Abstract
Purpose :
Müller cells, critical macroglial cells spanning the entire retina, establish vital connections with neuro-retinal, microglial, and endothelial cells, crucial for sustaining retinal function. However, the mechanisms facilitating communication between neuro-retinal cells and Müller cells in retinal degenerative conditions like diabetic retinopathy (DR) remain poorly understood. This study explores the novel roles of HuD and CRYAA in mediating intercellular regulation between Müller and neuro-retinal cells.
Methods :
R-28 cells were transfected with siRNA against HuD or Cryaa for 48 h, followed by incubation of rMC-1 cells in media collected from these transfected R-28 cells for 24 h. The secretome from transfected R-28 cells was analyzed using the Proteome Profiler Cytokine Array. Expression levels of RNA and proteins in rMC-1 or R-28 cells were evaluated using RT-qPCR and Western blotting, respectively. The association between HuD and a novel target mRNA was assessed using ribonucleoprotein complex-immunoprecipitation. Cell viability was measured using the MTT assay.
Results :
Our initial investigation into HuD expression in Müller glia rMC-1 cells revealed alterations in HuD and CRYAA expression levels under glycemic conditions. To demonstrate intercellular regulation between neuro-retinal cells and Müller cells, rMC-1 cells were treated with media cultured from HuD- or Cryaa-silenced R-28 cells. Subsequent incubation of rMC-1 cells with media from these silenced R-28 cells resulted in decreased cell viability. Correspondingly, down-regulation of both HuD and Cryaa mRNA and protein expressions was observed in these rMC-1 cells. Furthermore, levels of inflammatory cytokines, including IL-1β, IL-6, and TNFα, increased, while the expression of the antiangiogenic factor OPTICIN significantly decreased in rMC-1 cells. Notably, Opticin mRNA was identified as a novel binding target of HuD.
Conclusions :
Our study reveals an intricate interplay involving HuD, CRYAA, and the cellular environment in retinal cells. These insights hold potential for informing the development of targeted therapeutic strategies aimed at preserving retinal integrity and ameliorating the impact of diabetic retinopathy and similar conditions on vision and ocular health.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.