, Japan.
Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA.
Autophagy. 2021 Mar;17(3):743-760. doi: 10.1080/15548627.2020.1731270. Epub 2020 Feb 27.
In an rat glaucoma model using dissected retinas, the neurosteroid allopregnanolone (AlloP) protects retinal ganglion cells (RGCs) via GABR/GABA receptors. To determine the involvement of macroautophagy/autophagy in neuroprotection by AlloP, we examined the effects of autophagy activators, rapamycin and torin 2, and autophagy inhibitors, bafilomycin A and SAR405, on retinal retinal morphology and expression of MAP1 LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) and SQSTM1 (sequestosome 1). Administration of rapamycin or torin 2 exerted partial histological neuroprotection, while combined administration of AlloP with bafilomycin A or SAR405 induced severe degeneration in a hyperbaric condition. Electron microscopic analyses showed that the addition of AlloP significantly increased autophagosomes and degenerative autophagic vacuoles in the retinal nerve fiber layer. Immunoblotting showed that the addition of AlloP or autophagic activators increased the lipidated form of LC3B (LC3B-II) and suppressed SQSTM1. Moreover, bafilomycin A increased LC3B-II and SQSTM1 protein levels in the presence of AlloP without changes in corresponding mRNAs compared to AlloP-treated retinas in a hyperbaric condition. These data indicate that AlloP likely induces a protective form of autophagy in this model. In an rat model of glaucoma, we also observed neuroprotective effects of AlloP. Injection of polystyrene microbeads into the anterior chamber increased intraocular pressure about 3-fold and induced RGC apoptosis. A single intravitreal injection of AlloP or autophagy activators prevented apoptosis and protected RGCs with autophagy activation. We conclude that AlloP may serve as a potential therapeutic agent for the treatment of glaucoma via diverse mechanisms. 2HBCD: 2-Hydroxypropyl)-β-cyclodextrin; 3-MA: 3-methyladenine; AlloP: allopregnanolone; AP: autophagosome; AVd: degradative autophagic vacuoles; GCL: ganglion cell layer; INL: inner nuclear layer; IOP: intraocular pressure; IPL: inner plexiform layer; LC3B-I: cytosolic form of LC3B; LCB-II: lipidated form of LC3B; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mPTP: mitochondrial permeability transition pore; NDS: neuronal damage score; NFL: nerve fiber layer; OH: ocular hypertension; ON: optic nerve; ONL: outer nuclear layer; OPL: outer plexiform layer; p-STR: scotopic threshold response; RGC: retinal ganglion cells; RT-PCR: real-time reverse transcription polymerase chain reaction; SQSTM1: sequestosome 1; TUNEL: TdT-mediated dUTP Nick End Labeling.
在使用解剖视网膜的大鼠青光眼模型中，神经甾体别孕烯醇酮（AlloP）通过GABR/GABA受体保护视网膜神经节细胞（RGCs）。为了确定巨自噬/自噬在AlloP神经保护中的作用，我们研究了自噬激活剂雷帕霉素和托林2以及自噬抑制剂巴弗洛霉素A和SAR405对视网膜形态以及微管相关蛋白1轻链3β（MAP1 LC3B/LC3B）和聚集体蛋白1（SQSTM1）表达的影响。雷帕霉素或托林2给药可发挥部分组织学神经保护作用，而在高压条件下，AlloP与巴弗洛霉素A或SAR405联合给药会导致严重退变。电子显微镜分析表明，添加AlloP可显著增加视网膜神经纤维层中的自噬体和退化性自噬泡。免疫印迹显示，添加AlloP或自噬激活剂可增加LC3B的脂化形式（LC3B-II）并抑制SQSTM1。此外，与高压条件下AlloP处理的视网膜相比，在存在AlloP的情况下，巴弗洛霉素A增加了LC3B-II和SQSTM1蛋白水平，而相应mRNA无变化。这些数据表明，在该模型中AlloP可能诱导一种保护性自噬形式。在大鼠青光眼模型中，我们还观察到了AlloP的神经保护作用。向前房注射聚苯乙烯微珠可使眼压升高约3倍并诱导RGC凋亡。单次玻璃体内注射AlloP或自噬激活剂可防止凋亡并通过自噬激活保护RGCs。我们得出结论，AlloP可能通过多种机制作为治疗青光眼的潜在治疗剂。2HBCD：2-羟丙基）-β-环糊精；3-MA：3-甲基腺嘌呤；AlloP：别孕烯醇酮；AP：自噬体；AVd：退化性自噬泡；GCL：神经节细胞层；INL：内核层；IOP：眼压；IPL：内网状层；LC3B-I：LC3B的胞质形式；LCB-II：LC3B的脂化形式；MAP1LC3B/LC3B：微管相关蛋白1轻链3β；mPTP：线粒体通透性转换孔；NDS：神经元损伤评分；NFL：神经纤维层；OH：高眼压；ON：视神经；ONL：外核层；OPL：外网状层；p-STR：暗视阈值反应；RGC：视网膜神经节细胞；RT-PCR：实时逆转录聚合酶链反应；SQSTM1：聚集体蛋白1；TUNEL：TdT介导的dUTP缺口末端标记
