ABSTRACT
The retinal pigment epithelium (RPE) acts as a metabolic gatekeeper between photoreceptors and the choroidal vasculature to maintain retinal function. RPE dysfunction is a key feature of age-related macular degeneration (AMD), the leading cause of blindness in developed countries. Inflammation is a key pathogenic mechanism in AMD and tumor necrosis factor-alpha (TNFα) has been implicated as a pro-inflammatory cytokine involved in AMD. While mitochondrial dysfunction has been implicated in AMD pathogenesis, the interplay between inflammation and cellular metabolism remains elusive. The present study explores how the pro-inflammatory cytokine, TNFα, impacts mitochondrial morphology and metabolic function in RPE. Matured human primary RPE (H-RPE) were treated with TNFα (10 ng/ml) for up to 5 days. TNFα-induced upregulation of IL-6 secretion and inflammatory genes (IL-6, IL-8, MCP-1) was accompanied by increased oxidative phosphorylation (OXPHOS) and reduced glycolysis, leading to an increase in cellular adenosine triphosphate (ATP) content. Transmission electron microscopy (TEM) revealed defects in mitochondrial morphology with engorged mitochondria and loss of cristae integrity following TNFα treatment. Pre-treatment with the anti-inflammatory drug, 80 µM dimethyl fumarate (DMFu), blocked TNFα-induced inflammatory activation of RPE (IL-6, IL-8, MCP-1, CFH, CFB, C3) and normalized their bioenergetic profile to control levels by regulating PFKFB3 and PKM2 gene expression. Furthermore, DMFu prevented TNFα-induced mitochondrial dysfunction and morphological anomalies. Thus, our results indicate that DMFu serves as a novel therapeutic avenue for combating inflammatory activation and metabolic dysfunction of RPE in AMD.
ABSTRACT
PGC-1α, a key orchestrator of mitochondrial metabolism, plays a crucial role in governing the energetically demanding needs of retinal pigment epithelial cells (RPE). We previously showed that silencing PGC-1α induced RPE to undergo an epithelial-mesenchymal-transition (EMT). Here, we show that induction of EMT in RPE using transforming growth factor-beta 2 (TGFß2) suppressed PGC-1α expression. Correspondingly, TGFß2 induced defects in mitochondrial network integrity with increased sphericity and fragmentation. TGFß2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFß2 reduced mitochondrial OXPHOS levels consistent with reduced expression of NDUFB5. The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic reserve, glucose uptake and gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA). Treatment with ZLN005, a selective small molecule activator of PGC-1α, blocked TGFß2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFß2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. ZLN005 effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment of subretinal fibrosis.
