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

Abstract

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

Antiapoptotic Properties of α-Crystallin–Derived Peptide Chaperones and Characterization of Their Uptake Transporters in Human RPE Cells

“…3A-E). Consistent with previous work, 5 we did not observe any cellular uptake of full-length aB-crystallin over this time period (Fig. 3F).…”

“…3,4 Furthermore, aB-crystallin was shown to be upregulated with several stress stimuli and to translocate to nuclei and mitochondrial compartments of RPE cells. 3,5 Microarray and proteomic analysis and histological studies have revealed that a-crystallin accumulates in drusen. 2,6,7 Subsequent studies demonstrated the expression of aB-crystallin in RPE cells overlying drusen.…”

“…5 Custom FITClabeled a-crystallin-derived peptides were purchased commercially (NeoPeptide). Serum-starved hfRPE cells were incubated with aB-crystallin mini-chaperone (1.7 lM) for 5 to 30 minutes.…”

“…TUNEL-positive cells were counted and data were expressed as percent of total cells undergoing cell death. 5 …”

“…8 Since overexpression of full length aB-crystallin reduced susceptibility to oxidative stress-induced apoptosis in RPE cells, 9,10 we suggested that aB-crystallin may have therapeutic potential. 5 Since full length aB-crystallin has limited entry into nonstressed cells, 2,5 we considered the possibility that peptides of aB-crystallin may retain antiapoptotic activity while having enhanced cellular uptake.…”