Retinal metabolic and immunological changes in the TSC1 photoreceptor knockout mouse model of AMD

Abstract

Purpose : AMD affects as many as 11 million adults in the United States alone, which will only increase as the population ages. Several mouse models of AMD exist, however none fully recapitulate the human disease. A recent model deleting the mTOR regulatory protein TSC1 from both rod and cone photoreceptors displays early and advanced features of AMD. We investigated the metabolic and immunological phenotype of this model to better understand the molecular mechanisms underlying AMD.

Methods : Mice harboring a floxed Tsc1 allele were crossed to animals carrying Cre under the control of either the Rho or both the Rho and Opn1mw promoters to delete TSC1 in rods (cKO) or both rods and cones (dcKO), respectively. Animals were aged up to 12 months. Serial OCT and fundus photography was performed from 2 months to 12 months of age. ERG and fluorescein angiography were performed. Neural retina and eyecups (RPE/choroid) were harvested at various timepoints for Western blotting, qRT-PCR, metabolomics, flow cytometry, oxygen consumption, pyruvate kinase (PK) activity, and histological analysis.

Results : Western blotting confirmed mTORC1 activation upon Tsc1 deletion in photoreceptors as observed by the increase in pS6K and p4EBP1 expression. OCT and fundus analysis showed characteristic outer retinal folds and hyper-reflective accumulations in the sub-retinal space with progressive ONL thinning as animals aged. Fluorescein angiography demonstrated choroidal neovascular membrane leakage[WT1] [WT2] which was confirmed via histology. LC/MS-based metabolomics showed several changes in both the cKO and dcKO animals with retinal cGMP significantly increased in both models. We also found L-2-hydroxyglutarate and N-acetylglucosamine-1-phosphate to be significantly increased in the retina and eyecups of dcKO animals. Also, a significant increase in retinal Y105 PKM2 phosphorylation was observed in both models with a corresponding decrease in PK activity. Ongoing studies are investigating the immune cell profile in the retinas and eye cups of cKO and dcKO animals using flow cytometry.

Conclusions : Deletion of TSC1 from either rods or both rods and cones causes significant biochemical and molecular changes in the retina and eye cup. Understanding which molecular and metabolic pathways are affected will allow for a better understanding of the pathological changes associated with AMD and for the development of targeted therapies.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.