Abstract
Purpose :
The blood-retinal barrier (BRB) protects the neural retina and maintains retinal homeostasis by mediating movement of molecules from the blood to the inner retina. We previously found that inhibition of retinoic acid (RA) signaling results in BRB disruption in zebrafish at larval and adult stages. Further, RNAseq analysis indicated that disrupted RA signaling affects expression of genes associated with mTOR signaling in the retina. The purpose of this study was to investigate how RA signaling inhibition affects mTOR signaling in the retina and how these two pathways may interact to regulate the BRB.
Methods :
Zebrafish larvae and adults were treated with varying concentrations of DEAB and BMS493, pharmacological inhibitors of RA signaling. BRB integrity was assessed in vivo using the l-fabp:DBP-EGFP transgenic zebrafish model, which expresses GFP-tagged vitamin D-binding protein in the blood plasma. In this model, BRB breakdown can be assessed using a fluorescence microscope to detect GFP signal outside the vasculature. To assess the effects of disrupted RA signaling on mTOR signaling in the retina and RPE, we compared expression of mTOR signaling-associated markers pS6K and p4EBP1 by western blot and immunofluorescence in RA-inhibitor-treated fish vs. untreated control sibling fish. To determine whether inhibition of mTOR signaling can prevent RA-inhibitor-induced BRB breakdown, lfabp:DBP-EGFP transgenic zebrafish were co-treated with DEAB or BMS493 and mTOR inhibitor rapamycin. Following treatment, BRB integrity was assessed by fluorescence microscopy.
Results :
Pharmacological inhibition of RA signaling resulted in disruption of the BRB in zebrafish larvae and adults, which was rescued by co-treatment with all-trans retinoic acid. Co-treatment with rapamycin significantly reduced BRB breakdown in DEAB and BMS493-treated zebrafish larvae. Expression of mTOR signaling markers pS6K and p4EBP1 was increased in retinas and RPE of DEAB-treated adult zebrafish. Immunofluorescence experiments indicated altered localization of these markers in retinas of RA-inhibitor-treated zebrafish larvae and adults.
Conclusions :
The mTOR signaling pathway is affected in the retina and RPE of RA-inhibitor-treated fish and inhibition of mTOR signaling prevents RA-inhibitor-mediated BRB breakdown. Thus, RA signaling may maintain BRB integrity via crosstalk with the mTOR pathway.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.