Müller glia-mediated protection of retinal ganglion cells from ethambutol-induced neurotoxicity through brain-derived neurotrophic factor

Ethambutol (EMB)-associated optic neuropathy is the most common toxic optic neuropathy and poses a significant visual threat worldwide. This study aimed to investigate the neuroprotective roles of Mu¨ller glia, the most abundant macroglia in the retina, and their associated growth factors in supporting retinal ganglion cells (RGCs) under EMB-induced toxicity. Induced RGC-like cells (iRGCs), differentiated from human induced pluripotent stem cells, and Müller glial cell line MIO-M1 were used as cellular models. RNA sequencing revealed that EMB significantly disrupted neurotrophin and BDNF-associated pathways in iRGCs. Co-culture with MIO-M1 cells markedly enhanced the survival of iRGCs during EMB exposure. mRNA of BDNF and nerve growth factor (NGF) were elevated in MIO-M1 cells, while only BDNF improved the survival and morphological integrity of iRGCs under EMB treatment. Administration of BDNF also improved the survival of RGC in mouse retinal explants. The pro-survival effect of BDNF was suppressed by GNF5837, a pan-TRK inhibitor. Among the three downstream effectors of BDNF signaling, AKT was most prominently activated by BDNF in EMB-treated iRGCs, compared to ERK and PLC?1. Furthermore, the protective effect of BDNF was blocked by the AKT inhibitor MK-2206 but not by the ERK inhibitor GDC-0094. Müller glia confer neuroprotection to RGCs under EMB-induced stress, primarily via BDNF signaling. AKT functions as the key downstream effector mediating this protective response. These findings suggest that the BDNF-AKT signaling may serve as a promising therapeutic target for EMB-associated optic neuropathy. Overall design: The response of week 4 iRGCs to EMB was investigated by using RNA sequencing.

乙胺丁醇（Ethambutol, EMB）相关性视神经病变是最常见的中毒性视神经病变，在全球范围内构成严重的视觉威胁。本研究旨在探讨视网膜内丰度最高的大胶质细胞——米勒胶质细胞（Müller glia）及其相关生长因子在乙胺丁醇诱导的毒性下对视网膜神经节细胞（retinal ganglion cells, RGCs）的神经保护作用。本研究采用人诱导多能干细胞（human induced pluripotent stem cells）分化得到的诱导型RGC样细胞（induced RGC-like cells, iRGCs）以及米勒胶质细胞系MIO-M1作为细胞模型。RNA测序（RNA sequencing）结果显示，乙胺丁醇可显著干扰iRGCs内的神经营养因子（neurotrophin）及脑源性神经营养因子（Brain-derived neurotrophic factor, BDNF）相关通路。与MIO-M1细胞共培养可显著提升乙胺丁醇暴露下iRGCs的存活率。MIO-M1细胞中BDNF与神经生长因子（nerve growth factor, NGF）的mRNA水平升高，且仅BDNF可改善乙胺丁醇处理后iRGCs的存活能力与形态完整性。向小鼠视网膜外植体（mouse retinal explants）中添加BDNF同样可提升RGC的存活率。BDNF的促存活效应可被泛TRK抑制剂（pan-TRK inhibitor）GNF5837抑制。在BDNF信号通路的三个下游效应因子（downstream effectors）中，相较于细胞外调节蛋白激酶（Extracellular regulated protein kinases, ERK）与磷脂酶Cγ1（PLCγ1），AKT在乙胺丁醇处理的iRGCs中被BDNF激活的程度最为显著。此外，BDNF的保护效应可被AKT抑制剂MK-2206阻断，但不受ERK抑制剂GDC-0094的影响。米勒胶质细胞可在乙胺丁醇诱导的应激下为RGCs提供神经保护，该作用主要通过BDNF信号通路介导。AKT作为关键的下游效应因子，介导了这一保护性应答。上述研究结果表明，BDNF-AKT信号通路有望成为乙胺丁醇相关性视神经病变的潜在治疗靶点。整体实验设计：通过RNA测序探究第4周诱导型RGC样细胞对乙胺丁醇的应答反应。