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Shinichi Fukuda, Tetsuhiro Yasuma, Benjamin J Fowler, Bradley D Gelfand, Nagaraj Kerur, Reo Yasuma, Brian Werner, Jayakrishna Ambati; Endogenous complementary DNA in age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5262.
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© ARVO (1962-2015); The Authors (2016-present)
The central dogma of molecular biology states that genetic information flows sequentially from DNA to RNA to proteins. The retrograde flow of genetic information via reverse transcription (RT) in the form of retroviruses is one of the exceptions to this rule. RT also occurs endogenously in retrotransposons. In geographic atrophy (GA), an untreatable late stage of age-related macular degeneration (AMD), DICER1 deficiency leads to the accumulation of retrotransposon-derived Alu RNA, a potential substrate for endogenous RT. Nucleoside reverse transcriptase inhibitors (NRTIs) are protective for Alu RNA induced RPE degeneration. The purpose of this study was to determine the presence and biological function of endogenous complementary DNA (cDNA) derived from Alu RNA via RT in GA.
Alu cDNA was detected by in situ hybridization and southern blotting. Alu cDNA or Alu RNA was subretinally transfected into wild-type or mice lacking innate immune signaling constituents NLRP3 or P2X7. RPE degeneration was assessed by fundus photography and ZO-1 staining. Human and mouse RPE cells were transfected with synthetic Alu RNA or Alu cDNA. Acitvation of the innate immune effector Caspase-1 was monitored by western blotting. A retrospective cohort study was performed on an insurance claims database and analyzed to determine the incident development of AMD in HIV or Hepatitis B diagnosed patients on the basis of their NRTI usage (n=7,548) compare to non-NRTI usage (n=21,267). Logistic regression analysis was performed to control for age, sex, race, BMI class, tobacco use, and Charlson Comorbidity Index.
We identified endogenous cDNA derived from Alu RNA via RT in human cells. RT of Alu RNA into cDNA occurred after heat stress, DICER1 depletion or Alu RNA transfection, and could be inhibited by lamivudine (3TC), indicating that this was mediated by endogenous RT activity. Surprisingly, Alu cDNA was observed in the macular but not peripheral RPE of human eyes with GA. Transfection of Alu cDNA caused RPE toxicity through P2X7 and NLRP3 inflammasome in mouse models of GA. NRTI usage was associated with reduced incident development of dry AMD (odds ratio = 0.13, 95% C.I. (0.12,0.15), p<0.0001).
These findings reveal RT-dependent endogenous Alu RNA-derived cDNA in human GA patients. Our finding also support the therapeutic activity of NRTIs for GA, and provide a novel mechanism of action for these drugs in dry AMD.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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