Role of extracellular vesicles in retinitis pigmentosa

Resum

Retinitis pigmentosa (RP), an inherited degenerative retinal disease, is associated with progressive photoreceptor degeneration, which leads eventually to blindness. To date, the precise mechanisms leading to cell death remain unknown and no adequate treatment is available. Poly ADP ribose polymerase (PARP) over activity is involved in photoreceptor degeneration and, in mice models, its pharmacological inhibition protects the retina. Additionally, retinal cell survival depends of adequate reception and processing of the information and appropriate cellular communication. Initially, the Extracellular Vesicles (EVs) were recognized as a mechanism for discharging useless cellular components. Growing evidence has elucidated their roles in cell–cell communication by carrying nucleic acids, proteins, and lipids that can, in turn, regulate behavior of target cells. Nevertheless, the role of EVs in blinding diseases, such a RP, is far from being understood. The present project aims to investigate the EVs implication in retinal degeneration, including their release and cargo, their influence in neighboring cells, and the relationship with PARP activity in the retina. Rd1 and Rd10 mice - two well-known animal model for RP, which hold a mutation in the beta subunit of the phosphodiesterase 6 gene (PDE6) – helped advanced the understanding of the retinal degeneration. Section from rd1 and rd10 mice and organotypic retinal explants from rd10 were used to investigate cellular communication by EVs. CD9 and CD81 tetraspanins were studied to investigate EVs activity at tissue level by immunostaining. Inhibition of PARP activity was performed using Olaparib. Immunohistochemistry was carried out to evaluate PARylated proteins and immunostaining was performed to determinate rhodopsin (rho) expression, Müller glia cell activity, and cyclic guanosine monophosphate (cGMP) levels after olaparib treatment. Also, inmunofluorescence was used to study EVs and their colocalization with cilia in rd10 retinae after PARP inhibition. EVs were isolated using ultrafiltration and size exclusion chromatography or a commercial isolation kit, depending on downstream applications. Nanosight analysis, electron microscope, Fluorescence-Activated Cell Sorting (FACs), dot blot, and proteomics were used to characterize the EVs. Moreover, rd10 retinas were treated with EV from wt and vice-versa. Inmunostaining assays against CD9, CD81, rho, and IBA-1 (microglia marker) were carried out after EVs treatments. TUNEL assay was used to evaluate cell viability, thickness, and row photoreceptor number in the outer nuclear layer (ONL) after Olaparib and EVs treatments. EVs release changes with the age in wt mice and also under retinal degeneration in rd1 and rd10 in different retinal layers. PARP inhibition by Olaparib rescues photoreceptors and also modify the EVs release and cargo in rd10 mice. The EVs release was increased in rd10 retinae and the protein cargo was modified under retinal degeneration. Moreover, EVs from rd10 retinae had the ability to damage wt retinas and something similar was produced after treated rd10 retinae with EVs from wt. This data strongly suggests the implication of EVs in retina development and degeneration.