Molecular and functional characterization of extracellular vesicles derived from Muller glia cells

Abstract

Purpose : Müller glia cells retain progenitor-like characteristics in the adult human eye and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Recently it has been demonstrated that intracellular communication is possible via the secretion of nano-sized membrane-bound extracellular vesicles (EV), which contain bioactive molecules capable of inducing phenotypic change when internalised by recipient cells. We conducted molecular and functional characterisation of EV populations secreted by Müller glia in culture, to evaluate their potential role in the neuroprotective signalling attributed to these cells.

Methods : EV were characterised by nanoparticle tracking and electron microscopy. Proteomic analysis was conducted by western blot, and RNA contents were profiled and evaluated by Illumina small-RNA sequencing, followed by bioinformatic analysis of differential enrichment and prediction of miRNA targets and function. Retinal organoids formed by pluripotent stem cells were incubated with and without Müller sEV, and expression of miRNA targets assessed by qPCR

Results : Protein analysis of small EVs (sEV) and large EV (lEV) indicated markers characteristic of endocytic membranes, from which EV originate. RNA profiling of sEV and lEV isolated from Müller glia culture supernatants revealed enrichment in several small-RNA species. Of particular interest was the detection of numerous microRNAs (miRNA) present in sEVs, which are known to directly regulate gene expression through interference with mRNA transcripts. Sequencing of miRNAs recovered from sEV indicated preferential enrichment of species that target genes involved in cell proliferation and survival, including PTEN, the master inhibitor of the AKT/mTOR pathway

Conclusions : Culture of Müller derived sEV with human retinal organoids resulted in downregulation of the expression of predicted miRNA targets, including PTEN, suggesting a putative mechanism for the neural pro-survival effect ascribed to Müller glia. Fluorescent labelling of sEV confirmed direct internalisation into primary RGC in culture. Since EV uptake has been shown to be cell type specific, it is possible that surface molecules expressed by Müller glia-derived sEV may facilitate their internalisation by retinal neurons, making them appealing for use as vehicles for the delivery of neuroprotective molecules to the retina

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.