Journal
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
Volume 54, Issue 12, Pages 7395-7401Publisher
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.13-12500
Keywords
AMD; inflammasome; NLRP3
Categories
Funding
- National Eye Institute/National Institutes of Health (NIH) [R01EY018350, R01EY018836, R01EY020672, R01EY022238]
- Doris Duke Distinguished Clinical Scientist Award
- Burroughs Wellcome Fund Clinical Scientist Award in Translational Research
- Ellison Medical Foundation Senior Scholar in Aging Award
- Dr. E. Vernon Smith and Eloise C. Smith Macular Degeneration Endowed Chair
- Carl Reeves Foundation
- Research to Prevent Blindness departmental unrestricted grant
- Beckman Initiative for Macular Research
- Programme for Advanced Medical Education
- Fundacao Calouste Gulbenkian
- Fundacao Champalimaud
- Ministerio da Saude and Fundacao para a Ciencia e Tecnologia, Portugal
- American Heart Association
- National Center for Research Resources
- National Center for Advancing Translational Sciences, NIH [UL1TR000117]
- Arnold and Mabel Beckman Foundation
- NIH [T32HL091812, UL1RR033173]
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PURPOSE. Accumulation of Alu RNA transcripts due to DICER1 deficiency in the retinal pigmented epithelium (RPE) promotes geographic atrophy. Recently we showed that Alu RNA activated the NLRP3 inflammasome, leading to RPE cell death via interleukin-18 (IL-18)-mediated MyD88 signaling. However, the molecular basis for NLRP3 inflammasome activation by Alu RNA is not well understood. We sought to decipher the key signaling events triggered by Alu RNA that lead to priming and activation of the NLRP3 inflammasome and, ultimately, to RPE degeneration by investigating the roles of the purinoreceptor P2X7, the transcription factor NF-kappa B, and the Toll-like receptors (TLRs) in these processes. METHODS. Human and mouse RPE cells were transfected with a plasmid encoding an Alu element (pAlu) or an in vitro-transcribed Alu RNA. Inflammasome priming was assessed by measuring NLRP3 and IL18 mRNA levels by real-time quantitative PCR. Using immunoblotting, we assessed NF-kappa B activation by monitoring phosphorylation of its p65 subunit, and inflammasome activation by monitoring caspase-1 cleavage into its active form. RPE degeneration was induced in mice by subretinal transfection of pAlu or Alu RNA. The NF-kappa B inhibitor BAY 11-7082, the P2X7 receptor antagonist A-740003, and the NLRP3 inflammasome inhibitor glyburide were delivered by intravitreous injections. We studied wild-type (WT) C57Bl/6J, P2rx7(-/-), Nfkb1(-/-), and Tlr23479(-/-) mice. RPE degeneration was assessed by fundus photography and zonula occludens-1 (ZO-1) staining of mouse RPE. RESULTS. Alu RNA-induced NF-kappa B activation, independent of TLR-1, -2, -3, -4, -6, -7, and -9 signaling, was required for priming the NLRP3 inflammasome. Nfkb1(-/-) and P2rx7(-/-) mice and WT mice treated with the pharmacological inhibitors of NF-kappa B, P2X7, or NLRP3, were protected against Alu RNA-induced RPE degeneration. CONCLUSIONS. NF-kappa B and P2X7 are critical signaling intermediates in Alu RNA-induced inflammasome priming and RPE degeneration. These molecules are novel targets for rational drug development for geographic atrophy.
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