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.

PurposeTo investigate the expression of humanin (HN) in human retinal pigment epithelial (hRPE) cells and its effect on oxidative stress–induced cell death, mitochondrial bioenergetics, and senescence.MethodsHumanin localization in RPE cells and polarized RPE monolayers was assessed by confocal microscopy. Human RPE cells were treated with 150 μM tert-Butyl hydroperoxide (tBH) in the absence/presence of HN (0.5–10 μg/mL) for 24 hours. Mitochondrial respiration was measured by XF96 analyzer. Retinal pigment epithelial cell death and caspase-3 activation, mitochondrial biogenesis and senescence were analyzed by TUNEL, immunoblot analysis, mitochondrial DNA copy number, SA-β-Gal staining, and p16INK4a expression and HN levels by ELISA. Oxidative stress–induced changes in transepithelial resistance were studied in RPE monolayers with and without HN cotreatment.ResultsA prominent localization of HN was found in the cytoplasmic and mitochondrial compartments of hRPE. Humanin cotreatment inhibited tBH-induced reactive oxygen species formation and significantly restored mitochondrial bioenergetics in hRPE cells. Exogenous HN was taken up by RPE and colocalized with mitochondria. The oxidative stress–induced decrease in mitochondrial bioenergetics was prevented by HN cotreatment. Humanin treatment increased mitochondrial DNA copy number and upregulated mitochondrial transcription factor A, a key biogenesis regulator protein. Humanin protected RPE cells from oxidative stress–induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. Humanin treatment inhibited oxidant-induced senescence. Polarized RPE demonstrated elevated cellular HN and increased resistance to cell death.ConclusionsHumanin protected RPE cells against oxidative stress–induced cell death and restored mitochondrial function. Our data suggest a potential role for HN therapy in the prevention of retinal degeneration, including AMD.

α-Crystallins are key members of the superfamily of small heat shock proteins that have been studied in detail in the ocular lens. Recently, novel functions for α-crystallins have been identified in the retina and in the retinal pigmented epithelium (RPE). αB-Crystallin has been localized to multiple compartments and organelles including mitochondria, golgi apparatus, endoplasmic reticulum and nucleus. α-Crystallins are regulated by oxidative and endoplasmic reticulum stress, and inhibit apoptosis-induced cell death. α-Crystallins interact with a large number of proteins that include other crystallins, and apoptotic, cytoskeletal, inflammatory, signaling, angiogenic, and growth factor molecules. Studies with RPE from αB-crystallin deficient mice have shown that αB-crystallin supports retinal and choroidal angiogenesis through its interaction with vascular endothelial growth factor. αB-Crystallin has also been shown to have novel functions in the extracellular space. In RPE, αB-crystallin is released from the apical surface in exosomes where it accumulates in the interphotoreceptor matrix and may function to protect neighboring cells. In other systems administration of exogenous recombinant αB-crystallin has been shown to be anti-inflammatory. Another newly described function of αB-crystallin is its ability to inhibit β-amyloid fibril formation. α-Crystallin mini-chaperone peptides have been identified that elicit anti-apoptotic function in addition to being efficient chaperones. Generation of liposomal particles and other modes of nanoencapsulation of these minipeptides could offer great therapeutic advantage in ocular delivery for a wide variety of retinal degenerative, inflammatory and vascular diseases including age related macular degeneration and diabetic retinopathy.

Endoplasmic reticulum (ER) stress is linked to several pathological conditions including age-related macular degeneration. Excessive ER stress initiates cell death cascades which are mediated, in part, through mitochondrial dysfunction. Here, we identify αB crystallin as an important regulator of ER stress-induced cell death. Retinal pigment epithelial (RPE) cells from αB crystallin (−/−) mice, and human RPE cells transfected with αB crystallin siRNA, are more vulnerable to ER stress induced by tunicamycin. ER stress-mediated cell death is associated with increased levels of reactive oxygen species, depletion of glutathione in mitochondria, decreased superoxide dismutase activity, increased release of cytochrome c and activation of caspases 3 and 4. The ER stress signaling inhibitors, salubrinal and 4-(2-aminoethyl) benzenesulfonyl fluoride, decrease mitochondrial damage and reduce RPE apoptosis induced by ER stress. Prolonged ER stress decreases levels of αB crystallin, thus exacerbating mitochondrial dysfunction. Overexpression of αB crystallin protects RPE cells from ER stress-induced apoptosis by attenuating increases in Bax, CHOP, mitochondrial permeability transition, and cleaved caspase 3. Thus, these data collectively demonstrate that αB crystallin provides critical protection of mitochondrial function during ER stress-induced RPE apoptosis.

This study evaluated the role of crystallins in retinal degeneration induced by chemical hypoxia. Wild type, αA-crystallin (−/−), and αB-crystallin (−/−) mice received intravitreal injection of 12 nmol (low dose), 33 nmol (intermediate dose) or 60 nmol (high dose) cobalt chloride (CoCl 2 ). Hematoxylin and eosin and TdT-mediated dUTP nick-end labeling (TUNEL) stains were performed after 24 hours, 96 hours, and 1 week post-injection, while immunofluorescent stains for αA-and αB-crystallin were performed 1 week post-injection. The in vitro effects of CoCl 2 on αB-crystallin expression in ARPE-19 cells were determined by real time RT-PCR, Western blot, and confocal microscopy and studies evaluating subcellular distribution of αB-crystallin in the mitochondria and cytosol were also performed. Histologic studies revealed progressive retinal degeneration with CoCl 2 injection in wild type mice. Retinas of CoCl 2 injected mice showed transient increased expression of HIF-1α which was maximal 24 hours after injection. Intermediate dose CoCl 2 injection was associated with increased retinal immunofluorescence for both αA-and αB-crystallin; however, after high dose injection, increased retinal degeneration was associated with decreased levels of crystallin expression. Injection of CoCl 2 at either intermediate or high dose in αA-crystallin (−/−) and αB-crystallin (−/−) mice resulted in much more severe retinal degeneration compared to wild type eyes. A decrease in ARPE-19 total and cytosolic αB-crystallin expression with increasing CoCl2 treatment and an increase in mitochondrial αB-crystallin were found. We conclude that lack of α-crystallins accentuates retinal degeneration in chemically-induced hypoxia in vivo.