Distinct protective cell type-specific responses induced by chronic hypoxia in a mouse model of retinal hypovascularization revealed by single-cell RNAseq and metabolic profiling

Abstract

Purpose : The inner retina in the Norrin knockout (Ndp-KO) mouse is relatively preserved despite being severely hypovascularized and hypoxic, suggesting the presence of protective cellular responses. Moreover, the major angiogenic factor Vegfa was shown to be produced predominantly by Muller glia in the Ndp-KO retina, raising the possibility of distinct hypoxic responses in different cell types. We sought to characterize these different cellular responses using single-cell RNAseq (sc-RNAseq) and metabolic profiling.

Methods : Single cells from dissociated whole retinas of 8-week old male Ndp-KO (N=2) and wild-type (WT)(N=2) mice were profiled by droplet-based sc-RNAseq (10X Genomics). Cell type assignment and differential gene expression analysis were performed using Monocle2. Differentially expressed genes were validated using (i) in-situ hybridization and/or immunofluorescence and (ii) Muller glia-specific Ribotag RNAseq for Muller glia-enriched genes. For metabolic profiling, intact Ndp-KO and WT retinas were fixed in mixed aldehydes and resin-embedded for computational molecular phenotyping (CMP). CMP utilizes a panel of metabolite-specific antibodies to detect and evaluate small molecule concentrations with single-cell resolution in the intact retina.

Results : A total of 21,578 cells profiled by sc-RNAseq included all major retinal cell types as indicated by known markers. Differential gene expression analysis confirmed known cell type-specific transcriptional changes e.g. increased Vegfa in Muller glia, decreased Cldn5 in endothelial cells. Inner retinal cells displayed significant upregulation of genes that inhibit mitochondrial respiration, promote anaerobic glycolysis, and mitigate against oxido-reductive stress. There were distinct glial versus neuronal gene expression changes. Metabolic profiling revealed widespread changes in metabolites (e.g. aspartate, glutamate, glutathione) in the Ndp-KO inner retina that were likely due to protective anaplerotic adaptations.

Conclusions : sc-RNAseq and metabolic profiling in a mouse model of retinal hypovascularization revealed distinct neuronal-glial responses and general protective cellular responses induced by chronic retinal hypoxia. Strategies to augment or complement such protective responses could offer novel treatments for retinal diseases involving chronic hypoxia.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.