Huang, Hu1; Jeevan, Varun1; Rajooldezfuly, Ali1; Mays, Logan1; Elahi, Fazle1
1Department of Ophthalmology, The University of Missouri, School of Medicine
Purpose: Retinal neurodegeneration associated with neuroinflammation occurs in various retinal diseases, including age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy (DR), and glaucoma. This study aims to investigate the role and mechanism of interferon regulatory factor 1 (IRF1) in retinal neurodegeneration. Although IRF1 is a well-characterized inflammatory regulator, whether and how it is involved in the development of retinal neurodegeneration has not been explored.
Methods: scRNA-sequencing and QRT-PCR analyses determine gene expression in retinal cells. Immunofluorescence staining and immunoblotting examine protein expression. The animal models of retinal neurodegeneration model were generated using the chemicals sodium iodate and NMDA. Spectral domain-optical coherence tomography (SD-OCT) and fundus imaging with Micron OCT system (Phoenix) examine retinal structures of Irf1 gene knockout mice vs. C57 wild-type control mice. ERG determines the retinal visual function. PI stain and TUNEL Assay quantifies apoptotic cell death. SeaHorse XF96 Mito Stress test examines the mitochondrial metabolic function of retinal explants. Statistical analysis was performed using GraphPad Prism and Excel.
Results: IRF1 is primarily expressed by glial cells in the retina, such as Müller cells and microglia. IRF1 acts as a stress sensor, and its expression levels are upregulated in retinal tissues and glial cells under various stress conditions, such as IFNγ/TNFα, LPS, H2O2, and NMDA. Genetic deletion of the IRF1 gene in mice protects against retinal degeneration (e.g., photoreceptors) induced by the oxidizing agent sodium iodate. Additionally, IRF1 gene knockdown via siRNA reduces neuroinflammation related to microglial activation in the retina. IRF1 siRNA prevents NMDA-induced retinal ganglion cell (RGC) loss and visual dysfunction. Mechanistically, IRF1 mutation or knockout prevents microglia from shifting from a homeostatic state to an activated one both in vivo and in vitro. IRF1 mutation affects mitochondrial metabolic function and gene expression. Conditioned culture medium (CM) from IRF1-mutant microglia reduces retinal cell death compared to CM from IRF1 wild-type microglial cells.
Conclusions: IRF1 contributes to retinal neurodegeneration by regulating microglia-mediated neuroinflammation and mitochondrial metabolic function.
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