Analysis of Adult Neural Retina Extracellular Vesicle Release, RNA Transport and Proteomic Cargo

Abstract

Citation: Mighty J, Zhou J, Benito-Martin A, et al. Analysis of adult neural retina extracellular vesicle release, RNA transport and proteomic cargo. Invest Ophthalmol Vis Sci. 2020;61(2):30. https://doi.org/10.1167/iovs.61.2.30 PURPOSE. Extracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin. Adult neural retina EV release, RNA transfer, and proteomic cargo are the focus of this study. METHODS. Adult wild-type mouse retinae were cultured and released EV diameters and concentrations quantified using Nanosight. Immunogold transmission electron microscopy (TEM) was used to image EV ultrastructure and marker protein localization. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze retinal cell transcripts present in EVs. Super-resolution microscopy was used to image fluorescent (green) RNA and (red) lipid membrane labeled EVs, released by adult retina, and internalized by isolated retinal cells. Mass spectrometry was used to characterize the proteomes of adult retina and EVs. RESULTS. Adult neural retina released EVs at a rate of 1.42 +/− 0.08 × 10 8 /mL over 5 days, with diameters ranging from 30 to 910 nm. The canonical EV markers CD63 and Tsg101 localized to retinal EVs. Adult retinal and neuronal mRNA species present in both retina and EVs included rhodopsin and the neuronal nuclei marker NeuN. Fluorescently labeled RNA in retinal cells was enclosed in EVs, transported to, and uptaken by cocultured adult retinal cells. Proteomic analysis revealed 1696 protein species detected only in retinal cells, 957 species shared between retina and EVs, and 82 detected only in EVs.CONCLUSIONS. The adult neural retina constitutively releases EVs with molecular cargo capable of intercellular transport and predicted involvement in biological processes including retinal physiology, mRNA processing, and transcription regulation within the retinal microenvironment.

Human retinal organoids release extracellular vesicles that regulate gene expression in target human retinal progenitor cells

“…This aligns with our recent findings showing that mRNA and miRNA are packed into retinal progenitor cell EVs, and the content is functional following transfer to target retinal cells [34]. Additional recent work suggests that EV transfer of genetic material occurs between differentiated adult neural retina and multipotent retinal progenitor cells [20]. In this study, early differentiating 3D retinal organoid EVs were uptaken into multipotent hRPCs.…”

“…Both, 3D retinal organoids and EVs were confirmed to contain the 6 selected miRNAs ( Figure 6A). To demonstrate cell-to-cell cargo transfer by EVs released from retinal organoids, we labeled EV RNA with the green (FITC) fluorescent dye Syto RNASelect and, EV lipid envelopes with red (TRITC) fluorescent dye PKH26, and performed live imaging on their uptake into non-labeled human retinal progenitor cells (hRPCs) [20,21]. Following 15 hours of co-incubation of unlabeled hRPCs and fluorescently labeled EVs, media was replaced with fresh media without labeled EVs to quantify uptake, retention and reduction of EV-transferred RNA in hRPCs.…”

Human retinal organoids release extracellular vesicles that regulate gene expression in target human retinal progenitor cells

“…6 A). To demonstrate cell-to-cell cargo transfer by EVs released from retinal organoids, we labeled EV RNA with the green (FITC) fluorescent dye Syto RNASelect and, EV lipid envelopes with red (TRITC) fluorescent dye PKH26, and performed live imaging on their uptake into non-labeled human retinal progenitor cells (hRPCs) 20 , 21 . Following 15 hours of co-incubation of unlabeled hRPCs and fluorescently labeled EVs, media was replaced with fresh media without labeled EVs to quantify uptake, retention and reduction of EV-transferred RNA in hRPCs.…”

Extracellular Vesicles and MicroRNA: Putative Role in Diagnosis and Treatment of Diabetic Retinopathy

“…This feature makes these vesicles very interesting candidates as biomarkers and therapeutic drug delivery systems for a variety of chronic diseases, including cancer and degenerative diseases, such as DR [59]. A recent study characterized the EVs released by adult neural retina in culture and this allowed to determine the cellular origin of different types of EVs through the analysis of their molecular cargo (RNA and proteins) [60]. The authors were able to detect EVs derived from photoreceptors which contain proteins such as rhodopsin, the photo-responsive receptor of rod cells, and cadherin related family member 1 (Cdhr1), an adhesion protein that is normally present in the outer and inner segments of photoreceptors.…”

“…They have also detected the presence of neuronal-specific nuclear protein (NeuN) in the isolated EVs, a marker for amacrine and retinal ganglion cells. Additionally, through their molecular content, they were able to conclude that the retinal-derived EVs are mainly related with processes such as phototransduction, synapse structure, RNA processing, and transcription regulation [ 60 ]. Furthermore, other studies have also demonstrated that EVs from distinct retinal cells, such as astrocytes and retinal pigment epithelial (RPE) cells present different protein profiles, which suffer alterations during pathophysiological processes [ 61 , 62 ].…”