Müller cell glutamine metabolism links photoreceptor and endothelial injury in diabetic retinopathy

We characterized the timeline of molecular dysfunction in diabetic retinopathy (DR) and diabetic retinal disease (DRD) by studying the streptozotocin (STZ)-induced mouse retina over the course of 6 months of diabetes. We performed bulk RNA-Seq on endothelial and retinal cells, separately, at 1, 3 and 6 months of diabetes and single-cell RNA-Seq (scRNA-Seq) at 3 months. Transcriptomic changes were validated by in vitro, and ex vivo assays and immunohistochemistry of mouse and human tissue. Bulk RNA-Seq revealed inflammation in endothelial cells at 1 month. At 3 months, scRNA-Seq identified glutamine-driven anaplerotic dysfunction in Müller cells, confirmed by retinal culture. We posited this glutamine deficiency would impact the photoreceptors and endothelial cells. We validated this hypothesis using endothelial cells in vitro, and immunohistochemistry of disrupted photoreceptor ribbon synapses in mouse and human diabetic retinas. Additionally, glutamine deprivation increased expression of apoptotic genes in endothelial cells. At 6 months, we observed significant downregulation of angiogenic pathways and elevated profibrotic markers. Our results suggest that dysfunction of the metabolic ecosystem linking the Müller-photoreceptor-endothelial cells is central to the early stages of DRD pathogenesis, impacting photoreceptor synapses and endothelial cells, before the appearance of the classic microvascular features of DR. Overall design: Bulk RNA-seq on endothelial and retinal cells (analyzed separately) from sham and STZ-diabetic mice at 1, 3, and 6 months of diabetes, and single-cell RNA-seq at 3 months of diabetes (sham vs. STZ)