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Updated: Sep 13, 2025
Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples
Published on: August 4, 2021
ENO1 Dysfunction-Mediated Glycolytic Attenuation Exacerbates Oxidative Stress-Induced Retinal Ganglion Cell Death via
Naoki Takahashi1, Hiroshi Tawarayama1,2, Yuri Chida3
1Department of Ophthalmology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Purpose:
The effects of enolase dysfunction-mediated glycolytic attenuation on oxidative stress-induced retinal ganglion cell (RGC) death were investigated.
Methods:
Retinal expression of Enolase (ENO) 1 and 2 was detected using immunohistochemistry. Exogenous genes were introduced into mouse RGCs using viral vectors, and into the rat-derived retinal progenitor cell line R28 using lipofection. The effects of enolase dysfunction were evaluated using N-methyl-D-aspartate (NMDA)-induced RGC death and H2O2-induced death assays. Cell viability and gene expression were investigated using the alamarBlue, quantitative RT-PCR, and Western blotting, respectively. Reactive oxygen species (ROS) were detected using CM-H2DCFDA dye. 2-deoxyglucose and oligomycin were used to attenuate adenosine triphosphate (ATP) production in glycolysis and oxidative phosphorylation (OXPHOS), respectively.
Results:
ENO1 and ENO2 were expressed in the RGCs. Enolase overexpression inhibited NMDA-induced mouse RGC death, whereas deficiency of ENO1 but not ENO2 enhanced cell death. Additionally, ENO1 overexpression prevented the CDKN2B-induced enhancement of RGC death. H2O2 treatment increased ENO1 expression in R28 cells and inhibited H2O2-induced cell death. ATP production was immediately enhanced in ENO1 wild-type cells treated with H2O2. 2-deoxyglucose and oligomycin prevented enhanced ATP production, but ATP levels were still higher than, or similar to, those in untreated wild-type cells; in contrast, in knockdown cells, oligomycin-mediated OXPHOS inhibition led to lower ATP production in H2O2-treatment than in untreated cells. H2O2 treatment increased ROS production in knockdown R28 cells.
Conclusions:
ENO1 dysfunction leads to glycolytic attenuation, resulting in an excessive dependence of ATP production on OXPHOS under oxidative stress, contributing to excitotoxicity-induced RGC death. Preventing glycolytic attenuation may represent a promising treatment for RGC degeneration.
Insights
Enolase 1 (ENO1) dysfunction impairs glycolysis, increasing oxidative stress and retinal ganglion cell (RGC) death. Maintaining glycolytic function offers a potential treatment for RGC degeneration.
Area of Science:
- Cell Biology
- Neuroscience
- Biochemistry
Background:
- Retinal ganglion cells (RGCs) are crucial for vision and vulnerable to oxidative stress.
- Glycolysis and oxidative phosphorylation (OXPHOS) are key cellular energy pathways.
- Enolase enzymes (ENO1, ENO2) play roles in glycolysis.
Purpose of the Study:
- To investigate the impact of enolase dysfunction on RGC death under oxidative stress.
- To explore the role of glycolytic attenuation in RGC survival.
- To assess the potential of targeting glycolysis for RGC protection.
Main Methods:
- Detected enolase expression in RGCs using immunohistochemistry.
- Manipulated enolase levels in RGCs and R28 cells via gene introduction.
- Assessed RGC death using NMDA and H2O2-induced assays.
- Measured cell viability, gene expression, ATP production, and reactive oxygen species (ROS).
Main Results:
- Enolase overexpression protected RGCs from NMDA-induced death; ENO1 deficiency exacerbated cell death.
- H2O2 treatment increased ENO1 expression and inhibited cell death in R28 cells.
- ENO1 dysfunction led to increased reliance on OXPHOS for ATP production under oxidative stress, elevating ROS.
Conclusions:
- ENO1 dysfunction causes glycolytic attenuation, making ATP production overly dependent on OXPHOS and contributing to RGC death.
- Preventing glycolytic attenuation is a promising therapeutic strategy for RGC degeneration.
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