Extracellular vesicles and their miRNA cargo in retinal health and degeneration: mediators of homeostasis, and vehicles for targeted gene therapy

Abstract

Photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases, however the molecular mechanisms underlying these biological processes are largely unknown. Exosomes are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. Exosomes encapsulate and transfer nucleic acids, including microRNA (miRNA), to recipient cells which in disease may result in dysfunctional immune responses and a loss of homeostatic regulation. In this work we investigated the role of retinal exosomes in both the healthy and degenerating retina. Isolated exosomes from normal retinas were characterized using dynamic light scattering, transmission electron microscopy and western blotting, and quantified across 5 days of photo-oxidative damage-induced degeneration using nanotracking analysis. Small RNAseq was used to characterize the miRNA cargo of retinal exosomes isolated from healthy and damaged retinas. Finally, the effect of exosome inhibition on cell-to-cell miRNA transfer and immune modulation was conducted using systemic daily administration of exosome inhibitor GW4869 and in situ hybridization of exosome-abundant miRNA, miR-124-3p. Electroretinography and immunohistochemistry was performed to assess functional and morphological changes to the retina as a result of exosome depletion.

Inhibition of microRNA-155 Protects Retinal Function Through Attenuation of Inflammation in Retinal Degeneration

“…S-mEVs were isolated from papain-digested retinas by sequential ultracentrifugation following the MISEV2018 guidelines for studies of extracellular vesicles [55] and adapted for retina [56]. Briefly, retinas were mechanically and enzymatically dissociated in a digestion solution of 2.5 mg/ml papain (Worthington Biochemicals), 200 U DNAse I (Roche Applied Science, Penzberg, Germany) 5 μg/ml catalase (Sigma-Aldrich), 10 μg/ml gentamycin (Sigma-Aldrich) and 5 μg/ml superoxide dismutase (Worthington Biochemicals) in Hank's Balanced Salt Solution (HBSS; Gibco; Thermo Fisher Scientific).…”

MicroRNA-223 Regulates Retinal Function and Inflammation in the Healthy and Degenerating Retina

“…For RNA extraction from retinal extracellular vesicles, each sample contained two pooled retinas (from one mouse). Isolation of small-medium extracellular vesicles (s-mEVs) from papain-digested retinas was performed according to previously published methodology (Wooff et al, 2020a) and in accordance with the MISEV2018 guidelines (Théry et al, 2018). Characterization of the properties of isolated retinal s-mEVs (which include exosomes) has been published, detailing transmission electron microscopy, size distribution analysis, and western blot analysis of these s-mEVs (Wooff et al, 2020a).…”