Department of Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland.
School of Medicine and Medical Science, University College Dublin, Dublin, Ireland.
Invest Ophthalmol Vis Sci. 2020 Nov 2;61(13):4. doi: 10.1167/iovs.61.13.4.
The lamina cribrosa (LC) is a key site of damage in glaucomatous optic neuropathy. We previously found that glaucoma LC cells have an increased profibrotic gene expression, with mitochondrial dysfunction in the form of decreased mitochondrial membrane potential. Altered cell bioenergetics have recently been reported in organ fibrosis and in cancer. In this study, we carried out a systematic mitochondrial bioenergetic assessment and measured markers of alternative sources of cellular energy in normal and glaucoma LC cells.
LC cells from three glaucoma donors and three age-matched normal controls were assessed using VICTOR X4 Perkin Elmer (Waltham, MA) plate reader with different phosphorescent and luminescent probes. adenosine triphosphate levels, oxygen consumption rate, and extracellular acidification were measured and normalized to total protein content. RNA and protein expression levels of MCT1, MCT4, MTFHD2, and GLS2 were quantified using real-time RT-PCR and Western blotting.
Glaucoma LC cells contain significantly less adenosine triphosphate (P < .05) when supplied with either glucose or galactose. They also showed significantly diminished oxygen consumption in both basal and maximal respiration with more lactic acid contribution in ECA. Both mRNA and protein expression levels of MCT1, MCT4, MTHFD2, and GLS2 were significantly increased in glaucoma LC cells.
We demonstrate evidence of metabolic reprogramming (The Warburg effect) in glaucoma LC cells. Expression of markers of glycolysis, glutamine, and one carbon metabolism are elevated in glaucoma cells at both the mRNA and protein levels. A better understanding of bioenergetics in glaucoma may help in the development of new therapeutics.
筛板（LC）是青光眼视神经病变损害的关键部位。我们之前发现，青光眼 LC 细胞的促纤维化基因表达增加，同时伴有线粒体功能障碍，表现为线粒体膜电位降低。最近有研究报道，器官纤维化和癌症中存在细胞生物能量的改变。在这项研究中，我们对正常和青光眼 LC 细胞进行了系统的线粒体生物能量评估，并测量了细胞能量替代来源的标志物。
使用 Perkin Elmer（马萨诸塞州沃尔瑟姆）VICTOR X4 平板读数仪，用不同的荧光和发光探针评估来自 3 位青光眼供体和 3 位年龄匹配的正常对照的 LC 细胞。测量并将三磷酸腺苷（adenosine triphosphate，ATP）水平、耗氧量和细胞外酸化率与总蛋白含量进行归一化。使用实时 RT-PCR 和 Western blot 定量测定 MCT1、MCT4、MTHFD2 和 GLS2 的 RNA 和蛋白质表达水平。
与葡萄糖或半乳糖相比，青光眼 LC 细胞的三磷酸腺苷（adenosine triphosphate，ATP）含量明显较低（P <.05）。它们在基础和最大呼吸时的耗氧量也明显减少，而 ECA 中乳酸的贡献更多。在青光眼 LC 细胞中，MCT1、MCT4、MTHFD2 和 GLS2 的 mRNA 和蛋白质表达水平均显著增加。
我们证明了青光眼 LC 细胞存在代谢重编程（瓦博格效应）的证据。在青光眼细胞中，糖酵解、谷氨酰胺和一碳代谢的标志物的表达在 mRNA 和蛋白质水平上均升高。更好地了解青光眼的生物能量可能有助于开发新的治疗方法。
