Abstract
Purpose :
αA-crystallin (cryAA) is protective against metabolic stress especially when phosphorylated on Threonine 148 (T148). We previously showed that overexpressing T148-phosphorylated cryAA in Müller glial cells (MGCs) decreases their stress-induced expression of inflammatory cytokines. As MGCs are vital for many aspects of retinal homeostasis regulation, the goal of this study was to determine whether cryAA affects MGC expression of trophic factors, a crucial aspect of neuron survival.
Methods :
Genes of interest were first identified from RNA-seq data of rat MGCs, with Vegfα, Fgf2, Tgfβ, and Igf2 shown to be highly expressed. Trophic factor expression was measured in WT and cryAA knock out (AKO) mice. Whole retina, laser microdissection (LMD)-isolated retinal layers, and flow cytometry-isolated MGCs were analyzed. For whole retina analysis, retinas were directly harvested into TriZol. For layer analysis, INL, ONL, and whole retina were collected by LMD from sections of fresh frozen eyes. For isolated cell analysis, retinas were first dissociated and filtered through a mesh. For characterization experiments, cells were fixed and permeabilized, then labeled with GS and PKCα antibodies to assess the dissociation efficiency. For MGC sorting, GFP+ MGCs were obtained by injecting pups with the shH10-CMV-GFP MGC-specific viral vector. MGCs were then sorted by flow cytometry and confirmed by microscopy. For all experiments, transcript expression was measured by qPCR using Taqman probes targeting trophic factors and retinal layer markers Thy-1, GS, and Rho.
Results :
While Vegfα, Fgf2, Tgfβ, and Igf2 were expressed in whole retina, only Fgf2 transcripts were differentially expressed in AKOs. Compared to whole retina, INL and ONL were characterized by enrichment in GS and Rho respectively. WT INL was enriched in Vegfα compared to other layers, while ONL was enriched in Fgf2, with significantly higher expression of Fgf2 in AKOs.
Conclusions :
Our findings confirm that Vegfα, Fgf2, Tgfβ, and Igf2 are expressed in the retina. We show that Vegfα is highly expressed in WT INL, the layer where MGC nuclei reside. Fgf2 is highly expressed in ONL, and further enriched in AKOs. Though the cell specificity of cryAA's effect on Fgf2 remains to be fully characterized, this change in expression supports an important role of cryAA in regulation of trophic support.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.