Single-cell transcriptome analysis of the Akimba mouse retina reveals cell-type-specific insights into the pathobiology of diabetic retinopathy

Abstract

Single-cell RNA sequencing characterizes molecular pathways underlying inflammatory, metabolic, oxidative stress-mediated changes in Akimba model of diabetic retinopathy and identifies distinct functional subtypes of inflammatory & macroglial cells @Oxurion @LambrechtsDlab 2 ABSTRACT AIM: Diabetic retinopathy is a common complication of diabetes and a leading cause of visual impairment and blindness. Despite recent advances, our understanding of its pathophysiology remains incomplete. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by systematically mapping the transcriptional changes that occur in the different cellular compartments of the degenerating diabetic mouse retina. METHODS: Single-cell RNA sequencing was performed on retinal tissue from 12-week-old wild-type and Akimba (Ins2 Akita xVEGF +/-) mice, which are known to replicate features of clinical diabetic retinopathy. This resulted into transcriptome data for 9474 retinal cells, which could be annotated to 8 distinct retinal cell types. Using STRING analysis, we studied differentially expressed gene networks in neuronal, glial and immune cell compartments to create a comprehensive view on the pathological changes that occur in the Akimba retina. Using subclustering analysis, we further characterized macroglial and inflammatory cell subpopulations. Prominent findings were confirmed on protein level using immunohistochemistry, Western blotting and ELISA. RESULTS: At 12 weeks, the Akimba retina was found to display degeneration of rod photoreceptors and presence of inflammatory cells, identified by subclustering analysis as monocyte, macrophage and microglial populations. Analysis of differential expressed genes in the rod, cone, bipolar cell and macroglial compartments indicated changes in cell metabolism and ribosomal gene expression, gliosis, activation of immune system pathways and redox and metal ion dyshomeostasis. Experiments on protein level supported a metabolic shift from glycolysis to oxidative phosphorylation (GAPDH), activation of microglia/macrophages (Isolectin-B4), metal ion and oxidative stress response (metallothionein and heme oxygenase-1) and reactive macroglia (GFAP and S100) in the Akimba retina, as compared to wild-type mice. Our single-cell approach also indicates macroglial subpopulations with distinct fibrotic, inflammatory and gliotic profiles. CONCLUSION: Our study identifies molecular pathways underlying inflammatory, metabolic and oxidative stress-mediated changes in the Akimba mouse model of diabetic retinopathy and distinguishes distinct functional subtypes of inflammatory and macroglial cells.

Characterizing the Retinal Phenotype of the Thy1-h[A30P]α-syn Mouse Model of Parkinson’s Disease

“…On each section, analysis was done over a distance of 300 μm at four locations per section. For α-syn, TH and GFAP, the immunopositive area was measured in the inner retina (from the retinal NFL until the INL included), while for AQP4 both the outer retina (from OPL to ONL) and inner retina were measured and for VGLUT1 and Homer1, only the IPL was included ( Van Hove et al, 2020 ). For cell counting, both on wholemounts and sections, Fiji “Cell Counter” plugin was used.…”

Microglia and Inflammatory Responses in Diabetic Retinopathy

“…The Akimba mouse was generated by crossing the non-diabetic Kimba mouse model of proliferative retinopathy with diabetic Ins2 Akita mouse (107). Single cell RNA sequencing of Akimba mouse retinas has uncovered microglia as an immune cell cluster involved in the pathogenesis of DR (108). Although the mouse models of pharmacologically induced diabetes have contributed to our understanding of the morphological changes and the secretome of activated microglia cells, they fail to illustrate the neuronal and vascular changes associated with the development of DR in humans.…”

Immune Cell Landscape of Patients With Diabetic Macular Edema by Single-Cell RNA Analysis

“…Increasing evidence indicates that cytokines such as interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), IL-6, and IL-8 were significantly upregulated in vitreous and serum of DR patients, resulting in a persistent chronic inflammation state in the retina ( Ben-Mahmud et al, 2004 ; Demircan et al, 2006 ; Boss et al, 2017 ; Feng et al, 2018 ; Khaloo et al, 2020 ). Innate immune cells, especially monocytes, are reported to play a pivotal role in promoting the pathogenesis of DR ( Van Hove et al, 2020 ; Wan et al, 2020 ). In peripheral blood of diabetic patients with microvascular or macrovascular complications, CD45 + CD14 + classical monocytes were increased, but CD16 + nonclassical monocytes were decreased, compared with patients without complications ( Min et al, 2012 ).…”