Differential effects of TNFα on Mitochondrial Function and Metabolic Activity in the Retinal Pigment Epithelium

Abstract

Purpose : 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.

Methods : Matured ARPE-19 were treated with TNFα (10 ng/ml) for up to 72h. Glycolysis and oxidative phosphorylation (OXPHOS) were examined by high-resolution respirometry on the Seahorse XF24. Gene expression of EMT and metabolic markers were assessed by qPCR. Mitochondrial morphology was assessed by confocal imaging and ImageJ processing of MitoTracker Orange-stained ARPE-19.

Results : TNFα induced ARPE-19 to elongate into spindle-shaped cells, reminiscent of epithelial-mesenchymal transition (EMT). However, qPCR showed that TNFα reduced EMT genes expression (Col1A1: 1.2 vs 0.39, p=0.015; α-SMA: 1.01 vs 0.66 p=0.001) indicating that the elongated cells were not mesenchymal. TNFα increased basal OXPHOS levels (OCR = 2.2 vs 3.7 pmol/min/μg, p=0.042) and increased glycolytic capacity (ECAR = 0.68 vs 0.92 mpH/mol/μg, p=0.03). Paradoxically, TNFα induced a reduced expression of OXPHOS genes (COX4I1: 1.02 vs 0.16, p < 0.0001; COX5B: 1.02 vs 0.24, p<0.0001). TNFα significantly upregulated expression levels of the mitochondrial antioxidant SOD2 (1.14 vs 14.8, p<0.0001) and disrupted mitochondrial network morphology exhibiting increased sphericity and fragmentation.

Conclusions : 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. 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 the intriguing TNFα-driven metabolic changes.

This is a 2021 ARVO Annual Meeting abstract.