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Benjamin Fowler, Younghee Kim, Yoshio Hirano, Nagaraj Kerur, Valeria Tarallo, Bradley D Gelfand, Jayakrishna Ambati; Dideoxy-nucleosides are anti-inflammatory and inhibit RPE degeneration independent of reverse transcriptase inhibition. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1735.
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© ARVO (1962-2015); The Authors (2016-present)
NLRP3 inflammasome activation induces RPE cell death in dry AMD, a devastating and currently untreatable blindness. In the dry AMD model based on Alu RNA accumulation in the RPE, cytotoxic non-coding Alu RNAs cause macular degeneration. Although the precise details of how Alu RNAs induce inflammasome activation are not fully understood, it is known that Alu RNA requires endogenous reverse transcriptase for its life cycle. Therefore, we hypothesized that nucleoside reverse transcriptase inhibitors (NRTIs) block inflammasome activation and RPE cell death in cell culture and animal models of dry AMD.
NLRP3 inflammasome activation was monitored by western blot for Caspase-1 and phospho-IRAK4 (primary human RPE cells), or western blot/ELISA for Interleukin (IL)-1 beta, IL-18, and Caspase-1 (mouse BMDM/THP-1 monocytes/Raji TK+/- cells). AZT phosphorylation was measured by LC-MS/MS. Inflammasome priming was assessed by real time-quantitative PCR. ATP release was assessed using a luciferase-based detection kit. P2X7 receptor function was assessed by uptake of YO-PRO-1 dye in HEK293 cells with stable expression of P2X7 after incubation with bzATP. For the mouse model of dry AMD, RPE degeneration was induced by subretinal injection of a plasmid expressing Alu RNA. Oral gavage of NRTIs or controls in wild-type male C57BL6/J mice was performed daily for one week after Alu administration. RPE degeneration was assessed by fundus photography and ZO-1 staining of RPE flat mounts.
Multiple NRTIs blocked RPE cell death and inflammasome activation induced by Alu RNA. Oral gavage delivery of the NRTI stavudine, at a similar equivalent dose typically administered in humans, prevented P2X7-dependent RPE degeneration in the Alu RNA-induced mouse model of dry AMD. NRTIs blocked ATP-mediated P2X7-dependent Caspase-1 and Interleukin-1 beta/-18 secretion, and YO-PRO-1 dye uptake. AZT inhibited inflammasome and was not phosphorylated in thymidine kinase-deficient cells.
We have identified a novel anti-inflammatory action of NRTIs characterized by P2X7 and NLRP3 inflammasome inhibition, and demonstrated their in vivo ability to prevent RPE cell death in a mouse model of dry AMD. The tested NRTI compounds have been FDA-approved and widely used clinically for decades, and are therefore ideal drug repurposing candidates in an unanticipated treatment strategy for dry AMD.
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