Analysis of metabolomic, proteomic and mitochondrial dysfunction in rd10 retinas

Abstract

Purpose : Mitochondrial dysfunction and metabolomic and proteomic profiles were compared in wild type (WT) and rd10 mice to identify early-stage events in cellular dysfunction in a model of autosomal recessive retinitis pigmentosa.

Methods : Mice were raised under normal cyclic light. A Seahorse XFe96 Flux Analyzer was used to measure the oxygen consumption rate (mitochondrial respiration) and extracellular acidification (glycolysis) in WT and rd10 retinas from P14 to P18. Untargeted LCMS metabolomics at P15 was performed using flash-frozen retinas that were analyzed by Ultra-High Performance Liquid Chromatography (UHPLC) followed by high resolution mass spectrometry (HRMS). Metabolite peaks were matched to compounds in an in-house library and pathway analysis was performed. Proteomics was performed on crude mitochondrial fractions at P15 and P18 using High Performance Liquid Chromatography (HPLC) followed by protein identification and quantification by mass spectrometry.

Results : Maximal respiration was significantly reduced in rd10 mice at P16. Both basal and maximal respiration were significantly reduced at P16 and P18 indicating progressive loss of mitochondrial function. At P18, basal glycolytic activity was also significantly reduced. At P15, both metabolomics and proteomics revealed significant differences between WT and rd10mice. Eleven pathways were significantly enriched based on analysis of the metabolites. These pathways include several linked to mitochondrial function, including Pantothenate and Coenzyme A biosynthesis, b-alanine metabolism and Purine metabolism. The Primary Bile Acid Synthesis pathway was also enriched consistent with a critical role for the amino acid taurine in the regulation of oxidative stress, cholesterol metabolism and apoptosis. Proteomic analysis identified 49 proteins with significant fold-changes at P15 and 35 proteins with significant fold-changes at P18. Among the differences detected were proteins involved in mitochondrial transport, electron transport, fission, apoptosis and glycolysis.

Conclusions : Cellular pathways altered in the rd10 mouse were identified prior to significant degeneration. These pathways are involved in mitochondrial function and other cellular functions such as DNA synthesis and cholesterol metabolism. Additional analyses such as western blot analysis, immunohistochemistry and electron microscopy will be performed to validate these affected pathways.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.