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.

Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.

Citation: Sreekumar PG, Chothe P, Sharma KK, et al. Antiapoptotic properties of a-crystallin-derived peptide chaperones and characterization of their uptake transporters in human RPE cells. Invest Ophthalmol Vis Sci. 2013;54:278754: -279854: . DOI:10.1167 PURPOSE. The chaperone proteins, a-crystallins, also possess antiapoptotic properties. The purpose of the present study was to investigate whether 19 to 20-mer a-crystallin-derived mini-chaperone peptides (a-crystallin mini-chaperone) are antiapoptotic, and to identify their putative transporters in human fetal RPE (hfRPE) cells. METHODS.Cell death and caspase-3 activation induced by oxidative stress were quantified in early passage hfRPE cells in the presence of 19 to 20-mer aA-or aB-crystallin-derived or scrambled peptides. Cellular uptake of fluorescein-labeled, a-crystallin-derived mini-peptides and recombinant full-length aB-crystallin was determined in confluent hfRPE. The entry mechanism in hfRPE cells for a-crystallin mini-peptides was investigated. The protective role of polycaprolactone (PCL) nanoparticle encapsulated aB-crystallin mini-chaperone peptides from H 2 O 2 -induced cell death was studied.RESULTS. Primary hfRPE cells exposed to oxidative stress and either aA-or aB-crystallin minichaperones remained viable and showed marked inhibition of both cell death and activation of caspase-3. Uptake of full-length aB-crystallin was minimal while a time-dependent uptake of aB-crystallin-derived peptide was observed. The mini-peptides entered the hfRPE cells via the sodium-coupled oligopeptide transporters 1 and 2 (SOPT1, SOPT2). PCL nanoparticles containing aB-crystallin mini-chaperone were also taken up and protected hfRPE from H 2 O 2 -induced cell death at significantly lower concentrations than free aB-crystallin minichaperone peptide.CONCLUSIONS. aA-and aB-crystallin mini-chaperones offer protection to hfRPE cells and inhibit caspase-3 activation. The oligopeptide transporters SOPT1 and SOPT2 mediate the uptake of these peptides in RPE cells. Nanodelivery of aB-crystallin-derived mini-chaperone peptide offers an alternative approach for protection of hfRPE cells from oxidant injury.Keywords: a-crystallin, chaperone peptides, oxidative stress, RPE protection, oligopeptide transporters T he superfamily of small heat shock proteins (sHSPs) has attracted considerable attention in recent years because of its multifunctional cellular properties. The human genome encodes 10 members of the sHSP family, among which aAcrystallin and aB-crystallin are considered important members. 1 Both aA-and aB-crystallins have been studied extensively in the lens for their chaperone and related functions. However, recent studies have identified several novel functions for aA-and aBcrystallins in retina and other tissues in addition to their wellrecognized chaperone function. 2 Both a-crystallins are expressed in RPE cells and in the retina; higher expression of aBcrystallin was found in the RPE while aA-crystallin was found mostly in photoreceptors and astroglial and Mül...

Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD and αB crystallin expression is increased in RPE and associated drusen in AMD. The purpose of this study was to investigate the role of αB crystallin in sodium iodate (NaIO3)-induced retinal degeneration, a model of AMD in which the primary site of pathology is the RPE. Dose dependent effects of intravenous NaIO3 (20-70 mg/kg) on development of retinal degeneration (fundus photography) and RPE and retinal neuronal loss (histology) were determined in wild type and αB crystallin knockout mice. Absence of αB crystallin augmented retinal degeneration in low dose (20 mg/kg) NaIO3-treated mice and increased retinal cell apoptosis which was mainly localized to the RPE layer. Generation of reactive oxygen species (ROS) was observed with NaIO3 in mouse and human RPE which increased further after αB crystallin knockout or siRNA knockdown, respectively. NaIO3 upregulated AKT phosphorylation and peroxisome proliferator–activator receptor–γ (PPARγ) which was suppressed after αB crystallin siRNA knockdown. Further, PPARγ ligand inhibited NaIO3-induced ROS generation. Our data suggest that αB crystallin plays a critical role in protection of NaIO3-induced oxidative stress and retinal degeneration in part through upregulation of AKT phosphorylation and PPARγ expression.