Cellular and molecular mechanisms of retinoic acid-mediated blood-retinal barrier regulation

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PROJECT SUMMARY The blood-retinal barrier (BRB) mediates movement of molecules from the blood to the retina, protecting the retinal neural tissue from potentially harmful molecules and maintaining retinal homeostasis. Breakdown of the BRB is associated with ocular diseases such as diabetic retinopathy and age-related macular edema. The BRB consists of both an inner barrier, formed by microvascular endothelial cells, and an outer barrier, formed by the RPE. Tight junctions between these cells are essential to barrier function. However, the molecular mechanisms regulating tight junction integrity in the BRB and the effects of BRB breakdown on retinal morphology and visual function are not fully understood. The zebrafish (Danio Rerio) is an ideal model to investigate the mechanisms of BRB breakdown and maintenance due to its rapid ex vivo visual development and the availability of a wide array of genetic tools. We have recently determined that retinoic acid (RA), a metabolite of Vitamin A, plays a critical role in the maintenance of the BRB. Disruption of RA signaling in zebrafish larvae and adults with a pan-retinoic acid receptor inhibitor (BMS493) results in BRB breakdown, disrupted expression of tight junction proteins in the retinal vasculature and RPE, and decreased visual acuity. Preliminary RNA sequencing and pathway analysis of differentially expressed genes indicates that the mTOR signaling pathway is significantly upregulated in retinas of fish treated with an inhibitor of RA signaling compared to untreated fish. Additionally, inhibition of mTOR signaling by treatment with rapamycin is sufficient to restore BRB integrity in RA-inhibitor-treated larvae. Therefore, we propose that RA maintains the BRB via crosstalk with the mTOR pathway and that RA-inhibitor-induced BRB breakdown leads to retinal damage and visual dysfunction. We will characterize the role of RA and the mTOR signaling pathway in BRB maintenance by assessing the pattern of mTOR activation in the inner and outer BRB and identifying the upstream and downstream regulators involved in BMS493- induced BRB breakdown. We will also determine which cells in the retina contribute to RA-inhibitor- induced BRB breakdown and characterize the cellular changes in the retina in response. Understanding the cellular and molecular mechanisms involved in RA-mediated BRB maintenance and the progression of retinal damage and vision loss following BRB breakdown will be critical to identify therapeutic approaches for preventing vision loss due to BRB disruption.