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.
Highlights
Age-related macular degeneration (AMD) is the most common cause of central vision loss in the elderly
Results aB Crystallin is secreted from human fetal retinal pigment epithelium (RPE) cells We used confluent, early passage human RPE cells (76106 cells/T75 Flask) to determine secretion. aB crystallin secreted to the medium in a 24 h period was measured by Western blot analysis (Figure 1A)
We provide evidence that polarized human RPE monolayers secrete aB crystallin preferentially to the photoreceptor facing apical side
Summary
Age-related macular degeneration (AMD) is the most common cause of central vision loss in the elderly. The retinal pigment epithelium (RPE) is regarded as a primary site of pathology in AMD [1,2]. The RPE forms a quiescent monolayer of nonproliferating cells, strategically located between the choriocapillaris/Bruch’s membrane complex and the light-sensitive photoreceptors. The interphotoreceptor matrix (IPM) is a carbohydraterich complex that occupies the extracellular compartment between the outer neural retina and the apical surface of the RPE [3]. The IPM regulates the interaction between RPE and photoreceptors by exchanging nutrients, signaling molecules, and metabolic end products [4]. Visual loss in early AMD is minimal, the RPE cells accumulate lipofuscin and are associated with the formation of extracellular deposits (drusen) in the macular region
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