April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Enhanced Alu RNA stability due to iron-mediated DICER1 impairment causes NLRP3 inflammasome priming
Author Affiliations & Notes
  • Charles B Wright
    Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY
  • Younghee Kim
    Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY
  • Tetsuhiro Yasuma
    Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY
  • Shengjian Li
    Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY
  • Benjamin J Fowler
    Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY
  • Mark Ellsworth Kleinman
    Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY
  • Jayakrishna Ambati
    Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY
  • Bradley D Gelfand
    Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY
  • Footnotes
    Commercial Relationships Charles Wright, None; Younghee Kim, None; Tetsuhiro Yasuma, None; Shengjian Li, None; Benjamin Fowler, None; Mark Kleinman, None; Jayakrishna Ambati, None; Bradley Gelfand, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 2187. doi:https://doi.org/
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      Charles B Wright, Younghee Kim, Tetsuhiro Yasuma, Shengjian Li, Benjamin J Fowler, Mark Ellsworth Kleinman, Jayakrishna Ambati, Bradley D Gelfand; Enhanced Alu RNA stability due to iron-mediated DICER1 impairment causes NLRP3 inflammasome priming. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2187. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

Geographic atrophy (GA) is an untreatable advanced form of nonexudative age-related macular degeneration that results in loss of central vision via retinal pigment epithelium (RPE) cell death. We previously demonstrated that downregulation of DICER1 in the RPE resulted in the accumulation of toxic Alu RNA and cell death via the NLRP3 inflammasome in human GA eyes. Here we explored whether iron overload can cause Alu RNA accumulation via interference with the DICER1 cofactor Poly(C)-binding protein 2 (PCBP2).

 
Methods
 

Iron overload was induced by supplementing culture media of human RPE cells and by subretinal injection in wild-type mice. Northern blotting and fluorescence in situ hybridization were used to determine whether iron overload affected abundance and localization of endogenous Alu RNA and mouse B1/B2 RNA. Time-resolved degradation of a synthetic labeled Alu RNA was used to assess Alu RNA stability upon exposure to iron. To determine whether iron overload induced inflammasome priming, qRT-PCR was used to measure NLRP3 mRNA abundance. Binding affinity of Alu RNA to PCBP2 was assessed by pulldown assay, and the effect of PCBP2 on DICER1-medieated Alu RNA processing was measured by in vitro cleavage assays. Finally, to determine whether Alu RNA stability mediated inflammasome priming, cells were subjected to siRNA-mediated knockdown of PCBP2, in the presence of an Alu RNA antisense oligonucleotide compared to non-targeting controls.

 
Results
 

Iron overload resulted in an increase in Alu RNA and B1/B2 RNA levels in human RPE cells and mouse retina, respectively, which was associated with increased stability of Alu RNA transcripts. Pulldown assays revealed iron-sensitive Alu RNA/PCBP2 binding. PCBP2 promoted dose-dependent, DICER1-dependent Alu RNA processing. Knockdown of PCBP2 expression resulted in higher Alu RNA stability and NLRP3 mRNA levels, which was prevented by Alu RNA targeted antisense oligonucleotide.

 
Conclusions
 

Alu RNA causes RPE cell death via the NLRP3 inflammasome, iron overload promotes Alu RNA accumulation. The addition of iron resulted in decreased Alu RNA binding by PCBP2, which enhances DICER1-mediated processing of Alu RNA. Alu RNA accumulation due to iron overload or PCBP2 targeting caused NLRP3 inflammasome priming. This work suggests that increased iron levels in human RPE might impair DICER1/Alu RNA metabolism in GA, thus contributing to RPE degeneration.

 
Keywords: 412 age-related macular degeneration • 701 retinal pigment epithelium  
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