αB Crystallin Is Apically Secreted within Exosomes by Polarized Human Retinal Pigment Epithelium and Provides Neuroprotection to Adjacent Cells

alpha B Crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether alpha B crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular alpha B crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that alpha B crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of alpha B crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced alpha B crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, alpha B crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that alpha B crystallin was localized predominantly in the apical compartment of RPE monolayers, where it colocalized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of alpha B crystallin to the basolateral side. In normal mouse retinal sections, alpha B crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous alpha B crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. alpha B Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for alpha B crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of alpha B crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for alpha B crystallin in ocular diseases.