Kamuvudine-9 Protects Retinal Structure and Function in a Novel Model of Experimental Rhegmatogenous Retinal Detachment.

Purpose: Rhegmatogenous retinal detachment (RRD) is a vision-threatening event that benefits from surgical intervention. While awaiting surgical reattachment, irreversible hypoxic and inflammatory damage to the retina often occurs. An interim therapy protecting photoreceptors could improve functional outcomes. We sought to determine whether Kamuvudine-9 (K-9), a derivative of nucleoside reverse transcriptase inhibitors (NRTIs) that inhibits inflammasome activation, and the NRTIs lamivudine (3TC) and azidothymidine (AZT) could protect the retina following RRD. Methods: RRD was induced in mice via subretinal injection (SRI) of 1% carboxymethylcellulose (CMC). To simulate outcomes following the clinical management of RRD, we determined the optimal conditions by which SRI of CMC induced spontaneous retinal reattachment (SRR) occurs over 10 days (RRD/SRR). K-9, 3TC, or AZT was administered via intraperitoneal injection. Inflammasome activation pathways were monitored by abundance of cleaved caspase-1, IL-18, and cleaved caspase-8, and photoreceptor death was assessed by TUNEL staining. Retinal function was assessed by full-field scotopic electroretinography. Results: RRD induced retinal inflammasome activation and photoreceptor death in mice. Systemic administration of K-9, 3TC, or AZT inhibited retinal inflammasome activation and photoreceptor death. In the RRD/SRR model, K-9 protected retinal electrical function during the time of RRD and induced an improvement following retinal reattachment. Conclusions: K-9 and NRTIs exhibit anti-inflammatory and neuroprotective activities in experimental RRD. Given its capacity to protect photoreceptor function during the period of RRD and enhance retinal function following reattachment, K-9 shows promise as a retinal neuroprotectant and warrants study in RRD. Further, this novel RRD/SRR model may facilitate experimental evaluation of functional outcomes relevant to RRD.

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity.

Long interspersed nuclear element-1 (L1)­mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA­induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA­induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Cytoplasmic synthesis of endogenous Alu complementary DNA via reverse transcription and implications in age-related macular degeneration.

Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1-reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493-0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

Voluntary Exercise Suppresses Choroidal Neovascularization in Mice.

Purpose: To determine the effect of voluntary exercise on choroidal neovascularization (CNV) in mice. Methods: Age-matched wild-type C57BL/6J mice were housed in cages equipped with or without running wheels. After four weeks of voluntary running or sedentariness, mice were subjected to laser injury to induce CNV. After surgical recovery, mice were placed back in cages with or without exercise wheels for seven days. CNV lesion volumes were measured by confocal microscopy. The effect of wheel running only in the seven days after injury was also evaluated. Macrophage abundance and cytokine expression were quantified. Results: In the first study, exercise-trained mice exhibited a 45% reduction in CNV volume compared to sedentary mice. In the replication study, a 32% reduction in CNV volume in exercise-trained mice was observed (P = 0.029). Combining these two studies, voluntary exercise was found to reduce CNV by 41% (P = 0.0005). Exercise-trained male and female mice had similar CNV volumes (P = 0.99). The daily running distance did not correlate with CNV lesion size. Exercise only after the laser injury without a preconditioning period did not reduce CNV size (P = 0.41). CNV lesions of exercise-trained mice also exhibited significantly lower F4/80+ macrophage staining and Vegfa and Ccl2 mRNA expression. Conclusions: These findings provide the first experimental evidence that voluntary exercise improves CNV outcomes. These studies indicate that exercise before laser treatment is required to improve CNV outcomes.

Chronic Dicer1 deficiency promotes atrophic and neovascular outer retinal pathologies in mice.

Degeneration of the retinal pigmented epithelium (RPE) and aberrant blood vessel growth in the eye are advanced-stage processes in blinding diseases such as age-related macular degeneration (AMD), which affect hundreds of millions of people worldwide. Loss of the RNase DICER1, an essential factor in micro-RNA biogenesis, is implicated in RPE atrophy. However, the functional implications of DICER1 loss in choroidal and retinal neovascularization are unknown. Here, we report that two independent hypomorphic mouse strains, as well as a separate model of postnatal RPE-specific DICER1 ablation, all presented with spontaneous RPE degeneration and choroidal and retinal neovascularization. DICER1 hypomorphic mice lacking critical inflammasome components or the innate immune adaptor MyD88 developed less severe RPE atrophy and pathological neovascularization. DICER1 abundance was also reduced in retinas of the JR5558 mouse model of spontaneous choroidal neovascularization. Finally, adenoassociated vector-mediated gene delivery of a truncated DICER1 variant (OptiDicer) reduced spontaneous choroidal neovascularization in JR5558 mice. Collectively, these findings significantly expand the repertoire of DICER1 in preserving retinal homeostasis by preventing both RPE degeneration and pathological neovascularization.

RF/6A Chorioretinal Cells Do Not Display Key Endothelial Phenotypes.

Purpose: The misuse of inauthentic cell lines is widely recognized as a major threat to the integrity of biomedical science. Whereas the majority of efforts to address this have focused on DNA profiling, we sought to anatomically, transcriptionally, and functionally authenticate the RF/6A chorioretinal cell line, which is widely used as an endothelial cell line to model retinal and choroidal angiogenesis. Methods: Multiple vials of RF/6A cells obtained from different commercial distributors were studied to validate their genetic, transcriptomic, anatomic, and functional fidelity to bona fide endothelial cells. Results: Transcriptomic profiles of RF/6A cells obtained either de novo or from a public data repository did not correspond to endothelial gene expression signatures. Expression of established endothelial markers were very low or undetectable in RF/6A compared to primary human endothelial cells. Importantly, RF/6A cells also did not display functional characteristics of endothelial cells such as uptake of acetylated LDL, expression of E-selectin in response to TNF-α exposure, alignment in the direction of shear stress, and AKT and ERK1/2 phosphorylation following VEGFA stimulation. Conclusions: Multiple independent sources of RF/6A do not exhibit key endothelial cell phenotypes. Therefore, these cells appear unsuitable as surrogates for choroidal or retinal endothelial cells. Further, cell line authentication methods should extend beyond genomic profiling to include anatomic, transcriptional, and functional assessments.