Single-nuclei RNA sequencing of the diabetic mouse retina reveals dysregulation of neurexins in early diabetic retinal disease

Abstract

Purpose : While increasing evidence from patients and animal models suggests that early diabetic retinal neurodegeneration may precede the retinal vascular abnormalities that currently define diabetic retinopathy (DR), the underlying pathophysiology and relevant cell types affected in early diabetic retinal disease (DRD) remain poorly understood. In order to identify molecular pathways dysregulated in specific retinal neuronal cell types in early DRD, we performed single-nuclei RNA sequencing of the retina in diabetic mice at two time points following disease onset.

Methods : To induce diabetes, male C57BL/6J mice were treated with five daily intraperitoneal injections of 60mg/kg streptozotocin (STZ) or vehicle. Animals with random blood glucose >250 mg/dL were considered diabetic and the early retinal phenotype was confirmed at 6 weeks after treatment using optical coherence tomography and dark-adapted electroretinogram. At either 6 or 36 weeks following STZ treatment, retinas from control and diabetic mice (n=8 mice per group per time point) were dissected and separated into two batches for methanol fixation, homogenization, and nuclei preparation. Nuclei were subjected to sequential combinatorial labeling using HiFseq and library sequencing was performed using the Illumina NovaSeq6000 S4 platform. Selected results were validated by multiplexed in situ hybridization using RNAscope.

Results : After preprocessing, we selected nuclei having >500 UMIs (unique molecular identifiers) and >250 genes, then normalized the count matrix in the Seurat package. In total, 3200 and 650 nuclei were identified at the 6- and 36-week time points, respectively. Unsupervised cell-type annotation analyses yielded 12 clusters, representing five retinal neuronal cell types as well as Muller glia. Components of the neurexin-neuroligin reactome were highly represented among the differentially-expressed genes. We confirmed decreased expression of these transcripts in retinal ganglion cells of STZ-treated diabetic mice compared with controls.

Conclusions : Single-nuclei RNA sequencing of the retina in the STZ-induced diabetes mouse model reveals dysregulation of neurexins in retinal neurons, suggesting a novel role for these synaptic proteins in the pathogenesis of early DRD.

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