Intercellular trafficking of Alu RNAs causes geographic atrophy expansion in AMD

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Abstract

Purpose

Age-related macular degeneration (AMD) accounts for the bulk of blindness in the elderly in Western societies. The hallmark of advanced atrophic AMD, geographic atrophy (GA), is loss of retinal pigment epithelium (RPE) cells that expands centrifugally over time. We previously identified decreased abundance of the enzyme DICER1 and accumulation of its substrate Alu RNA in RPE cells as a cause of GA (Kaneko et al. Nature, 2011). We hypothesized that disease progression is due to transfer of cytotoxic Alu RNA from dying RPE to neighboring healthy cells by means of bleb/microvesicle RNA trafficking.

Methods

Human RPE or 661W cells were transfected with DICER AS oligonuclotide. Blebs/exosomes were collected from conditioned media by differential centrifugation. RNA content of the isolated fractions was quantified by qPCR. For live cell imaging, FITC-Alu RNA transfected hRPE were stained with Fluorescent Cellvue®. Subretinally injected mouse eyes were stained (anti-ZO1), flatmounted and imaged with confocal microscopy.

Results

Blebs and microvesicles isolated from DICER1-deficient RPE cells show increased abundance of Alu RNA. Additionally, exposing healthy RPE or photoreceptors to conditioned medium from Alu-enriched cells leads to cell death. Live cell imaging identified the presence of FITC-Alu RNA in Alu-naïve cells co-cultured with cells transfected with the fluorescently tagged RNA. Further, we demonstrate that subretinally injected vesicles induce RPE degeneration in mice.

Conclusion

These data provide evidence for “neighborhood poisoning” as a mechanistic basis for progression of atrophic AMD. Targeting intercellular spread of Alu RNA holds therapeutic promise for inhibiting expansion of this devastating disease.

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