Abstract
Purpose :
Impaired glycolysis is a recent finding in age-related macular degeneration (AMD) pathogenesis. We use a genetically engineered mouse model to explore the role of mechanistic target of rapamycin (mTOR) complexes (mTORC1 and mTORC2). This study seeks to elucidate the impact of heightened mTOR levels on molecular pathways, like glycolysis, offering potential insights for targeting pathogenic mechanisms in AMD.
Methods :
RPE-specific mLST8 (mammalian lethal with SEC13 protein 8) overexpression was accomplished by integrating the mLST8 coding sequence following a T2A sequence within the last exon and 3' untranslated region (3'UTR) of the mouse Best1 gene. mLST8 overexpression was confirmed through western blotting (WB) using RPE and retina markers. mTORC1 and mTORC2 activation in mLST8 KI RPE cells was verified via phospho-protein arrays. The model underwent comprehensive characterization, including scotopic full-field electroretinography (ERG), fundus photography, hematoxylin-eosin (H&E) staining, transmission electron microscopy (TEM), WB and immunohistochemistry (IHC). Whole RPE transcriptome sequencing, proteomics, and metabolomics were conducted to analyse genetic and proteomic changes.
Results :
Mice overexpressing mLST8 displayed disrupted RPE (H&E) and reduced ERG activity at 9 and 10 months of age. Whole RPE metabolomics revealed dysregulation in the glycolytic pathway, with upregulated ATP synthase, elevated OxyHbA and increased glucose intake compared to controls. Dysregulated neutrophil degranulation proteins (decreased PGRN, increased FABP5) indicated hyperactivated immune cell infiltration. IHC revealed decreased rhodopsin and elevated Iba1, while WB showed increased LDHA suggesting elevated glycolysis, and upregulation of SOD2 and CAT, indicating oxidative stress.
Conclusions :
Elevated glycolysis in aged mLST8 KI mice induces lactate production, RPE barrier dysfunction, and oxidative stress. Metabolomic and proteomic analyses reveal substantial alterations in glycolysis, oxidative stress, and immune activation pathways, confirmed by IHC and WB. This unique mouse model, with heightened mTOR complex signalling impacting diverse pathways, is a valuable tool for investigating AMD pathogenic mechanisms. These findings offer promise for future research and therapeutic interventions in AMD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.