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Shinichi Fukuda, Benjamin J Fowler, Tetsuhiro Yasuma, Akhil Varshney, Kameshwari Ambati, Nagaraj Kerur, Bradley Gelfand, Jayakrishna Ambati; Non-integrated, reverse transcribed Alu complementary DNAs in Geographic Atrophy cause RPE toxicity. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2456. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Alu RNAs are ~300 nucleotide non-coding RNAs transcribed from repetitive retrotransposon elements, which are prevalent in the human genome. Geographic atrophy (GA) is an untreatable late stage of age-related macular degeneration (AMD). The accumulation of toxic retrotransposon Alu RNA transcripts in the retinal pigment epithelium (RPE) is involved in the pathogenesis of GA toxicity. The life cycle of Alu elements involves transcription into RNA transcripts, followed by reverse transcription into complementary DNA (cDNA) and genetic insertion processes which are mediated by endogenous LINE-1 (L1) encoded proteins. However, the fate of cDNA generated from endogenous RNA that does not become integrated in the genome is poorly understood. The purpose of this study was to determine whether L1-dependent Alu cDNA synthesis and integration are requisite intermediates of Alu RNA-mediated RPE toxicity in GA.
Free, non-integrated Alu cDNA was detected by in situ hybridization and Southern blotting. Alu RNA or a mutant Alu RNA with reduced ability to integrate into the genome was subretinally transfected into wild-type C57BL/6J mice. L1 was inhibited by siRNA. RPE degeneration was assessed by fundus photography and ZO-1 staining.
Non-genomic Alu cDNA was detected in the macular RPE of human eyes with GA. An siRNA targeting L1 prevented the formation of Alu cDNA in primary human RPE cells following Alu RNA transfection. L1 siRNA, but not control siRNA, also blocked Alu RNA-induced RPE degeneration in mice, indicating that Alu RNA toxicity was mediated by endogenous L1 reverse transcriptase activity. To determine whether insertion of Alu cDNA into the genome contributes to Alu RNA toxicity, we synthesized mutant Alu RNA. A mutant Alu RNA impaired in its genomic integration activity still underwent reverse transcription into Alu cDNA and induced RPE toxicity in mice.
Alu RNA can undergo reverse transcription into Alu cDNA that does not integrate into the genome. Non-integrated Alu cDNA induced RPE toxicity. These findings may reveal a novel mechanism of the life cycle of Alu RNAs in GA, and also provide molecular rationale for evaluating the potential therapeutic activity of nucleoside reverse transcriptase inhibitors for GA.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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