Paradoxical Effect of TNFα on Mitochondrial Function and Metabolic Activity in the Retinal Pigment Epithelium (RPE)

Abstract

The retinal pigment epithelium (RPE) acts as a metabolic gatekeeper between photoreceptors and the choroidal vasculature to maintain healthy retinal function. RPE dysfunction is a key feature of age‐related macular degeneration (AMD), the leading cause of blindness in developed countries. Tumor necrosis factor‐alpha (TNFα), a potent pro‐inflammatory cytokine, has been implicated in the pathogenesis of AMD. Growing evidence supports metabolic dysfunction as another key mechanism driving AMD. To date, there is no literature on the metabolic effects of TNFα on RPE and thus, this study sheds light on the impact of TNFα on mitochondrial morphology and metabolic function in RPE. Matured ARPE‐19 (human RPE cell line) were treated with TNFα (10 ng/ml) for up to 72h. TNFα induced ARPE‐19 to elongate into spindle‐shaped cells, reminiscent of epithelial‐mesenchymal transition (EMT). However, qPCR showed that TNFα reduced the expression of EMT genes (α‐SMA, Col1A1, fibronectin, MMP2, CTGF) indicating that the elongated cells were not mesenchymal in nature. To explore the effects of TNFα on metabolism, two major energy‐generating pathways, oxidative phosphorylation (OXPHOS) and glycolysis were assessed by Seahorse high‐resolution respirometry and qPCR. The Seahorse Mito Stress Test revealed an increase in basal respiration but reduced spare respiratory capacity following TNFα treatment. TNFα increased glycolytic reserve and capacity. Despite the increased OXPHOS and glycolysis, qPCR showed a significant reduction in expression of OXPHOS (ATP5O, COX4I1, COX5B, NDUFB5) and glycolysis (G6P, PFKL, PFKFB3, PGK1, LDHA) genes with TNFα treatment. ARPE‐19 were transfected with mitochondria‐tagged GFP and imaged using confocal microscopy. Parameters on mitochondrial morphology were extracted from the confocal images using automated image segmentation and revealed that TNFα disrupted mitochondrial network integrity. This was supported by qPCR data showing significantly reduced expression of genes regulating mitochondrial function (PGC‐1α, TFAM, POLG, MFN1, MFN2, FIS1, OPA1). TNFα significantly elevated SOD2, a mitochondrial antioxidant enzyme, but not SOD1. Taken together, we find that TNFα robustly disrupts mitochondrial function and morphology in RPE, although shifting the bioenergetic profile in a paradoxical manner, i.e. TNFα raised the levels of basal respiration and glycolysis despite the suppression of genes regulating OXPHOS and glycolysis. These findings highlight the potential of targeting metabolic pathways in RPE as a promising therapeutic avenue for AMD. Further research is required to elucidate the mechanisms underlying these intriguing TNFα‐driven metabolic changes.Support or Funding InformationGrimshaw‐Gudewicz Charitable Foundation; National Council of Scientific and Technological Development—Brazil (PDE 210474/2014‐9); The Iraty Award; NEI Core Grant P30EYE003790; Department of Defense, Spinal Vision Research Program under Award no. VR180132