Novel roles for α-crystallins in retinal function and disease

Abstract

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.

1. General background

The α-crystallins are molecular chaperones belonging to the small heat shock protein (sHSP) superfamily. The members of this family have been found throughout all animal kingdoms (Kappe et al., 2002). In spite of the differences in sequence and size, sHSPs share some features in common such as a highly conserved α-crystallin domain, a comparatively small molecular mass (12–43kDa), a dynamic quaternary structure, formation of large oligomers, induction by different stressors, and chaperone activity. The most widely studied sHSP α-crystallin, has two subunits, namely αA and αB-crystallin. Though once considered lens specific, α-crystallin has widespread non-lenticular distribution (Horwitz, 2003). It was once thought that sHSPs were exclusively intracellular molecules which functioned as intracellular molecular chaperones. Recent data reveal that certain HSPs are released from cells which are induced by endogenous and exogenous factors and exhibit extracellular functions. We have described the pathways of exosomal secretion of αB-crystallin from primary human RPE cells (Sreekumar et al., 2010) which have also been described in the ARPE-19 cell line (Gangalum et al., 2011). When released, HSPs have the potential to act on cell surface receptors; elicit autocrine signals; paracrine signals; or endocrine signals. In this respect, the sHSPs are considered akin to cytokines.

α-Crystallins are characterized by the presence of a conserved crystallin domain flanked by a variable N-terminal domain and C-terminal extension (Fig. 1). Each of the domains has chaperone function and this function can vary depending on post-translational modifications of the N-terminus such as oxidation, phosphorylation, deamidation, acetylation and truncation (Kapphahn et al., 2003; Asomugha et al., 2011). The N- and C-termini, together with part of the crystallin domain, are also involved in substrate binding. The α-crystallin domain consists of 6–8 β-strands organized in two β-sheets flanked by the NH₂- and COOH-terminal regions, and this domain is considered an important hallmark of sHSPs, independent of their origin and nature (Kampinga et al., 2009). Very often the NH₂-terminal region contains a number of sites phosphorylated by different protein kinases (Haslbeck et al., 2005; Vos et al., 2008). α-Crystallins have stable structures and are water soluble. In the lens, native α-crystallin is composed of subunits of αA and αB-crystallin (Horwitz, 2003). αA- and αB-crystallin occur as a heteroaggregate of variable size, ranging from 300,000 to over 1,000,000 daltons and each monomer is approximately 20 KDa in size. The proportion of αA-crystallin to αB-crystallin varies in each aggregate, but generally in a 3:1 ratio of αA- to αB-crystallin in the mammalian lens (Posner, 2003). Attempts to crystallize full-length human crystallins were unsuccessful until recently. In 2010, the structure of full length αB-crystallin was determined by means of solid-state NMR and small-angle X-ray scattering (Jehle et al., 2010). The NMR ribbon structure of αB-crystallin core domain dimer is shown in Figure 2.

Figure 1.

Structural organization of α-crystallin protein. α-Crystallins have a crystallin core domain flanked by variable N-terminal domain and C-terminal extension. Three known phosphorylation sites on αB-crystallin are located in the N-terminal domain and can be activated by p44/42 and p38 MAPkinases as well as other yet to be determined pathways.

Figure 2.

Diagramatic ribbon representation of the lowest-energy solid state NMR structure of the αB-crystallin dimer core domain and sites of the beta (β) sheet strands. Figure modified and redrawn from Jehle S., et al. Solid-state NMR an SAXS studies provide a structural basis for the activation of alphaB-crystallin oligomers. Nat Struct Mol Biol 2010; 17: 1037–1042.

In addition to their chaperone functions, α–crystallins and other sHSPs perform multiple additional tasks; they may assist in the efficient folding of newly translated proteins (Frydman, 2001), inhibit subcellular transport of some proteins (Liu et al. 2007; Mao et al. 2004), prevent cell death (Acunza et al, 2012; Adhikari et al., 2011; Andley 2000, 2007; Arrigo et al., 2007; Mao et al., 2001, 2004; Parcellier et al., 2005, Sreekumar et al., 2010, 2012, 2012b; Yaung et al., 2008, 2007; Whiston et al. 2008), inhibit inflammation (Arac et al., 2011; Arrigo et al., 2007; Masilamoni et al., 2005, 2006; Ousman et al., 2007; Rothbard et al., 2012; van Noort et al., 2012; Whiston et al., 2008), provide neuroprotection (Ousman et al., 2007; Sreekumar et al., 2010) and regulate proteosomal degradation of proteins (Adhikari et al., 2011; Boelens et al., 2001; den Engelsman et al., 2003; Kase et al., 2010; Singh et al., 2010). Further, sHSPs participate in angiogenesis (Dimberg et al., 2008; Kase et al., 2010), and interact with several proteins or cytokines and influence cell signaling (Andley, 2007; Ecroyd and Carver, 2009; Ghosh et al. 2007a, 2007b; Rekas et al., 2004; Kase et al., 2010; Sreekumar et al., 2011). Thus, these chaperones broadly shape protein homeostasis by controlling protein quality control and turnover during both normal and stress conditions. Consistent with these diverse activities, genetic and biochemical studies have implicated their role in a wide range of pathological conditions, including neurodegeneration, cancer, and inflammation. Thus new roles of sHSPs important for physiology and disease are emerging in this rapidly expanding field. This review summarizes our knowledge on α-crystallins in the retina with special emphasis on their function under normal and pathological conditions and their potential as therapeutic agents.

2. Evolutionary aspects

In humans, the αA-crystallin gene encodes a 173 amino acid residue protein, while the αB-crystallin gene encodes a 175 amino-acid residue protein. The amino-acid sequence homology between αA and αB is about 57% (de Jong et al., 1998). Both αA- and αB-crystallin are encoded by single copy genes located on chromosomes 21 and 11, respectively, in humans (Quax-Jeuken et al., 1985; Ngo et al., 1989) and contain two introns at homologous positions. However, in some species such as rat, rabbit and mouse an additional 63 bp are added from intron 1 resulting in a protein 23 amino acids longer than the regular αA crystallin, named αA insert (Derham and Harding, 1999; Graw, 2009). A variant without the insert is also present in these species. Although some evidence suggests decreased chaperone function for the insert (Derham et al., 2001), this phenomenon remains to be further explored. The genes for αA- and αB-crystallin originated by duplication of an ancestral α-crystallin gene, which occurred well before the divergence of the cartilaginous fishes, and the higher vertebrates (de Jong et al., 1993).

3. Tissue and cellular distribution of α-crystallin and factors affecting its expression

αA and αB-crystallin have a differential tissue distribution. αA-crystallin is prominently localized to the eye and is the most abundant in the lens. However, its non-lenticular expression is well described (Kato et al., 1991; Srinivasan et al., 1992). It was reported that a significant amount of αA-crystallin was present in rat spleen and thymus and low levels were detected in the rectum, cecum, liver, kidney, adrenal, cerebellum and brain stem (Kato et al., 1991; Srinivasan et al., 1992). In the retina, αA-crystallin is expressed mostly in the ganglion cell and inner nuclear layers and in photoreceptors (Rao et al., 2008; Xi et al. 2003; Yaung et al., 2008). αA-crystallin is significantly upregulated in the early phase of experimental autoimmune uveitis (EAU), especially in the photoreceptor inner segments (Rao et al., 2008). It is also one of the components of drusen and was found to increase in retinal degenerative diseases such as age-related macular degeneration (AMD) (Nakata et al., 2005). αB-crystallin is ubiquitously expressed and its expression has been well documented in lens, neural retina, retinal pigment epithelium (RPE), heart, skeletal muscle, kidney, placenta, skin, brain, peripheral nerves, spinal cord, and liver; however, the highest level of αB-crystallin is found in the lens (Bhat and Nagineni, 1989; de Jong et al., 1993; Horwitz et al., 1999; Inaguma et al., 1995; Iwaki et al., 1990, 1989; Lowe et al., 1992). Expression of αB-crystallin is highly regulated during development (Robinson and Overbeek, 1996), under some pathological conditions such as AMD (De et al., 2007; Nakata et al., 2005), ischemia (Pangratz-Fuehrer et al., 2011), diabetes (Kandpal et al., 2012; Kumar et al., 2005), retinal and choroidal neovascularization (Kase et al., 2010), multiple sclerosis (Ousman et al., 2007; Rothbard et al., 2012), inflammatory disorders (Hegen et al., 2010; Wanschitz et al., 2008) and myofibrillar myopathies (Selcen, 2011). An especially large increase in αB-crystallin expression was detected in cancer cells from patients with retinoblastoma (Pineda et al., 1993), breast cancer (Chan et al., 2011; Launay et al., 2010), and glioblastoma (Goplen et al., 2010). In the retina, both αA-and αB-crystallins were found to prominently expressed in the inner retina (ganglion cell layer and inner synaptic layer) and in photoreceptors and RPE (Piri et al., 2007; Xi et al., 2003; Yaung et al., 2008). Fig. 3 shows the distribution of αB-crystallin in the retina of a 10 week old 129 SvJ mouse. Expression is seen in the inner retina, the inner and outer segments of photoreceptors and in the RPE. The expression of α-crystallin is regulated by oxidative stress showing either an increase or a decrease depending on the severity of dose and duration. In general, αB-crystallin expression is increased with mild oxidative stress (Shin et al., 2009; Yaung et al., 2008, 2007); however, chronic oxidative stress with H₂O₂ causes a decrease in expression (Yaung et al., 2007). For instance, in mice treated with 33 nmol CoCl₂ for a week, increased expression of αB-crystallin was seen and expression was more diffusely distributed across the retina, whereas increased retinal degeneration and decreased levels of αB-crystallin were observed with a higher dose (Yaung et al., 2008). Further, in a intraocular hypertension induced ocular ganglion cell degeneration rat model, the mRNA and protein levels of α-crystallins showed an initial decrease at 2 weeks followed by recovery to levels found in control retina at 5 weeks post-IOP (Piri et al., 2007). Temperature also upregulated αB-crystallin expression and the temperature-dependent increase of αB-crystallin seems to be dependent on the activity of heat shock factors which interact with two regulatory heat shock elements (Kato et al., 1999). Intense green light exposure significantly upregulated αB-crystallin expression in rod outer segments of photoreceptor and RPE cells, and this could be a mechanism to protect photoreceptors from light damage (Sakaguchi et al., 2003). Recent evidence supports an important role for αB-crystallin in the pathogensis of AMD. In pathologic specimens from patients with both late “dry” and “wet” AMD, αB-crystallin was heterogeneously expressed by RPE cells and the expression level was significantly increased in the RPE when compared with control samples (De et al., 2007). As shown in Fig. 4, we have confirmed that there is prominent and increased expression of αB-crystallin in RPE associated with drusen. In addition, αB-crystallin has been identified as a component of drusen (Crabb et al., 2002; De et al., 2007; Nakata et al., 2005).

Figure 3.

Immunofluorescent localization of αB-crystallin in retina and in RPE (arrows) of a 129 S6/SvEvTac mouse (10 weeks of age). Abbreviations used are OS, outer segement; IS, inner segment, ONL, outer nuclear layer; OPL, outer plexiform layer, INL, inner nuclear layer and IPL, inner plexiform layer. Bar indicates 20 microns. For inset, bar indicates 10 microns.

Figure 4.

Expression of αB-crystallin in RPE associated with drusen from a patient with age-related macular degeneration. Confocal immunofluorescent image shows increased expression of αB-crystallin (FITC-green) in RPE (arrow), especially overlying drusen (*). Autofluorescent signal (eg. from lipofuscin) was digitally subtracted from the image. DAPI nuclear counterstain (blue).

Both αA- and αB-crystallin can be considered as soluble cytosolic proteins but they are also localized within subcellular organelles of unstressed cells including mitochondria and endoplasmic reticulum (Yaung et al., 2007). The cellular localization and distribution seems to be dependent on the physiological status of the cells. In the developing lens, there is a transient association of αB-crystallin with golgi-associated membrane (Gangalum and Bhat, 2009); an association between αB-crystallin and golgi in RPE cells and photoreceptors has also been shown (Deretic et al., 1994). In stressed cells, αB-crystallin also translocates to the nucleus (Adhikari et al., 2004; den Engelsman et al., 2005; Sreekumar et al., 2010; Verschuure et al., 2002) or to mitochondria (Adhikari et al., 2004; Jin et al., 2008; Whittaker et al., 2009; Yaung et al., 2007) or endoplasmic reticulum (Dou et al., 2010; Kase et al., 2010). Both HSP-27 and αB-crystallin localized to nuclear speckles in unstressed cardiac myocytes (den Engelsman 2004). Further it was shown that silencing of αB-crystallin sensitized ARPE-19 cells to a histone deacetylase inhibitor (HDAC1) SAHA by abolishing the association of HDAC1 with nuclear SC35 speckles (Noh et al. 2008). An upregulation of αB-crystallin in mitochondria with oxidative stress from H₂O₂ treatment is illustrated in Fig. 5. Some studies claimed that phosphorylation is required for the translocation (Golenhofen, 1998) while others showed translocation is independent of phosphorylation (Verschuure et al., 2002). However, a recent study demonstrated that during murine cardiac myocyte ischemia αB-crystallin translocates to the mitochondria and contributes to modulating mitochondrial damage upon reperfusion. In this model system αB-crystallin is, at least in part, phosphorylated in association with mitochondria (Whittaker et al., 2009). We studied the translocation of αB-crystallin using fluorescein labeled human recombinant αB-crystallin in the presence or absence of oxidative stress in RPE. Human RPE cells incubated with 500 μM H₂O₂ for 1 h showed a marked uptake of αB-crystallin into the nucleus when compared with unstressed cells (Sreekumar et al., 2010). Chemically induced hypoxia caused translocation of αB-crystallin to the ER where it colocalized with growth factors such as vascular endothelial growth factor (VEGF) (Kase et al., 2010). Hypoxia also caused translocation of αB-crystallin to the mitochondria (Yaung et al., 2008). Further work is needed to understand other mechanism(s) that may be involved in the subcellular translocation of α-crystallins such as role of putative transporters or phosphorylation.

Figure 5.

Localization of αB-crystallin in mitochondria of RPE. Confocal micrographs showing αB-crystallin expression in human RPE mitochondria with and without H₂O₂ treatment. Mitochondria tracker (Mito-Tracker; red) allowed visualization of mitochondria in the RPE (i-l). αB-Crystallin expression was upregulated with low dose H₂O₂ treatment indicated by an increase in fluorescent intensity. In composite images, the cellular distribution pattern of αB-crystallin was found to show an increase in mitochondrial localization (j). The staining for αB-crystallin decreased in mitochondria with increasing dose of H202 (k,l). Bar represents 50 μm. αB-Crystallin staining is green, mitochondria red, colocalization of α-crystallin, and mitochondria shown in yellow in merged panels. Reproduced with permission from Yaung et al. alpha-Crystallin distribution in retinal pigment epithelium and effect of gene knockouts on sensitivity to oxidative stress. Mol Vis. 2007, 13:566–77.

4. Knockout models

To facilitate understanding the in vivo functions of α-crystallins, animal models have been developed where one or both of the α-crystallin gene products have been knocked out. With an idea of studying the significance of αA-crystallin in lens function, Brady et al. (1997) generated αA-crystallin knockout (KO) mice. Targeted elimination of the αA-crystallin gene induced cataract and accumulation of dense cytoplasmic bodies including αB-crystallin, HSP25 and α-crystallin in lens fiber cells (Brady et al., 1997) without any apparent changes in the retinal layers. αA-crystallin KO lenses were 40% smaller in size than age matched wild type mice; a mild cataract begins to develop as early as 7 weeks and a fully opaque mature cataract ensues by 18 weeks (Brady et al., 1997; Kannan et al., 2001). Targeted disruption of αB-crystallin did not produce cataracts (Brady et al., 2001) as observed in αA-crystallin KO mice. αB-crystallin KO mice are viable and fertile, with no obvious prenatal defects and normal lens transparency (Brady et al., 2001). Until recently, a retinal phenotype had not been described in the αB-crystallin KO mice. The major phenotype in these mice, which lack both αB-crystallin and HspB2 (an adjacent gene located 1kb upstream of αB-crystallin) was that as the mice become aged, homozygous KO mice developed degeneration of the musculature with severe spine curvature (kyphosis) (Brady et al., 2001). Histology of the eyes, cardiac muscles and skeletal muscle from αB-crystallin KO revealed no apparent structural abnormalities. A study from our laboratory tested the hypothesis that deficiency of αB-crystallin during development could result in changes in retinal vasculature (Kase et al., 2010). Retinal vessel density was lower at the junction between the inner plexiform layer and the outer nuclear layer in adult αB-crystallin KO mice (Kase et al., 2010) leading to the suggestion that localized changes in retinal vessel formation could result from decreased expression of vascular endothelial growth factor (VEGF) during development. The observed phenomenon was due to αB-crystallin deletion rather than HspB2 since HspB2 was not expressed retina or RPE (Kase et al., 2010). Further, HspB2 is not heat-shock-inducible, and is thus unlikely to act as a general chaperone (Doerwald et al., 2004).

αB-Crystallin deficient mice or αB-crystallin mutants (Sacconi et al., 2012) are highly susceptible to cell death when subjected to oxidative stress (Yaung et al., 2008, 2007) or in inflammatory disease models (Arac et al., 2011; Ousman et al., 2007). As in the case of αB-crystallin KOs, RPE cells isolated from αA-crystallin KO mice are highly susceptible to inflammation or oxidative stress-induced apoptosis (Rao et al., 2008; Yaung et al., 2007). In αA-crystallin KO mice, αB-crystallin could compensate for some of the functions of the missing protein, particularly in the lens, where both αA and αB are abundantly expressed. Therefore, to study the combined function(s) of α-crystallins, double deficient mice were generated which had gross abnormalities in the fiber cell structure that was not evident in single crystallin KO mice (Boyle et al., 2003). The lack of cellular organization and uniform cell shape at the equatorial region observed in α-crystallin double KO lenses suggests a role for α-crystallins in cytoskeletal organization.

5. Function and properties of α-crystallin

Mammalian sHSPs, including αA-crystallin and αB-crystallin, are constitutively expressed during development. Though chaperone activity is the first described function of α-crystallins in the eye, the importance of α-crystallins in cellular function is further highlighted by their participation in a growing number of fundamental cellular processes such as prevention of apoptosis, protection against oxidant insults, and neuroprotection (Andley, 2007; Arrigo et al., 2007). These are discussed in detail below.

5.1 Chaperone function

Molecular chaperones are a class of unrelated families of proteins that assist the proper assembly of other polypeptide-containing structures in vivo, but are not components of these assembled structures when they are performing their normal biological function (Ellis, 1993). Molecular chaperones consist of different classes of proteins that can assist in stabilization of native protein conformations, protein folding, mediation and stabilization of correct oligomeric assembly of proteins, translocation of proteins, and protection of proteins from multiple stresses (Derham and Harding, 1999). The chaperone activity of α-crystallins was first characterized under in vitro experimental conditions using purified bovine α-crystallin where they inhibited aggregation of several enzymes, prevented heat-induced aggregation of β and α-crystallins and refolding of α-crystallins in the lens (Horwitz, 1992). The fundamental cellular processes, like protein synthesis, transport and turnover involve unfolded or partially denatured proteins, exposed hydrophobic regions which may interact incorrectly, even at low concentration (Jaenicke, 1987). The chaperone activity is generally studied by measurement of light scattering of proteins incubated at high temperature or to reduction induced denaturation. The protective properties of α-crystallin have been demonstrated with other in vitro assays including prevention of aggregation of insulin B by reduction of disulfide bonds (Das and Surewicz, 1995; Farahbakhsh et al., 1995), by refolding of guanidine hydrochloride (or urea)-denatured proteins (Horwitz, 1992; Raman et al., 1995), by prevention of UV-induced aggregation of proteins (Raman and Rao, 1994) and prevention of inactivation of enzymes by small molecules (Ganea and Harding, 1995, 1996; Heath et al., 1996; Hook and Harding, 1996). The ability of α-crystallin to confer thermotolerance in vivo which supported the in vitro findings has also been described (Plater et al., 1996; van den Ijssel et al., 1994).

The mechanism by which α-crystallin prevents protein aggregation or its chaperone activity has been studied by several investigators. These studies suggest that there are multiple substrate binding sites in each α-crystallin oligomer and the substrates that bind with are in a molten globular state (Carver and Lindner, 1998; Carver et al., 2002). The substrate–α-crystallin complex is very stable. α-Crystallins do not interfere with intermediates of protein folding pathway but are specific for the destabilized, precipitation-bound, relatively unstructured intermediates that have the potential to aggregate (Carver et al., 2002). Thus, kinetic factors could be one of the major determinants in regulating the interaction of a protein with molecular chaperones; transient intermediates associate with protein folding pathway chaperones, e.g. GroEL and DnaK, whereas long-lived intermediates that are potentially aggregating, associate with off-folding pathway chaperones, e.g. sHSPs (Carver et al., 2002). Further, the association and dissociation of oligomer subunits play an essential role in the chaperone functions (Laskowska et al., 2010). A number of parameters such as temperature, phosphorylation, ionic strength, pH, Ca²⁺ concentration, tissue age, and protein concentration lead to the subunit exchange and dissociation of oligomers (Bova et al., 2000, 1997; Regini et al., 2004). Several in vitro studies suggest that α-crystallin activity does not require ATP hydrolysis and therefore, α-crystallin becomes particularly significant under stress conditions when the level of ATP is very low (Horwitz, 1992). However, ATP effects have been reported by others, primarily with α-crystallin (Barton et al., 2009; Biswas and Das, 2004; Muchowski and Clark 1998; Wang and Spector, 2000). The identification of β4-β8 groove as the ATP interactive region in αB-crystallin, is in contrast to the existing paradigm that classifies sHSPs as ATP-independent chaperones (Dutta et al., 2010; Ghosh et al., 2006). However, it should be noted that many sHSPs are not constitutively active; they are specifically activated in the presence of stress. Further studies are needed to clarify the importance of ATP in the chaperone activity of α-crystallins.

Different methods are utilized by investigators to identify potential sequences of αcrystallins showing chaperone activity. Biophysical studies (Berengian et al., 1999; Bova et al., 2000) and subunit interaction studies using peptide array (Sreelakshmi et al., 2004) and pin array (Ghosh et al., 2005) showed extensive contact and interaction occurs between the subunits. Regions 42–57 and 60–71 in αB-crystallin function as subunit interaction sites (Sreelakshmi et al., 2005; 2004). The residues 73–92 in αB-crystallin, called mini-αB-crystallin, can independently prevent the aggregation of denatured substrate proteins similar to the action of native αB-crystallin (Bhattacharyya et al., 2006). Using protein pin arrays Ghosh et al. (2005) identified seven interactive sequences (9–20, 43–58, 75–82, 113–120, 131–138, 141–148, and 157–164) in the human αB-crystallin using endogenous target proteins β_H/γD crystallins and non-physiological targets alcohol dehydrogenase and citrate synthase for the chaperone activity. Two of the seven chaperone sequences were in the N-terminus, four in the conserved αcrystallin core domain, and one was in the C-terminal extension containing the highly conserved I-X-I/V motif. The authors also suggested that point mutations within the interactive domains could modify the chaperone activity of αB-crystallin and those outside the interactive domains could have little or no effect on chaperone activity. Oligomerization of sHsp subunits is essential in order for α-crystallins to function as molecular chaperones. Two interactive sequences, ₇₃DRFSVNLDVKHFS₈₅ and ₁₃₁LTITSSLSDGV₁₄₁, belonging to the α-crystallin core domain were synthesized as peptides and assayed for chaperone activity in vitro (Ghosh et al., 2005). Both synthesized peptides inhibited the thermal aggregation of βH crystallin, alcohol dehydrogenase, and citrate synthase in vitro. Moreover, it was found that specific αB-crystallin interactive peptides were capable of protecting key regulatory proteins from unfolding and aggregation. Several studies implicate that structure, hydrophobicity, and ionic charge are the major factors determining the chaperone activity of α-crystallins (Carver et al., 1995; Sharma et al., 2000, 1997; Santhoshkumar and Sharma, 2001). The chaperone efficiency of α-crystallin depends on its ability to recognize substrates undergoing denaturation, interact with the substrate, and keep the complex in soluble form and any alterations of these factors would result in a protein with reduced or altered chaperone function. For αA-crystallin Sharma et al. (2000) identified a 19-mer sequence which has similar chaperone activity to that of αA-crystallin by in vitro assays. The peptide sequence corresponded to the β3 and β4 region present in the α-crystallin domain (Sharma et al., 2000). Further studies revealed that the 19-mer peptide sequence of αA-crystallin as well as the region 42–57 derived from αB-crystallin elicited antiapoptotic properties in RPE cells challenged with oxidative stress (Kannan et al., 2010; Santoshkumar and Sharma, 2004). On the other hand, Santhoshkumar et al. (2011) found that a different αA-crystallin-derived peptide, (₆₆SDRDKFVIFLDVKHF₈₀), which accumulates in the aging lens, could potentially inhibit the chaperone activity of α-crystallin and cause aggregation and precipitation of lens crystallins. They proposed that the αA-(66–80) peptide generated in human lenses interacts with α-crystallin and causes its aggregation and precipitation and suppresses the chaperone activity of α-crystallins. The direct functional demonstration of α-crystallin sequences provides support for the hypothesis that the chaperone function of α-crystallin is not limited to stress conditions and has an important function in regulating normal cellular growth and development. The physiologic significance of the chaperone function of α-crystallin is further substantiated by the findings that mutations in α-crystallin have a dramatic effect on its chaperone activity (Sun and MacRae, 2005). Although most of the chaperone activity studies are based on in vitro findings, in vivo studies using double heterozygous α-crystallin knock-out [αA (+/−) αB (+/−)] mice suggests that the total chaperone activity of α-crystallins is critical for maintaining lens transparency (Cheng et al., 2010). However, we can’t rule out that loss of lens transparency in these mice is a non-chaperone effect resulting from reduced expression of these abundant structural lens proteins. The role of overall chaperone activity of α-crystallin in retinal function and disease is yet to be delineated.

5.2. Angiogenesis & vascular remodeling

Angiogenesis and vascular remodeling are normal physiologic processes occurring during embryonic development, wound healing, and the development of compensatory collateral blood vessels in response to different diseases. Pathological angiogenesis or pathological neovascularization is the development of abnormal blood vessels or blood vessel formation in response to inflammation, ischemia and other pathologies. The role of αB-crystallin in regulating angiogenesis has attracted a lot of attention. αB-crystallin and HSP27 have been shown to participate in the regulation of angiogenesis (Dimberg et al., 2008; Kase et al., 2010; Keezer et al., 2003). We found that αB-crystallin KO resulted in attenuation of neovascularization in oxygen induced retinopathy while prominent neovascularization was observed in the wild type mouse (Fig. 6). αB-Crystallin regulates angiogenesis and vasculogenesis through multiple mechanisms, depending on the cell and tissue type. αB-Crystallin could act either directly on endothelial cells and/or indirectly on other cell types such as RPE (Kase et al., 2010). It has been shown that αB-crystallin directly acts as a chaperone for VEGF and other growth factors such as fibroblast growth factor-2 (FGF-2) (Ghosh et al., 2007a). Our laboratory demonstrated coimmunoprecipitation of αB-crystallin with VEGF in cultured RPE cells and extended the significance of this finding to αB-crystallin KO mice exposed to oxygen-induced retinopathy or laser induced choroidal neovascularization (CNV) (Kase et al., 2010). The stability of VEGF in these models depended on αB-crystallin and showed a decrease in the absence of αB-crystallin (Kase et al., 2010). Inner retinal vascular density was slightly lower in αB-crystallin KO, suggesting that αB-crystallin also plays a role in the development of the retinal vasculature (Kase et al., 2010). Recent in vitro studies show the importance of αB-crystallin in maintaining the intracrine stimulation of VEGF by endothelial cells (Ruan et al., 2011), while, interestingly, in vivo studies using αB-crystallin KO mice did not produce any major vascular defects, suggesting that αB-crystallin may not be strictly essential for intracrine VEGF signaling. However, it is not clear whether αB-crystallin phosphorylation or extracellular αB-crystallin could play a role in angiogenesis. In the mouse model of oxygen-induced retinal angiogenesis (Smith et al., 1994), phosphorylated serine 59 increased in WT mice which showed increased VEGF and retinal angiogenesis, suggesting a possible role for αB-crystallin phosphorylation in angiogenesis (Kase et al., 2010). This was further supported by additional evidence that under the same experimental conditions αB-crystallin KO mice showed decreased cellular and plasma VEGF levels and significant reduction in retinal angiogenesis. Similar findings were observed for the laser-induced choroidal angiogenesis model (Kase et al., 2010). Therefore, it can be argued that phosphorylation at serine 59 is essential for maintaining the chaperone function and retaining the stability of VEGF given that αB-crystallin binds to VEGF (Kase et al., 2010). Extracellular αB-crystallin could have a role in angiogenesis since αB-crystallin in normal blood vessel walls immediately responds to mechanical stress and is released from injured arterial wall into circulation (Kozawa et al., 2001). In the plasma, αB-crystallin inhibits aggregation of platelets which has crystallin binding sites on its surface (Enomoto et al., 2009). Extracellular αB-crystallin may inhibit release of granules from platelets and it has been reported that platelets contain both pro- and anti-angiogenic molecules (Enomoto et al., 2009; Sabrkhany et al., 2011). Therefore, it can be hypothesized that extracellular αB-crystallin could play a role in angiogenesis either by modulating platelet aggregation or by inhibiting granule release occurring during injury or inflammation. Further studies are needed to define the role of αB-crystallin phosphorylation (for eg. role of other phosphorylation sites, see below) on angiogenesis. Further the effect of role of extracellular αB-crystallin on retinal angiogenesis either under physiological or pathological conditions is a useful area for further research. In this context, a recent study on the role of αA-crystallin in suture or chemical burn induced corneal neovascularization in mice is of interest (Zhu et al., 2012). The authors found that subconjunctival injection of αA-crystallin significantly attenuated suture-induced corneal neovascularization and this inhibition was shown to be mediated by the expression of soluble VEGFR1 (Zhu et al., 2012).Thus, it appears that the effect and mechanism of α-crystallins on neovascularization may vary depending on the crystallin isoform studied, the tissue type and the model of neovascularization.

Figure 6.

Attenuation of retinal neovascularization in oxygen-induced retinopathy (OIR) in αB-crystallin knockout (KO) mice. In (a–h), FITC-Isolectin-B4 angiography in wild-type (a–d) and αB-crystallin KO (e–h) mice at P12 (a,e) and P17 (b–d, f–h) with OIR are shown. At P12, avascular areas are identified with arrows. In panel H, the αB-crystallin KO flat mount is seen at greater magnification with additional DAPI nuclear staining. Bars indicate 1 mm in panels a,b,e, and f; 200 μm in panels c, d, and g; and 50 μm in panel h. In panels i-l, hematoxylin and eosin staining in wild-type (i,j) and αB-crystallin KO (k,l) mice at P12 (i,k) and P17 (j,l) with OIR. At P12, occluded existing retinal vessels identified with white arrows. At P17, neovascular tufts identified with black arrows. Bars indicate 100 μm in panels i-l. In panels m and n, immunofluorescent staining with isolectin-B4 (green) demonstrates prominent neovascularization (white arrows) in wild-type mice (M) and infrequent neovascularization in αB-crystallin KO mice (n). Bar indicates 50 μm in panels m-p.. “This research was originally published in Blood. Kase et al. alphaB-crystallin regulation of angiogenesis by modulation of VEGF. Blood 2010; 115: 3398–3406, © The American Society of Hematology.”

Taking into consideration that αA-crystallin plays a significant role in ocular inflammation (Rao et al., 2008) and the fact that there is molecular crosstalk between inflammation and angiogenesis (Arroyo and Iruela-Arispe, 2010; Costa et al., 2007), it can be postulated that proteins that alter inflammation could also play a role in angiogenesis. Future studies will unravel the mechanisms of αA-crystallin participation in angiogenesis.

5.3. Phosphorylation and cell survival

Phosphorylation is one of the most important post-translational modifications controlled either enzymatically (e.g. phosphorylation and glycosylation) or occurring non-enzymatically (eg. oxidation). Post-translational modifications from phosphorylation have been studied extensively for αB-crystallin. Protein phosphorylation in eukaryotic cells takes place on serine, threonine, and tyrosine residues, with serine phosphorylation being the most common (McLachlin and Chait, 2001). αB-Crystallin is characterized by its capacity to be phosphorylated in response to a variety of stimuli originating from physiological changes or environmental insults (Hoover et al., 2000). For example, αB-crystallin undergoes multisite phosphorylation catalyzed by different protein kinases, and serine residues 19, 45, and 59 are the major sites that are phosphorylated in vivo (Smith et al., 1992). As illustrated in Fig. 1, the three serine residues in αB-crystallin are phosphorylated by different protein kinases, p44/42 MAP kinase (ERK), and MAPKAP kinase-2 (p38) targeting Ser-45 and Ser-59, respectively (Kato et al., 1998; Li and Reiser, 2011; Shu et al., 2005). Upstream of p38, RhoK, PKC and PKA are selectively involved in the activation of p38 and phosphorylation of αB-crystallin at serine 59 (Launay et al., 2006). The signaling pathway that results in phosphorylation of serine 19 is presently unknown. Under conditions of different stresses such as heat (Djabali et al., 1997), ischemia (Golenhofen et al., 1998), and hypoxia (Kase et al., 2010) the level of phosphorylated αB-crystallin increases. Phosphorylation of αB-crystallin at Ser-45 is enhanced in dividing cells (Kato et al., 1998), whereas phosphorylation at Ser-59 is high in centrosomes and midbodies (Inaguma et al., 2001). Human glioma cells treated with proteasome inhibitors, MG-132 or lactacystin, show enhanced phosphorylation of αB-crystallin suggesting that αB-crystallin (phosphorylated and total crystallin) could participate in the degradation of proteins via the ubiquitin-proteasome system (Ito et al., 2002). Studies using phosphorylation mimics demonstrated that an increase in phosphorylation leads to a decrease in the oligomerization of the protein (Ito et al., 2001) and a disruption in dimeric substructure within the oligomer (Aquilina et al., 2004). A slight decrease in the ability of a triple phosphorylation mutant (Ser-19D/Ser-45D/Ser-59D) to prevent the thermal aggregation of lactate dehydrogenase or to refold denatured firefly luciferase, compared with the wild-type recombinant protein was reported (Ito et al., 2001). However, individual mutations to mimic either phosphorylation or dephosphorylation of αB-crystallin showed that pseudophosphorylation of αB-crystallin at Ser-45 or Ser-59 provided powerful protection of astrocytes against oxidative stress (Li and Reiser, 2011). In addition, unphosphorylation of αB-crystallin at Ser-45 or Ser-59 resulted in the loss of protective function supporting a significant role for serine phosphorylation at position 45 and 59 in cell protection (Li and Reiser, 2011). It has to be noted that the phosphorylation/unphosphorylation at Ser-19 does not affect the protection status, demonstrating that only Ser-45 and Ser-59 participate in cytoprotection.

As further evidence on the regulation of the chaperone activity of αB-crystallin by phosphorylation (Ecroyd et al., 2007), our laboratory has recently found that in mouse models of retinal and choroidal angiogenesis, phosphorylation of αB-crystallin at Ser-59 increased significantly suggesting increased and sustained chaperone activity throughout angiogenesis (Kase et al., 2010). These results suggest that the highly prevalent post-translational modification of α-crystallins may play an important role in alleviating the pathogenic effects associated with protein conformational diseases. Whether a direct link between phosphorylation and chaperone activity exists is unresolved because of the contradictory reports in the literature using phosphorylation-mimicking mutants of αB-crystallin (Ahmad et al., 2008; Ecroyd et al., 2007; Ito et al., 2001; Koteiche and McHaourab, 2003). For instance, it has been reported that the phosphorylation-mimicking mutant has decreased chaperone activity towards heat-induced aggregation and refolding of luciferase (Ito et al., 2001). On the other hand, an increase in the chaperone activity of αB-crystallin in binding to the destabilized T4 lysozyme mutants was found in another study (Koteiche and Mchaourab, 2003). In support, Ecroyd et al. (2007) reported that the phosphorylation-mimicking mutation leads to increased chaperone activity of αB-crystallin towards amorphous reduction-induced aggregation or heat stress-induced aggregation. Other studies demonstrated similar chaperone activity irrespective of the phosphorylation status of αB-crystallin (Wang et al., 1995). The discrepancies in results could arise partly from differences in the substrate-dependent behavior of αB-crystallin, the phosphorylation-mimicking mutants, experimental conditions and or changes the structure and stability of αB-crystallin. Therefore, to understand the role of phosphorylation in the structure and function of αB-crystallin one has to consider the chaperone activity of the phosphorylated αB-crystallin, differences in the structure and stability of phosphorylated αB-crystallin and/or the possibility of phosphorylated αB-crystallin forming mixed oligomers with αB-crystallin. At the functional level, ectopic expression of a pseudophosphorylated αB-crystallin mutant with three target serine residues (Ser-19E,Ser-45E,Ser-59E) substituted for aspartate did not have any anti-apoptotic activity (Kamradt et al., 2002). Further studies showed that phosphorylation of αB-crystallin at Ser-59 provided cytoprotection in stressed cardiac myocytes (Morrison et al., 2003). p38-MAPK and its downstream substrate MSK1 have shown to be involved in H₂O₂-induced αB-crystallin (Ser-59) phosphorylation and in cytoprotection (Aggeli et al., 2008). As mentioned earlier, our work has shown significant phosphorylation of Ser-59 in experimental models of retinal angiogenesis (Kase et al., 2010). It is of interest that a recent study demonstrated that Ser-59 phosphorylation of αB-crystallin down regulates its anti-apoptotic function by binding and sequestering Bcl-2 in breast cancer cells (Launay et al., 2010). In summary, while phosphorylation contributes to apoptosis prevention, the exact nature and phosphorylation sites involved in other functions of crystallins such as inflammation and angiogenesis remain to be determined.

Unlike αB-crystallin, detailed functional studies for αA-crystallin phosphorylation are scarce. Earlier studies had shown in vivo phosphorylation sites of αA-crystallin in the lens at Ser-122 and Ser-148 (Chiesa et al., 1987; Voorter et al., 1986). The major phosphorylation of αA-crystallin at Ser-122 appears to be an age regulated event in eye lenses (Takemoto, 1996). Later studies identified eight (Thr-4, Ser-20, Ser-45, Ser-59, Ser-122, Ser-148, Ser-155 and Ser-172 or 173) different phosphorylation sites of αA-crystallin in in vivo mouse lenticular samples (Schaefer et al., 2006). Nine phosphorylated sites (Ser-20, Ser-59, Ser-62, Ser-66, Ser-81, Ser-86, Ser-148, Ser-155, Ser-173) of αA-crystallin were identified in vivo from porcine lens by phosphoproteomic analysis (Chiou et al., 2010). It is noteworthy that Chiou et al. (2010) identified some reported phosphorylation sites but could not find some other sites from previous studies. Thus, there are discrepancies in the literature regarding sites of αA-crystallin phosphorylation. Further, very little is known about the functional significance of these phosphorylation sites. Therefore, site-directed mutagenic studies by replacing phosphorylated sites of Ser- or Thr- residue to mimic the phosphorylation status of chaperoning αA-crystallin are needed to clearly understand the role of phosphorylation in relation to chaperone activity and biological significance. The precise mechanism of phosphorylation is also unclear. Serine-specific phosphorylation of α-crystallins is believed to occur via cAMP-dependent protein kinase pathways (Spector et al., 1985). An alternative pathway of phosphorylation is provided by the finding that αA-crystallin possesses autokinase activity (Kantorow and Piatigorsky, 1994); conversion from oligomers to tetramers enhances this activity (Kantorow et al., 1995). It is unknown which one of the phosphorylation pathways for αA-crystallin operates in RPE cells or in retina.

5.4. Cytoprotection

Although chaperone function was thought to be the only major function of α-crystallins, the ubiquitous tissue distribution (especially that of αB-crystallin) and studies in multiple tissues discovered several other functions which are of equal importance. One such function is its cytoprotective property (Andley, 2007, 2000; Arrigo et al., 2002; Kamradt et al., 2005, 2002, 2001, Liu et al., 2004a, 2004b; Ousman et al., 2007; Pasupuleti et al., 2010; Rao et al., 2008; Sreekumar et al., 2012, 2010; Yaung et al., 2007). The finding of increased expression of α-crystallin in different retinal disorders may be for neuroprotective purposes. Apoptosis is mediated by multiple signaling pathways and regulators such as the mitogen activated protein kinases (MAPKs) and or RAF/MEK/ERK or AKT Kinases (Liu et al., 2004a). αA-crystallin was reported to provide higher level of protection against cell death than αB-crystallin in cultured lens epithelial cells (Andley et al., 2000). However, RPE isolated from αA-crystallin KO mice were just as susceptible as αB-crystallin KO RPE to oxidative stress, despite the relatively low abundance of αA-crystallin in RPE (Yaung et al., 2007). Further, we found RPE cells overexpressing either αA- or αB-crystallin provided similar protection against H₂O₂–induced cell death (Sreekumar et al., 2012). This phenomenon of augmentation of apoptosis in αA- or αB-crystallin deficiency and suppression of apoptosis in αA- or αB-crystallin overexpressing ARPE-19 cells is shown in Figure 7. Similar supportive data were also reported in lens epithelial cells irradiated with UVA light (Liu et al., 2004a) or staurosporine, etoposide and sorbitol (Mao et al., 2004). In vivo, in CoCl₂ induced hypoxia, retinas from αA- or αB-crystallin KO mice exhibited similar, rapid and more severe degeneration as compared to WT retinas, supporting in vitro findings (Yaung et al., 2008). The retinal layers, especially inner and outer nuclear layers from α-crystallin KO retina under hypoxia suffered severe degeneration while wild type retinas were not severely affected (Fig. 8). Based on these studies, one can conclude that the two α-crystallins display similar anti-apoptotic properties in the retina, irrespective of their relative abundance. However, the mechanisms associated with the protection may be different and may vary depending upon the stress stimulus (Mao et al., 2004). Accumulating evidence in several cell types including RPE suggests that αB-crystallin can provide potent anti-apoptotic protection against multiple stress stimuli such as staurosporine, tumor necrosis factor, ultraviolet A irradiation, okadaic acid, H₂O₂, hypoxia and ceramide (Andley et al., 1998; Bluhm et al., 1998; Ito et al., 1997; Li and Reiser, 2011; Martin et al. 1997; Ousman et al., 2007; Sreekumar et al., 2012, 2010; Yaung et al., 2007). Accordingly, overexpression of αB-crystallin offers protection against a wide array of apoptotic stimuli while silencing renders cells susceptible to apoptosis (Bousette et al. 2010; Kamradt et al., 2005, 2002, 2001; Kase et al., 2010; Liu et al., 2004b; Mehlen et al., 1996; Nagaraj et al., 2005; Sreekumar et al., 2012). Deficiency of αB-crystallin increased levels of reactive oxygen species (ROS) and consequently, cells exposed to oxidative stress exhibited significant changes in mitochondrial permeability transition, and subsequent downstream activation of caspase 3 (Noh et al., 2008; Yaung et al., 2007). Oxidative stress also caused mitochondrial translocation of αB-crystallin (Jin et al., 2008; Saraswathy and Rao, 2009; Shin et al., 2009; Whittaker et al., 2009; Yaung et al., 2008, 2007). Further, αB-crystallin interacts directly with the pro-apoptotic members Bax and Bcl-X_S and P53 polypeptides in vitro and in vivo, with sequestration of these proteins preventing translocation to mitochondria and hence suppressing apoptosis (Liu et al., 2007; Mao et al., 2004). Interestingly, studies in cardiomyocytes and cancer cells found that phosphorylation of αB-crystallin at the level of Ser-59 may be sufficient to provide maximal protection against apoptosis (Launay et al., 2010; Morrison et al., 2003). In association to its anti-apoptotic effects, αB-crystallin protects multiple cell types by augmenting expression of antioxidants, such as glutathione (Kannan et al., 2001; Mehlen et al., 1996; Sreekumar et al., 2012). In αA- and αB-crystallin overexpressing ARPE-19 cells, levels of cellular GSH were >2 fold higher than corresponding WT cells which was accompanied by increased resistance to oxidative stress-induced cell death (Sreekumar et al., 2012). Our work also demonstrated that exogenously added recombinant human αB-crystallin was taken up by stressed cells and protected these cells from apoptosis by inhibiting caspase 3 and by Poly (ADP-ribose) polymerase activation (Sreekumar et al., 2010). As shown in Fig. 9, while H₂O₂ alone caused significant apoptosis as evidenced by increase in TUNEL positive cells and cleaved caspase 3, co-treatment with recombinant αB-crystallin nearly abolished this effect. The role of αB-crystallin in mitochondria, given its antiapoptotic function, could be to uphold or maintain mitochondrial function by protecting protein folding and to restore and prevent subsequent downstream activation of apoptotic events (McGreal et al., 2012). Inhibition of apoptosis could occur through an increase in mitochondrial GSH or activation of transcription factors such as NF-kB (Adhikari et al., 2011; Sreekumar et al., 2012). Munemasa et al. (2009) found that overexpression of αB-crystallin increased the number of retinal ganglion cells (RGCs) by 40% and 75% 1 week and 2 weeks after optic nerve axotomy–induced RGC degeneration, respectively. In experimental ischemic optic neuropathy, intravenous treatment with αB-crystallin enhanced the endogenous, post-ischemic response by decreasing microglial activation and promoting optic nerve oligodendrocyte survival (Pangratz-Fuehre et al., 2011). Studies in a cardiac ischemia-reperfusion model demonstrated that mitochondria may be among the key components in stressed cells with which S59 phospho-αB-crystallin interacts (Jin et al., 2008). Mitochondrial localization of αB-crystallin may protect the myocardium, in part, by modulating mitochondrial permeability transition pore opening and, thus, reducing ischemia/reperfusion injury (Jin et al., 2008). Other pathways of αB-crystallin’s antiapoptotic function have also been described. For instance, genotoxic stress induced αB-crystallin by a p53-dependent mechanism whereby the p53 family inhibitor ΔNp73, activated αB-crystallin gene expression (Evans et al., 2010). In another study, exogenous VEGF or VEGF overexpressing myoblasts were resistant to cell death from oxidative or hypoxic-like stress and this protection was linked to the phosphorylation of VEGF receptor (Flk-1), activation of NF-kB and/or the overexpression of αB-crystallin (Mercatelli et al., 2010). The involvement of NF-kB pathway was further supported by the recent findings that αB-crystallin-dependent NF-κB activation protected myoblasts from TNF-α induced cytotoxicity by enhancing the expression of the anti-apoptotic protein, Bcl2 (Adhikari et al., 2011).

Figure 7.

Increased apoptosis in RPE cells from α-crystallin KO mice (A,B) and attenuation of apoptosis in ARPE-19 cells overexpressing αA- or αB-crystallin (C). RPE from αB-crystallin KO mice grown to confluency were treated with 100 μM or 200 μM H₂O₂ for 24 h and stained for apoptosis by TUNEL and quantified by flow cytometry. Cell death of RPE increased in a dose dependent manner and αA-crystallin KO (A) and αB-crystallin KO (B) RPE showed an increased sensitivity to apoptosis from H₂O₂ treatment compared to wild type cells. C. Protection from cell death in α-crystallin overexpressing ARPE-19 cells. Cells were incubated in serum free medium and cell death was analyzed by TUNEL staining. Blue: DAPI nuclear staining; Red: TUNEL positive cells. Bar indicates 20 microns. Fig. A & B reproduced with permission from Yaung et al. alpha-Crystallin distribution in retinal pigment epithelium and effect of gene knockouts on sensitivity to oxidative stress. Mol Vis. 2007;13:566–77. Fig. C reproduced with permission from Sreekumar et al. Mechanism of RPE Cell Death in α-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux. PLoS ONE 7(3): e33420. doi:10.1371/journal.pone.0033420.

Figure 8.

Accelerated CoCl2-induced retinal degeneration in αA-crystallin KO and αB-crystallin KO mice, 96 hour post-injection. With 12 nmol (low dose) CoCl2 αA-crystallin KO and αB-crystallin KO retina revealed no apparent change compared to wild type retina (panels A,D,G). At 33 nmol (intermediate dose), αA-crystallin KO and αB-crystallin KO retina showed selective degeneration in both inner nuclear layer and outer nuclear layers while the wild type retina appeared comparatively intact (panels B,E,H). At 60 nmol (high dose), retina from both αA-crystallin KO and αB-crystallin KO mice exhibited extensive degeneration in all layers, while the wild type retina showed early changes of retinal degeneration (panels C,F,I).

GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, reinal pigment epithelium. Bar indicates 75 microns. Reproduced with permission from Yaung et al. Exacerbation of retinal degeneration in the absence of alpha crystallins in an in vivo model of chemically induced hypoxia. Exp Eye Res. 2008; 86:355–65.

Figure 9.

Exogenously added recombinant human (rh) αB-crystallin protects RPE cells from apoptosis. (A) Human RPE cells were treated with 500 μM H₂O₂ or 500 μM H₂O₂ plus 25 μg/ml rhαB-crystallin for 3 h, and apoptosis was assessed by TUNEL staining and cleaved caspase 3 staining. Apoptosis and caspase 3 staining were significantly higher in H₂O₂-only treated cells when compared with cells co-treated with rhαB-crystallin and H₂O₂. (B) Quantification of TUNEL-positive cells. One and two asterisks indicate *p<0.05, ** p<0.01. Reproduced with permission from Sreekumar et al. αB Crystallin Is Apically Secreted within Exosomes by Polarized Human Retinal Pigment Epithelium and Provides Neuroprotection to Adjacent Cells. PLoS ONE 2010: 5(10): e12578.

The antioxidant function of α-crystallin is linked to the cellular GSH level (Mehlen et al., 1996; Pal et al., 1998; Sreekumar et al., 2012a, 2012b). A direct correlation between αA-crystallin and GSH was found in lens epithelial cells; overexpression of αA-crystallin increased GSH, whereas lack of αA-crystallin caused a decrease in GSH expression (Kannan et al., 2001). Our recent study revealed that in α-crystallin (αA- or αB-crystallin) overexpressing cells GSH levels increased significantly particularly in RPE mitochondria, while in murine retina lacking α-crystallin (αA-crystallin KO or αB-crystallin KO) GSH decreased by 50% (Sreekumar et al., 2012). A corresponding increase in antiapoptotic function in α-crystallin overexpressing cells and an exacerbation of cell death in α-crystallin deficient cells was observed. In another study, the antiapoptotic function of αA-crystallin was shown to be directly interlinked with the chaperone function by enhancing P13K activity and reducing PTEN activity in HeLa and Chinese hamster ovary cells (Pasupuleti et al., 2010).

As has been mentioned earlier, distinct regions of αA- or αB-crystallin have been identified that exhibit chaperone functions. In experiments to test the hypothesis that some of these minipeptides are antiapoptotic, rat pheochromocytoma cells co-treated with β-amyloid peptide and αA-crystallin minipeptides were resistant to cell death (Santoshkumar and Sharma, 2004). However, the precise mechanism of this protection remains to be explored. Our preliminary observations in oxidatively stressed RPE cells suggest that peptides derived from both αA-crystallin and αB-crystallin have similar antiapoptotic properties to that of parent crystallins and this protection was through the inhibition of caspase 3 activation (Kannan et al., 2010). In additional studies, we found that poly(lactic-co-glycolic acid (PLGA) encapsulated αA- or αB-crystallin peptides were significantly more efficient in protection of RPE cells against oxidative stress than the free peptide in a dose and time dependent fashion. This suggests that nanocoated particle delivery of α-crystallin peptides to RPE could be an efficient approach in preventing RPE cell death and preserving cell function. We believe that conducting detailed investigation using in vitro and in vivo models with α-crystallin-derived chaperone peptides may prove to be valuable in developing therapeutic agents for retinal degenerative diseases.

5.5. Autophagy

Autophagy is an intracellular catabolic process which involves degradation of cell organelles and proteins through lysosomal pathways (Kaarniranta et al., 2009; Ryhanen et al., 2009; Wang et al., 2009a) and can also lead to non-apoptotic cell death (type 2 programmed cell death). Thus autophagy can either protect cells or promote cell death depending on the micro-environment of the cell. Among the three known autophagic mechanisms (Mizushima et al., 2008), we focus our comments mainly on chaperone-mediated autophagy (CMA) although the autophagic systems are not completely separated from each other. CMA is different from macroautophagy in that no vesicular trafficking is involved; instead a pentapeptide motif in substrate proteins allows their specific translocation to the lysosomal membrane (Cuervo, 2010; Dice, 2007).

Chaperone-mediated autophagy is involved in cellular stress response through selective targeting of cytosolic proteins for lysosomal degradation (Cuervo, 2010; Dice, 2007). Misfolded proteins and protein aggregates are linked to several pathological conditions associated with cardiovascular, oncological, and neurodegenerative disorders (Balch et al., 2008). The ability of αB-crystallin to promote either the destruction or renaturation of damaged proteins or unfolded proteins under diverse stress conditions potentially confers on them a major role in protein quality control and in the regulation of protein triage. Interestingly, α-crystallin expression is increased in neurodegenerative diseases such as AMD where it is a component of drusen (Crabb et al., 2002; De et al., 2007; Nakata et al., 2005). It has been hypothesized that increased autophagy, and exocytic activities in aged RPE could provide extracellular materials for the formation of drusen (Wang et al., 2009a). Consistent with this postulate, the presence of autophagic and exosomal markers in drusen from AMD patients has been reported (Wang et al., 2009a). We have presented experimental evidence that αB-crystallin is released from RPE cells through the exosomal route and co-localized with exosomal markers in RPE monolayers (Sreekumar et al., 2010). In polarized RPE monolayers, we evaluated secretion of αB-crystallin in exosomes by immunogold labeling and by immunofluorescence with CD63, an exosomal marker and found that the secretion was predominantly apical (Fig. 10). In severe oxidative stress causing RPE barrier breakdown, αB-crystallin containing exosomes accumulated in the basolateral side of RPE, indicating that exosomes may be a possible source for αB-crystallin accumulation in the drusen (Sreekumar et al., 2010). Drusen also contain potentially toxic components such as β-amyloid (Isas et al. 2010; Luibl et al., 2006; Wang et al., 2009b). Thus we speculate that the presence of αB-crystallin in drusen could be to decrease the toxic protein aggregation and to increase autophagy-mediated clearance. This notion is supported by the finding that a missense mutation (CryAB^R120G) in the αB-crystallin gene results in protein aggregation and aggresome formation (Sanbe et al., 2004), mitochondrial toxicity (Maloyan et al., 2005), disruption of proteasome function (Chen et al., 2005), and induces a state of reductive stress (Rajasekaran et al., 2007). The mutation of αB-crystallin decreased the expression of Atg7, a mediator of autophagosomal biogenesis and reduced the autophagic function of rat cardiomyocytes (Pattison et al., 2011). These authors further observed that induction of autophagy using Atg7 overexpression can rescue the accumulation of misfolded mutant αB-crystallin and aggregates (Pattison and Robbins, 2011; Pattison et al., 2011)

Figure 10.

Evidence for secretion of αB-crystallin via exosomes. (A) Western blot analysis showing preferential secretion of αB-crystallin into the apical domain of RPE cells. Secretion was measured in polarized human RPE monolayers with high trans-epithelial resistance in apical and basolateral medium. (B) Evidence by transmission electron microscopy showing that αB crystallin is present in exosomes of RPE cells. Exosomes averaged 100 nm in size. Immunogold labeling of αB-crystallin (15 nm gold particles) within exosomes is shown by arrows. (C) Immunofluorescence staining of αB-crystallin in RPE monolayers showing predominant apical staining as compared to central and basal regions. (D) Co-localization of αB-crystallin (green) with the exosomal marker CD63 (red) in polarized RPE. Panels A, C and D are reproduced with permission from Sreekumar et al. αB Crystallin Is Apically Secreted within Exosomes by Polarized Human Retinal Pigment Epithelium and Provides Neuroprotection to Adjacent Cells. PLoS ONE 2010: 5(10): e12578.

Few studies have addressed the role of autophagy in the retina in vivo, reporting contrasting results on both cytoprotective and prosurvival effects in photoreceptors in Drosophila (Wang et al., 2009c), in mouse models of axonal degeneration (Knoferle et al., 2010) and optic nerve axotomy (Rodríguez-Muela et al., 2012). Detrimental effects of autophagy have also been described in a mouse model of retinitis pigmentosa (Punzo et al., 2009) and in a rat model of ischemia (Piras et al., 2011). The importance of autophagy during early stages of retinal development has been documented; inhibition of autophagy resulted in accumulation of apoptotic cells during neurogenesis and the autophagic machinery provides the retina with the energy required for proper removal of cell remains and further degradation of apoptotic cells (Mellén et al., 2008). Thus, while the role of autophagy in retinal development and degeneration is clear, the link between αB-crystallin and autophagy remains speculative but should be an important area of future investigation. Support for this notion is provided by the recent demonstration that mutations in βA3/A1-crystallin impairs phagosome degradation and and results in a defect in autophagy in the RPE (Zigler et al., 2011).

5.6. Interaction with other proteins

Knowledge of protein-protein interactions is necessary to understand the functions of proteins and how these functions are regulated. Protein–protein interactions are decided by different factors, including hydrophobicity, solvent accessibility, and residue pairing preferences, which in turn are determined by the amino acid sequence and aspects of protein structure. The multiple β-strands of the β-sheet domain of αcrystallin have been suggested as one of the sites of protein–protein interactions (Hoskins et al., 2006). In addition to stabilizing different structurally compromised proteins during multiple pathological conditions, α-crystallins have been found to interact with several other proteins such as β-crystallins, α-crystallin, actin filaments, vimentin, CP49, filensin, intermediate filaments, VEGF, HSP27, galectin-related interfiber protein, apoptotic mediators, mitochondrial proteins and nuclear proteins (Barton et al., 2009; FitzGerald et al. 1991; Ghosh et al., 2007a; Nicholl and Quinlan 1994; Singh et al. 2007). A detailed list of α-crystallin interacting partners based on functional categories is presented in Table 1. Two examples of α-crystallin interaction, one with an antioxidant enzyme and the other with an angiogenic factor are shown in Figure 11. The interaction of αA- and αB-crystallin with methionine sulfoxide reductase A (MsrA) in the retina of a patient with age-related macular degeneration is shown in Fig. 11A. The binding of αB-crystallin to VEGF in RPE cells from two hypoxia animal models (OIR and CoCl₂-induced hypxoxia) is shown in Figure 11B. It is believed that sHSPs participate in the dynamic assembly and stabilization of several filamentous cytoskeletal elements and some of these associations protect against multiple stress conditions (Singh et al., 2007). αB-crystallin interacts directly with an array of filament proteins such as phakinin (CP49), filensin (CP119), desmin, glial fibrillary acidic protein (GFAP), vimentin, and actin where it functions in the organization and stabilization of the filament networks formed by these proteins (Djabali, et al,. 1999; Head et al., 2000; Launay et al., 2006; Muchowski et al., 1999; Prescott et al., 1996; Tomokane et al., 1991). Pin array analysis determined that the sequence, ₅₇APSWFDTG₆₄ of αB-crystallin had strong interactions with partially unfolded actin and this interactive sequence contains Ser-59, a key residue that is critical during several stress conditions (Ghosh et al., 2007b; Morrison et al., 2003). Phosphorylation of Ser-59 results in increased interactions between αB-crystallin and actin that is believed to protect actin filaments from unfolding and aggregation (Launay et al., 2006). αB-crystallin regulates actin filament dynamics in vivo and its association with actin helps maintenance of pinocytosis, a physiological function essential for survival of cells (Singh et al., 2007).

Table 1.

Proteins interacting with α-crystallins

Functional categories	Crystallin	Interacting protein	Study Type (Cell/tissue)	References
Apoptosis – related	αA	Caspase-3	ARPE-19, Lens epithelial cells	Hu et al., 2012; Mao et al., 2004
		Caspase 6	Lens extract	Morozov and Wawrousek, 2006
		Bax	ARPE-19, Lens epithelial cells	Mao et al., 2004
		Bcl-X(S)	ARPE-19, Lens epithelial cells	Mao et al., 2004
		Protease-activated receptor-2 (PAR-2)	Astrocytes	Li et al., 2009
		Methionine sulfoxide reductase (MsrA)	Retina	Sreekumar et al., 2011
	αB	Caspase-3	ARPE-19, Lens epithelial cells	Hu et al., 2012; Kamradt, et al., 2001; Mao et al., 2001, 2004
		Bax	ARPE-19, Lens epithelial cells	Hu et al., 2012; Mao et al., 2004
		Bcl-X(S)	ARPE-19, Lens epithelial cells	Mao et al., 2004
		Bcl-2-associated athanogene (BAG)	Cardiomyocytes	Hishiya et al., 2011
		Protease-activated receptor-2 (PAR-2)	Astrocytes	Li et al., 2009
		Methionine sulfoxide reductase (MsrA)	Retina	Sreekumar et al., 2011
		HSP22	COS7 cells	Fontaine et al., 2005
		Hsp27	HeLa cells	Fu and Liang, 2002.
Apoptosis – related	αA and αB crystallin	Methionine sulfoxide reductase (MsrA)	Human lens epithelial cells	Brennan et al., 2009
Cytoskeletal	αA	Actin	Lens Epithelial cells	Maddala and Rao, 2005
		Fiber cell cytoskeleton complex	Bovine lens cortex	FitzGerald and Graham, 1991
	αB	Actin	Cardiac tissue; Physico-chemical	Bennardini et al., 1992; Ghosh et al., 2007b; Singh et al., 2007
		Peripherin	Lens cell homogenate; Optic nerve head-astrocytes	Djabali et al., 1997
		Vimentin	Lens cell homogenate; Optic nerve head-astrocytes	Djabali et al., 1997; Muchowski et al., 1999 Salvador-Silva et al., 2001
		Filensin (CP115)	Lens cell homogenate	Muchowski et al., 1999
		Desmin filaments	Cardiac tissue; Physico-chemical	Bennardini et al., 1992; Ghosh et al., 2007a
		α-synuclein	Physico-chemical	Rekas et al., 2004; Waudby et al., 2010
		Glial fibrillary acidic protein (GFAP)	Astrocytoma cells, Optic nerve head astrocytes	Salvador-Silva et al., 2001; Wisniewski and Goldman, 1998
		Microtubules	L6E9 myoblasts cells, Lens epithelial cells, Oligodendroglial cell line	Bauer and Richter-Landsberg, 2006; Fujita et al., 2004; Xi et al., 2006
		Phakinin (CP49)	Lens cell homogenate	Muchowski et al., 1999
Cytoskeletal	αA and αB crystallin	Vimentin	Physiochemical	Nicholl and Quinlan, 1994
Growth factors	αB	Fibroblast growth factor 1	Physico-chemical	Edwards et al., 2001
		Platelet-derived growth factor (PDGF)	Cardiac myocytes	Shu et al., 2005
		FGF-2	Physico-chemical	Ghosh et al., 2007a
		NGF-β	Physico-chemical	Ghosh et al., 2007a
		VEGF	Physico-chemical, RPE cells	Ghosh et al., 2007a; Kase et al., 2010
Signaling	αA	α6 integrin receptor complexes	Lens fractions	Menko and Andley, 2010
		E-cadherin	Lens epithelial cells	Menko and Andley, 2010
	αB	β-catenin	Len epithelial cells, physico-chemical	Ghosh et al., 2007a; Maddala and Rao, 2005
		Protein kinase Cδ (PKC δ)	Retina	Kim et al., 2007
		F-box protein FBX4	HeLa cells	den Engelsman et al., 2003
		Cyclin D1	NIH3T3 cells/293T cells	Lin et al., 2006
		Kidney-specific cadherin-16	Kidney lysates	Thedieck et al., 2008
Amyloid associated	αA	Amyloid-β (Aβ) peptides	Physiochemical, PC12 cells	Santhoshkumar and Sharma, 2004; Tanaka et al., 2008
	αB	Amyloid-β (Aβ) peptides	Physico-chemical, Brain	Ecroyd and Carver, 2009; Raman et al., 2005; Shammas et al., 2011
Other Crystallins	αA	βA3-crystallin	HeLa cells	Gupta and Srivastava, 2009
		βB2 crystallin	HeLa cells	Fu and Liang, 2002
		C-crystallins	HeLa cells	Fu and Liang, 2002
	αB	αA crystallin	Physico-chemical	Sreelakshmi et al., 2004
		βA3-crystallin	HeLa cells	Gupta and Srivastava, 2009
		βB2 crystallin	HeLa cells	Fu and Liang, 2002
		βH crystallin	Physico-chemical	Ghosh et al., 2005
		γD crystallin	Physico-chemical	Ghosh et al., 2005
		δC-crystallins	HeLa cells	Fu and Liang, 2002
Others	αA	Galectin-related interfiber protein (GRIFIN)	Intact lens	Barton et al., 2009
	αB	Insulin	Physico-chemical	Ghosh et al., 2007a
		Alcohol dehydrogenase	Physico-chemical	Ghosh et al., 2005
		Citrate synthase	Physico-chemical	Ghosh et al., 2005
		HSP22	COS7 cells	Fontaine et al., 2005
		Survival motor neuron (SMN) protein	HeLa cells	den Engelsman et al., 2005
		Intranuclear lamin A/C speckles	Myoblasts	Adhikari et al., 2004
		Splicing factor SC-35	Myoblasts	Adhikari et al., 2004
		IKKβ	Myoblasts	Adhikari et al., 2011
	αA and αB crystallin	Vimentin	Physico chemical	Nicholl and Quinlan, 1994

Figure 11.

Protein-protein interaction of α-crystallin with methionine sulfoxide reductase A (MsrA) in retinas of patients with age-related macular degenetration and vascular endothelial growth factor (VEGF) in oxygen-induced retinopathy (OIR). (A) Immunohistochemical localization of αA-crystallin (red), αB-crystallin (red) and MsrA (green). Arrows indicate co-localization (yellow) of αA-crystallin or αB-crystallin and MsrA. *: Drusen. Bar indicates 50 microns. (B). Western blot for αB-crystallin, VEGF, and VEGF-R2 in anti–VEGF-A immunoprecipitates from wild-type and αB-crystallin KO mice at P12 and P17 in OIR. αB-Crystallin binds to VEGF-A at P12 and P17 in wild-type mice but not in αB-crystallin KO mice. Knockout of αB-crystallin does not affect VEGF-R2 binding to VEGF-A. (C). Localization of αB crystallin and VEGF in cultured RPE cells after hypoxia (a–d) -Expression of αB-crystallin and VEGF in cultured RPE cells after cobalt chloride (150 μM) treatment for 6 hr (b–d). Nuclei are identified with DAPI (a; blue); along with staining for αB-crystallin (b; red) and VEGF (c; green). Double staining for αB-crystallin and VEGF is seen in merged (d; yellow). Bar indicates 20 microns. Panel (A). Reproduced with permission from Sreekumar et al. Methionine sulfoxide reductase A: Structure, function and role in ocular pathology. World J. Biol. Chem.2011 2(184–192). Panels (B& C) “This research was originally published in Blood. Kase et al. alphaB-crystallin regulation of angiogenesis by modulation of VEGF. Blood 2010; 115: 3398–3406, © The American Society of Hematology.”

In addition, αB-crystallin interacts with several growth factors that are important for cell proliferation, differentiation and development such as fibroblast growth factor-2, nerve growth factor-β, VEGF, insulin, and β-catenin further suggesting its significance during normal development (Andley, 2007; Djabali et al., 1997; Gusev et al., 2002; Sun and Macrae, 2005). Although αB-crystallin is a cytosolic protein, under pathological situations or oxidative stress, αB-crystallin migrates to the nucleus, mitochondria or endoplasmic reticulum (Dou et al., 2010; Kase et al., 2010; Sreekumar et al., 2010; Yaung et al., 2008, 2007) and interacts with other structural or regulatory proteins (Ghosh et al., 2007a, 2007b). Cell migration is a physiological or pathological process and conformational changes in proteins have been associated with migration and the migrated proteins require special protective assistance to prevent protein aggregation or protein misfolding. For instance, αB-crystallin colocalizes with β-catenin during cell migration and is likely the chaperone for β-catenin (Maddala and Rao, 2005). The identification of chaperone sequence in αB-crystallin that mediates interactions with β-catenin during oxidative stress may protect and inhibit β-catenin degradation thereby enabling cell migration. A study by Ghosh et al. (2007a) provides in vitro evidence that fibroblast growth factor-2, nerve growth factor-β, VEGF, insulin, and β-catenin interact with specific sequences of αB-crystallin, previously reported to have chaperone activity (Ghosh et al., 2005) and further supports the known role of αB-crystallin in the pathways associated with these growth factors (Andley et al., 2000; Civil et al., 2000; Edwards et al., 2001; Maddala and Rao, 2005). In lens epithelial cells, FGF-2 treatment significantly induced the expression of all major crystallins, particularly α-crystallins (Mailankot et al., 2010) supporting the finding that αB-crystallin has specific interacting sequences for FGF-2 (Ghosh et al., 2007a). The overlap between the interactive sequences in αB-crystallin for chaperone activity and binding with VEGF is of high biological significance given the known role of αB-crystallin in VEGF related functions such as angiogenesis (Kase et al., 2010), vascular tube formation (Dimberg et al., 2008) and protecting VEGF from intracellular degradation (Ruan et al., 2011). The latter finding was reported in a recent study in which knockdown of αB-crystallin significantly increased the proteolytic degradation of VEGF in endothelial cells (Ruan et al., 2011). Further, VEGF levels were significantly lower in αB-crystallin KO mice exposed to hypoxia followed by minimal retinal angiogenesis probably due to VEGF proteolysis (Kase et al., 2010). However, it can be argued that since αB-crystallin is intracellular, the possibility of the interaction with growth factors will be minimal. However, recent evidence shows extracellular release of αB-crystallin (Gangalum et al., 2011; Sreekumar et al., 2010). Since αB-crystallin protects VEGF against unfolding, aggregation and proteolysis, it has therapeutic potential as a VEGF stabilizing molecule.

In addition, there are other α-crystallin binding partners and their number will increase as the research on finding new functions of crystallins intensifies. Methionines of αA- and αB-crystallins are oxidized in aged human lenses (Lund et al., 1996). Methionine oxidation damages α-crystallin chaperone activity, but methionine sulfoxide reductase A (MsrA) can repair oxidized methionines in α-crystallin and restore the chaperone function (Brennan et al., 2009). We have previously reported strong immunoreactivity for MsrA in sub-RPE macular drusen from patients with age-related macular degeneration (Sreekumar et al., 2011, 2005). Both αA- and αB-crystallins co-localize with MsrA in AMD retinas, particularly in the drusen, suggesting potential interaction between the two types of proteins (Fig. 11A). A recent study identified a direct interaction between αB-crystallin and Bcl-2-associated athanogene 3 (BAG3) in myoblast cells and the interaction could inhibit the aggregation and toxicity (Hishiya et al., 2011). Stidies on peptides that inhibit this interaction in an in vitro binding assay indicate that two conserved Ile-Pro-Val regions of BAG3 are involved in the interaction with αB-crystallin, which is similar to results showing BAG3 binding to HspB8 and HspB6. Further, BAG3 overexpression increased αB-crystallin R120G solubility and inhibited its intracellular aggregation (Hishiya et al., 2011). The importance of metal ions in regulating chaperone activity has been well documented (Biswas and Das, 2008). It is known that αA- and αB-crystallins are involved in the maintenance of redox homeostasis (Ahmad et al., 2008; Sreekumar et al., 2012) and their interaction with metal ions such as Cu²⁺ modulate the chaperone activity (Ganadu et al., 2004; Moschini et al., 2006). A recent study demonstrated that αA-crystallin chaperone site is also a Cu²⁺-binding site (Raju et al., 2011). We anticipate that further investigation will identify new interacting partners of these two major chaperone proteins and will reveal potential therapeutic approaches for retinal and other neurodegenerative and other aggregation diseases.

5.7. Interaction of α-crystallin with other small heat shock proteins

Substantial evidence exists on the interaction of α-crystallins between themselves and with other sHSPs. Studies show that the interaction between αA- and αB-crystallin is required to form structurally stable complexes (Srinivas et al., 2002). Although peptide scan studies provide evidence for two regions in αB-crystallin, spanning from residues 42–57 and 60–71, to be involved in interaction with αA-crystallin, mutation studies revealed that sequence 42–57 is the only major interacting region in αB- and αA-crystallin oligomer formation (Sreelakshmi et al., 2004). αB-crystallin is copurified with Hsp27, these proteins form tight complexes (Kato et al., 1992; Zantema et al., 1992) and the complexes formed by Hsp27 and αA- or αB-crystallin are detected both under in vitro and in vivo conditions (Bova et al., 2000; Kato et al., 1992; Zantema et al., 1992). It is hoped that a detailed analysis of the sHSP interactions will contribute to a better understanding of the biological role of sHSP complexes. The interactions of sHSPs with one another may be of significant medical relevance since mutations in sHSPs including αA-crystallin and αB-crystallin are associated with human cataracts, and desmin-related myopathy.

5.8. Amyloid β-αB-crystallin interaction and neurodegenerative diseases

Given the known similarity between brain degenerative disease (Alzheimer Disease) and retinal degenerative disease (AMD) with regard to the presence of amyloid β (Aβ), the interaction between Aβ and α-crystallins is of considerable interest with the objective of developing a common therapeutic strategy (Ohno-Matsui, 2011). Visual problems have been observed during the initial stages of Alzheimer Disease (AD) (Sadun et al., 1987) with subsequent reduction in the number of ganglion cells and in the thickness of the nerve-fiber layer, increase in optic disc cupping, retinal vascular thinning and visual function impairment (Guo et al., 2010; Hinton et al., 1986). Evidence for the potential role of Aβ in the pathogenesis of AMD has been shown in in vitro studies (Bruban et al., 2009; Luibl et al., 2006; Wang et al., 2008) as well as in different animal models (Bruban et al., 2009; Ding et al., 2011; Wang et al., 2008; Yoshida et al., 2005). Beta amyloid, a 39–43 amino acid residue cleavage product of the amyloid precursor protein (APP), has been identified in the retinas of AD patients (Koronyo-Hamaoui et al., 2011) and is also one of the components of drusen (Anderson et al., 2004; Dentchev et al., 2003; Isas et al., 2010; Luibl et al., 2006; Wang et al., 2008). The aggregation of these peptides is thought to be critical to the pathogenesis of the diseases (Ohno-Matsui, 2011), however, α-crystallins are also found colocalized with Aβ p eptides (Liang, 2000; Muchowski and Wacker, 2005; Stege et al., 1999). Of special interest, αB-crystallin is associated with Aβ deposition in supranuclear cataracts in lenses from patients with AD (Goldstein et al., 2003).

The observation that Aβ and αB-crystallin are colocalized in vivo (Liang, 2000; Stege et al., 1999) has led to detailed investigations into the effect of αB-crystallin on Aβ aggregation under in vitro experimental conditions. Several independent studies indicated that α-crystallin is capable of interacting with Aβ (Liang, 2000; Raman et al., 2005; Santhoshkumar and Sharma, 2004; Shammas et al., 2011; Stege et al., 1999; Wilhelmus et al., 2006). Some of these studies showed that upon interaction, αB-crystallin enhanced β-sheet structure in Aβ (Liang, 2000; Stege et al., 1999) and stabilized Aβ into toxic β-sheet rich oligomers as evidenced by increased toxicity in cultured rat neuronal cells (Stege et al., 1999). In support, Narayanan et al. (2006) found that instead of preventing the cell from toxicity of Aβ, αB-crystallin induced an increased toxicity which could arise from the oxidation of the side chain of the Met35 in Aβ by αB-crystallin. However, several other studies reported that αB-crystallin inhibited fibril formation (Dehle et al., 2010; Raman et al., 2005; Shammas et al., 2011; Wilhelmus et al., 2006) and presented evidence for a protective effect of αB-crystallin in Aβ-induced cytotoxicity (Dehle et al., 2010; Wilhelmus et al., 2006). Similarly, αA-crystallin also inhibited fibril formation and protected neuronal cells from cytotoxicity (Santhoshkumar and Sharma, 2004). Controversy exists regarding the ability of αB-crystallin to inhibit the toxicity associated with Aβ fibril formation (Ecroyd and Carver, 2009). The results are highly dependent on how the experiments are conducted, including the method of preparation, purification and handling of the Aβ peptide, the incubation conditions and in particular whether Aβ and αB-crystallin are pre-incubated before addition to the cell culture media. In a recent study Dehle et al. (2010) pre-incubated both αB-crystallin and Aβ species prior to addition to the culture media and observed a correlation between the inhibition of cell toxicity and fibril inhibition. However, in previous studies both species have been simultaneously added to the cell culture media (Stege et al., 1999). Similar findings have been documented for kappa-casein (which forms fibrils) and αB-crystallin, implying that αB-crystallin’s generic ability to inhibit fibril formation is coupled with its inhibition of cell toxicity (Dehle et al., 2010). It has to be noted that all the above studies were performed in different cell types and hence in addition to variations in experimental protocols, the observed effects may also be dependent on the type of cell line used. While αB-crystallin could prevent β-amyloid fibril formation, in a very recent study, Laganowsky et al. (2012) found that a segment of αB-crystallin called ‘cylindrin’ peptide formed a toxic amyloid small oligomer. However, it is intriguing that treatment with αB-crystallin for 3 weeks did not produce apparent toxicity either in in vitro RPE monolayer (Sreekumar et al., unpublished observations) or in animal models (Arac et al., 2011; Ousman et al., 2007; Pangratz-Fuehrer et al., 2011; Velotta et al., 2011). Thus, defining the precise beneficial role of αB-crystallin in β-amyloid fibril formation would require further study.

In vivo, the upregulation of α-crystallins has been reported in a variety of protein misfolding and neurodegenerative diseases (De et al., 2007; Guo et al., 2007; Kase et al., 2011; Kumar et al., 2005; Lowe et al., 1992; Nakata et al., 2005; Pountney et al., 2005; Rao et al., 2008; Renkawek et al., 1994; Shinohara et al., 1993; Wilhelmus et al. 2006), and αB-crystallin co-localization to amyloid deposits (Lowe et al., 1992; Pountney et al., 2005; Renkawek et al., 1994; Shinohara et al., 1993; Wilhelmus et al., 2006) may represent an important protective effect to mitigate fibril formation and prevent the progression of some of these diseases involving Aβ-αB-crystallin interaction. Aβ is known to induce inflammation (Johnson et al., 2000; Wang et al., 2009b), alter mitochondrial functions (Bruban et al., 2009), regulate growth factors (Wang et al., 2009b; Yoshida et al., 2005), alter RPE tight junction proteins and RPE hypertrophy (Bruban et al., 2009; Ding et al., 2011; Luibl et al., 2006). αB-crystallin on the other hand, is anti-inflammatory (Arac et al., 2011; Ousman et al., 2007; Rothbard et al., 2012), and is antiapoptotic, in part, through elevation of mitochondrial antioxidants (Sreekumar et al., 2012), and growth factor regulation (Ghosh et al., 2007a; Kase et al., 2010) so that it can be hypothesized that αB-crystallin could be a potential therapeutic molecule for the treatment for Aβ associated retinal diseases and other neurodegenerative diseases.

5.9. Interaction of α-crystallin with ubiquitin-proteasome system

The quality control and maintenance of proteasome homeostasis is achieved mostly by molecular chaperones which assist in de novo protein folding or refolding and the ubiquitin-proteosome (UPS) and autophagy systems which mediate timely removal of aggregated or misfolded proteins (Hartl et al., 2011). Accordingly, the proteasome complex is involved in the cell cycle, signal transduction pathways, transcriptional regulation, stress responses, cell differentiation, and metabolic adaptation (Gottesman et al., 1997). Participation of αB-crystallin in protein degradation pathways has been described. αB-Crystallin is reported to interact both in vitro and in vivo with C8/α7, one of the 14 subunits of the 20S proteasome (Boelens et al., 2001). The interaction is highly specific as C8/α7 does not interact with αA-crystallin or HSP27 (Boelens et al., 2001). αB-Crystallin can also contribute to the degradation of some misfolded proteins which cannot be converted into their native state after repeated cycling through the chaperone systems by the proteasome (den Engelsman et al., 2004; Lin et al., 2006). When αB-crystallin is phosphorylated at Ser-19 and Ser-45, it can interact with FBX4, which is a component of ubiquitin-ligase SCF (den Engelsman et al., 2003). This combination is of particular interest because these serines are mainly phosphorylated during mitosis (Kato et al., 1998), suggesting that the interaction with FBX4 might play a role during cell division. Both FBX4 and αB-crystallin are required for rapid ubiquitination and degradation of cyclin D1 in vivo and that purified SCF-FBX4-αB-crystallin complexes can direct cyclin D1 ubiquitination in vitro (Lin et al., 2006). Loss of αB-crystallin resulted in cyclin D1 stabilization showing the requirement of the SCF-FBX4-αB-crystallin ligase for cyclin D1 proteolysis (Lin et al., 2006). Another target for αB-crystallin is VEGF. Selective disruption of UPR pathways (IRE1/XBP-1, ATF6) or knockdown of αB-crystallin significantly increased VEGF proteolytic degradation (Ruan et al., 2011). We have shown that in the absence of αB-crystallin, VEGF is degraded, resulting in decreased secretion (Kase et al., 2010). Inhibition of the 26S proteasome partially rescues VEGF secretion leading to increased retinal angiogenesis, suggesting that in the absence of αB-crystallin, misfolded, mono-ubiquitinated VEGF could be exported to the cytoplasm and undergoes ubiquitination and degradation (Kase et al., 2010).

Another recently discovered target for αB-crystallin ubiquitination is NF-kB and this interaction is phosphorylation dependent and promotes cell survival (Adhikari et al., 2011). Oxidative stress by TNF-α caused phosphorylation of αB-crystallin at Ser-59, and phosphorylated αB-crystallin associates and enhances the kinase activity of the IKK complex, thereby facilitating phosphorylation and subsequent degradation of IkB (Adhikari et al., 2011). Thus, the mutation and or absence/decreased expression of αB-crystallin could inhibit the proteasomal pathways and result in the accumulation of denatured proteins or lead to toxicity/cell death. Further understanding of these processes may allow the development of promising targets in the therapy of degenerative diseases.

5.10. Mechanism for the export of αB-crystallin

Identification of HSPs and sHSPs in extracellular fluids has been a puzzling finding since the majority of these proteins lack the consensus signal sequence required for secretion via the classical endoplasmic reticulum-golgi pathway. α-Crystallins are synthesized in the cytosol and exported to extracellular space by an active mechanism that is not blocked by typical inhibitors of the ER-Golgi pathway, such as brefeldin or tunicamycin. Consequently, we hypothesized that αB-crystallin could be released by an alternative mechanism mediated by the non-classical secretory or unconventional pathway. To test the hypothesis, we collected exosomes from RPE cultured in exosome-free medium, and isolated exosomes using high speed centrifugation (Thery et al., 2006) and characterized exosomes by immunogold labeling using known exosomal markers such as CD63 (Fig. 10). Our studies revealed that αB-crystallin localized to exosomes. This was confirmed by immunoblot analysis that verified positivity for CD63 and αB-crystallin. Exosomes are rich in cholesterol and sphingolipids (Simpson et al., 2008; Thery et al., 2009) and inhibition of lipid rafts caused a significant decrease in exosome release and extracellular αB-crystallin suggesting participation of exosomes in αB-crystallin secretion in RPE cells (Sreekumar et al., 2010). In addition, we investigated the polarized nature of αB-crystallin secretion since αB-crystallin was reported to be neuroprotective (Ousman et al., 2007). Our results using highly polarized human RPE monolayers revealed preferential secretion of αB-crystallin toward the apical photoreceptor facing neural retina which supported its neuroprotective function (Fig. 12A,B). A recent report using ARPE-19 cells showed similar results on exosomal release of αB-crystallin (Gangalum et al., 2011). Further, exogenous αB-crystallin is taken up by RPE cells and photoreceptors with oxidative stress (Fig. 12C,D). As is well known, exosomes have diverse functions in the immune response, antigen presentation, intracellular communication, and the transfer of RNA and proteins (Simpson et al., 2009). Exosomes also contain inactive forms of both mRNA and microRNAs that can be transferred to a neighboring cell, conferring new functional properties to the recipient cell (Mathivanan et al., 2012, 2010). Therefore, detailed studies on microRNA profiling of diseased cell-derived exosomes and exosomes circulating in blood and their possible regulation of crystallins are needed in order to assess their potential as diagnostic biomarkers of different neurodegenerative disorders.

Figure 12.

Extracellular localization of αB-crystallin in murine interphotoreceptor matrix in retinal tissue sections, and uptake of exogenous αB crystallin in RPE and photoreceptors in vitro. (A). Double staining is shown for interphotoreceptor retinoid-binding protein (IRBP, red) and αB-crystallin (green) demonstrating partial colocalization in the merged image. Arrows indicate yellow staining in the merged image for αB crystallin and IRBP in the inter photoreceptor matrix (IPM). (B) Transmission electron microscopy of murine retinal sections shows distribution of αB-crystallin (15 nm gold particles, arrows in the figure and in inset) in the IPM and photoreceptor inner (IS) and outer segments (OS). (C) Uptake of fluorescein labeled recombinant human (rh) αB-crystallin in the presence or absence of oxidative stress (150 or 500 μM H₂O₂). Nuclear and cytoplasmic uptake of αB-crystallin (green) is seen after 1 h treatment which is more pronounced with 500 μM H₂O₂. (D) Uptake of fluorescein labeled 25 μg/ml rhαB-crystallin in photoreceptors of mouse retinal explant cultures co-treated with 500 μM H₂O₂ for 30 min (arrows in right panel and in inset). Unstressed control is shown in the left panel. INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment; OS, outer segment. Reproduced with permission from Sreekumar et al. αB Crystallin Is Apically Secreted within Exosomes by Polarized Human Retinal Pigment Epithelium and Provides Neuroprotection to Adjacent Cells. PLoS ONE 2010: 5(10): e12578.

5.11. Extracellular functions of α-crystallin

We will now turn our attention to the hypothesis that secreted sHSPs can act as signaling molecules in different cell types (eg. microglia, leukocytes and vascular endothelial cells) and can regulate immune and inflammatory mechanisms. Before addressing the biological relevance of extracellular sHSPs, it should be noted that a number of studies have demonstrated the extracellular presence of several HSPs (Campisi et al., 2003; Gangalum et al., 2011; Lai et al., 2006; Oh et al., 2012; Rothbard et al., 2012; Sreekumar et al., 2010). Furthermore, their release could be induced by endogenous and exogenous factors such as cell polarity and stress stimuli. As mentioned earlier, we have provided evidence that αB-crystallin is secreted by RPE cells via a non-classical pathway that involves exosomes (Sreekumar et al., 2010).

Several studies suggest that α-crystallins promote anti-inflammatory rather than pro-inflammatory innate immune response profiles (Arac et al., 2011; Masilamoni et al. 2006, 2005; Ousman et al., 2007; Pangratz-Fuehrer et al., 2011; Rao et al., 2012). Also α-crystallin has been shown to be upregulated in diseases such as AMD, and diabetic retinopathy (De et al., 2007; Kumar et al., 2005; Losiewicz and Fort, 2011; Nakata et al., 2005). However, in spite of the upregulation of α-crystallin, chronic inflammation persists or disease progression continues. Although oxidative stress resulted in the upregulation of α-crystallins (Kumar et al., 2005; Rao et al., 2008; Yaung et al., 2007), the amount of α-crystallins synthesized and/or released may not be adequate for eliciting their anti-inflammatory role or protein modifications could compromise their functions resulting in inflammation to persist. For example, in diabetic retinopathy, αcrystallins showed significant upregulation, but the upregulated crystallins could not stop inflammation and subsequent apoptosis (Kumar et al., 2005). This hypothesis was further verified in multiple in vivo animal models such as experimental autoimmune encephalomyelitis (Ousman et al., 2007), ischemia-reperfusion (Pangratz-Fuehrer et al., 2011; Velotta et al., 2011), stroke (Arac et al., 2011) and experimental optic neuropathy (Pangratz-Fuehrer et al., 2011). Systemic or exogenous delivery of αB-crystallin had broad anti-inflammatory activity that counteracted the pro-inflammatory environment thereby providing a stable cellular homeostasis. In experimental autoimmune uveitis, systemic delivery of αA-crystallin ameliorated the disease by decreasing infiltration of immune cells into the retina and suppression of immune cell function (Rao et al., 2012). These authors showed that intravenous αA-crystallin administration caused a marked decrease in Th1 cytokines and inhibition of the expression of toll-like receptors (Rao et al., 2012). Intraperitoneal delivery of α-crystallin (a combination of αA- and αB-crystallin) in a silver nitrate-induced inflammatory model inhibited neuroinflammation by inhibiting synthesis and release of inflammatory cytokines and nitric oxide synthesis (Masilamoni et al., 2006, 2005). The above findings suggest that the effects of exogenous administration of α-crystallins are not limited to a single disease model but can be effective in multiple disease models where inflammation is involved in disease initiation or progression. Of note, irrespective of the models studied, either αA- or αB-crystallin or their combination inhibited cell death whether it is in the brain, retina or heart, demonstrating that exogenous delivery offers a unique approach for preventing diseases involving multiple cell/tissue types. To explore how αB-crystallin exerts its therapeutic effect, Rothbard et al. (2012) studied the proinflammatory proteins that bind with αB-crystallin. Binding of αB-crystallin in the plasma occurred with an array of proinflammatory proteins which influence both innate and adaptive immunity in a temperature dependent manner, thereby modulating inflammation. In experimental autoimmune uveitis, systemic injection of αA-crystallin resulted in the downregulation of Toll-like receptors; and NFkB-related genes, including Ccl2, IFN-γ, NFkB, TNF-α, and TNF-α receptors and retinas were free of inflammatory cell infiltration and were well preserved (Rao et al., 2009). It is also important to note in this context that αA-crystallin has been reported to be an endogenous ligand for TLR4 (Roelofs et al., 2006). While systemic delivery of either - αA or αB-crystallin resulted in the inhibition of inflammation and apoptosis, the mechanism is different for the two crystallins. αA-crystallin acts through the TLR4 –NFkB pathway (Rao et al., 2012) and αB-crystallin by binding and reducing the amount of inflammatory plasma proteins (eg. IL-6) (Rothbard et al., 2012).

Receptor-mediated signaling by HSPs has been a subject of debate because the receptors (Toll-like receptors) can be activated by bacterial products that may be present in the recombinant preparations used in the experiments. This could lead to activation of NF-κB and increased production of TNFα, IL-6 and several chemokines (Takeda and Akira, 2005). Despite earlier controversy, TLR2 and TLR4 are still considered candidate receptors for HSP60, HSP70 and αA-crystallin (Habich et al., 2006; Roelofs et al., 2006). Whether exogenous αA-crystallin is primarily responsible for induction or inhibition of inflammatory mediators is not quite clear. Dendritic cells treated with αA-crystallin induced secretion of IL-6, TNF-α, and to a lesser extent IL-10, which resembled the cytokine pattern induced by lipopolysaccharide stimulation. This may involve TLR4 since with blockade of TLR4 or in TLR4 KO mice this effect was clearly abrogated (Roelofs et al., 2006). This phenomenon appears to be specific for αA-crystallin since HSP27 and αB-crystallin did not induce cytokine production (Roelofs et al., 2006). In another study, activated retinal microglial cells treated with α-crystallin (mixture of αA- and αB-crystallin) in in vitro or intravitreal injection of α-crystallin after optic nerve crush, significantly reduced inflammation by downregulating the expression and release of TNF-α and inducible nitric oxide synthase (Wu et al., 2009). However, it has to be noted that incubation of non-activated microglia with α-crystallin resulted in an increased production of TNF-α and inducible nitric oxide synthase (Wu et al., 2009). Therefore, the function of αA-crystallin as a pro- or anti-inflammatory protein depends among other things, on the stage or status of the cells, form of α-crystallin used, and the disease type.

Several questions remain unanswered. Further research is needed to define precisely the physiological functions of α-crystallins in the extracellular space. Second, it is not clear whether extracellular α-crystallins are capable of acting as chaperones. Finally, how extracellular α-crystallins enter cytosol from outside would need further investigation.

6. Changes in αB-crystallin expression in retina and relationship to retinal diseases

All the functions described for αB-crystallins above have distinct implications in the pathogenesis of AMD, DR and related disorders. In AMD tissue samples, αB-crystallin has been identified in drusen and is prominently expressed in the RPE associated with drusen (Fig. 4) (Crabb et al., 2002; Johnson et al., 2005; Nakata et al., 2005; De et al., 2007). This has led to the concept that increased αB-crystallin expression in RPE may be a “biomarker” of AMD (De et al., 2007). An increase in αB-crystallin expression was also seen in Muller glial cells, cones, and cone terminals overlying and adjacent to drusen (Johnson et al., 2005); this may be a stress response in the photoreceptors and Muller cells but could also represent increased uptake in retinal cells from exosomal secretion of αB-crystallin from RPE (Sreekumar et al, 2010). The localization of αB-crystallin in synaptic regions may be regulated by post-translational modifications as has been shown for Ser-59 phosphorylation of αB-crystallin in hippocampal neurons (Schmidt et al., 2012)

Kumar et al. (2005) showed that in streptozotocin (STZ) –induced diabetic rats both αA- and αB-crystallin increased in the retina with considerable degradation of αB-crystallin. An increase in α-crystallin expression was also reported by Fort et al. (2009) using retinal proteomics and immunostaining in the STZ rats. Elevated α-crystallin expression was found around the blood vessels of the ganglion cell layer as well as in activated Mueller cells. Kase et al. (2011) reported increased expression of αA-crystallin in the retina of diabetic retinopathy (DR) patients. This group also described an increased expression of αB-crystallin in the cytoplasm of endothelial cells of epiretinal membranes in proliferative DR where it co-localized with VEGF (Dong et al., 2012). Several other reports in animal models of DR confirm an increase in αA- and/or αB-crystallin in the retina (VanGuilder et al., 2011; Kandpal et al., 2012).

These observations suggest the importance of α-crystallins in retinal disorders. A compilation of several ocular and other neurological disorders that involve changes in α-crystallins is presented in Table 2.

Table 2.

Pathological Disorders Associated with α αα-Crystallin

Disease	Species	Tissue/crystallin	Change in level	Reference (s)
Ocular Disorders
Age-related macular degeneration	Human	Retina/αA & α B crystallin	Increase	Nakata et al., 2005
Age-related macular degeneration	Human	Retina/αB crystallin	Increase	De et al., 2007; Johnson et al., 2005
Diabetic retinopathy	Human	Retina/αA crystallin	Increase	Kase et al., 2011
Proliferative diabetic retinopathy	Human	Epiretinal membranes/αB crystallin	Increase	Dong et al., 2012
Retinoblastoma	Human	Retina/αA crystallin	Increase	Kase et al., 2009; Mallikarjuna et al., 2010
Retinoblastoma	Human	Retina/αB crystallin	Increase	Pineda et al.,1993
Leber Congenital Amaurosis	Human	Retina/αA crystallin	Increase	Vorum et al., 2007
Sympathetic Ophthalmia	Human	Retina αA crystallin)	Increase	Kase et al., 2012
Experimental diabetes	Rat	Retina/αA and αB crystallin	Increase	Fort et al., 2009; Kumar et al., 2005; VanGuilder et al., 2011
Experimental diabetes	Rat	Retina/αA crystallin	Increase	Wang et al., 2007
Experimental diabetes	Mouse	Retina/αA crystallin, αB crystallin	Increase	Losiewicz and Fort, 2011
Retinal degeneration	rd Mouse	Retina/αB crystallin	Increase	Jones et al., 1998
Retinitis pigmentosa	RCS Rat	Retina/αA crystallin	Decrease	Maeda et al., 1999
Staphylococcus aureus-induced endophthalmitis	Mouse	Retina/αB crystallin	Increase	Whiston et al., 2008
Experimental Autoimmune uveitis	Mouse	Retina/αA crystallin	Increase	Rao et al., 2008
Retinal ganglion cell (RGC) degeneration model	Rat	Retina/αA & αB crystallin	Decrease	Munemasa et al., 2009
Retinal neovascularization	Mouse	Retina/αA & αB crystallin	Decrease	Zhou et al., 2011
Retinal neovascularization	Mouse	Retina/αB crystallin	Increase	Kase et al., 2010
Experimental axotomy induced retinal ganglion cell degeneration	Rat	Retina/αA & αB crystallin	Decrease	Munemasa et al., 2009
Spontaneous equine recurrent uveitis	Horse	Retina/αA crystallin	Increase	Hauck et al., 2007
Steroid induced ocular hypertension model	Rat	Retina/αA crystallin	Decrease	Miyara et al., 2008
CoCl2-induced retinal degeneration (intermediate dose)	Mouse	Retina/αA & αB crystallin	Increase	Yaung et al., 2008
CoCl2-induced retinal degeneration (High dose)	Mouse	Retina/αA & αB crystallin	Decrease	Yaung et al., 2008
Light Injury	Rat	Retina/αA & αB crystallin	Increase	Organisciak et al., 2011; Sakaguchi et al., 2003
Neurologic disorders
Alexander disease	Human	Brain/αB crystallin	Increase	Iwaki et al., 1989; Kato et al., 2001
Creutzfeldt-Jacob disease	Human	Brain/αB crystallin	Increase	Rekas et al., 2004; Renkawek et al., 1992
Alzheimer disease	Human	Brain/αB crystallin	increase	Björkdahl et al., 2008; Renkawek et al., 1994
Parkinson disease	Human	Brain/αB crystallin	Increase	Waudby et al., 2010
Olivary hypertrophy	Human	Brain/αB crystallin	Increase	Fukushima et al., 2006
Pediatric Epilepsy	Human	Brain/αB crystallin	Increase	Sarnat and Flores-Sarnat 2009; Sarnat et al., 2011
Amyloidotic polyneuropathy	Human	Brain/αB crystallin	Increase	Magalhães et al., 2010
Multiple sclerosis	Human	Brain/αB crystallin	Increase	Sinclair et al., 2005; van Noort et al., 1995
Experimental autoimmune encephalomyelitis (EAE)	Mouse	Brain/αB crystallin	Increase	Ousman et al., 2007

7. α-Crystallin as a therapeutic molecule

Current therapies for ocular degenerative diseases such as AMD, diabetic retinopathy, and other neuroinflammatory diseases may be useful only in a small fraction of the patients and are ineffective in most patients thus underscoring a vital need for new therapies. α-Crystallins, especially αB-crystallin has shown to have pluripotent functions stemming from cell protection through preventing inflammation. Recombinant αB-crystallin or αA-crystallin or a combination of α-crystallin therapy was shown to be effective in multiple disease models such as experimental autoimmune encephalomyelitis, stroke, cardiac ischemia-reperfusion, optic neuropathy, experimental autoimmune uveitis, and other inflammation-induced toxicity models. The route of administration of α-crystallin in these studies was either intravenous, intravitreous or intraperitoneal and irrespective of the route of administration, α-crystallin showed anti-inflammatory and neuroprotective functions and rescued the phenotype in all the models studied. However, the amount and duration of treatment varied based on the route of delivery; lower and single dose regimens were used in localized delivery while multiple and extended treatment was required for systemic or intraperitoneal delivery. Recombinant human α-crystallins were used in all published in vitro and in vivo studies. The major advantages of recombinant proteins are related to their higher specificity and to their safety since they are produced by recombinant DNA technology. However, there are disadvantages such as low bioavailability and short half-life, conformational changes and tendency to irreversible aggregation on long-term storage. Because of these limitations, different techniques have been developed for stabilizing the protein drugs using carriers whether in entrapped form, encapsulated in a semipermeable membrane, covalently bonded to a carrier or adsorbed to the carrier. As stated earlier, in the case of α-crystallin, specific regions within the parent proteins have similar chaperone, antiapoptotic properties and antifibrilogenic functions (Bhattacharyya et al., 2006; Ghosh et al., 2005; Kannan et al., 2010; Santhoshkumar and Sharma, 2004; Sharma et al., 2000). The 20-mer mini-chaperone peptide derived from either αA- crystallin or αB-crystallin protected human RPE cells from oxidant-induced stress by inhibiting caspase 3 activation (Kannan et al., 2010). Further, PLGA nanoparticles loaded with either αA- or αB-minichaperone peptide protected RPE cells from oxidative stress-induced cell death and more effectively than the peptide alone for the same doses, suggesting the efficacy of carrier mediated delivery. Polymeric nanoparticles have more advantage over other carrier molecules as they can be easily modified and conjugated to targeting proteins or peptides. For example, it has been reported that PLGA nanoparticles containing superoxide dismutase, effectively prevented H₂O₂ induced neuronal cell death when compared to superoxide dismutase alone (Reddy et al., 2008). An area of great interest to our laboratory is determining the therapeutic efficacy of free and nanoparticle coated α-crystallin minichaperone peptides in arresting disease progression in established animal models of ocular disease. However, more information needs to be gathered on establishing the best route and duration for administration of α-crystallins and their derivatives, as well as the selection of the ideal formulation of crystallins that is safe and non-toxic. In addition, whether αB-crystallin based therapy would inhibit or promote angiogenesis and whether α-crystallin combination therapy provide greater protective benefit than α-crystallin monotherapy are some other issues to consider before initiating clinical therapy.

8. Future Perspectives

From the extensive nature of the disorders listed in Table 2, one can appreciate how the initial research which was confined to the cataract-preventing and chaperone properties of α-crystallins in the lens has expanded greatly in the last decade. Our emphasis in the present review is on the multifaceted functions of α-crystallins in non-lenticular tissues particularly the retina. In the retina, participation of α-crystallins in a variety of cellular processes has been characterized using mainly RPE as the cell system. The multiple functions of α-crystallins known to date are shown schematically in Fig. 13. The basic and mechanistic findings outlined in Fig. 13 have laid the foundation and opened up new avenues for applied research in some or all of the categories listed with particular emphasis on the therapeutic potential of α-crystallins in ocular pathophysiology. Studies using human RPE and the retina have revealed that α-crystallins possess antiapoptotic properties and whether this function is independent of their chaperone activity or works in tandem with the antioxidative enzymes remains to be fully explored. In this context, the fact that αB-crystallin was found to be localized in mitochondria and is regulated by stress is relevant and suggests that mitochondrial drug targeting has potential in arresting cell death. The localization of αB-crystallin in other organelles including the golgi and the endoplasmic reticulum opens up new areas for investigation. An important feature of αB-crystallin is that it readily interacts with a wide array of substrates as discussed earlier and summarized in Table 2. It is likely that this list will grow with the identification of more and more binding partners. The exact nature of this protein-protein interaction is important to understand since varying binding sites can have differing and contrasting cellular functions based on the amino acid sequence and chain length of the peptides involved. While αB-crystallin interacts with several growth factors, it will be of interest to determine the topology of this interaction and whether other membrane proteins, such as membrane receptors participate in this interaction. Autophagy has emerged as an important protective and pathologic mechanism in retinal development and disease. Future investigations will determine the nature and significance of the link between αB-crystallin and autophagy.

Figure 13.

Cartoon depicting novel roles of αB-crystallin. Several cellular functions of αB-crystallin are highlighted followed by the mechanism of action of αB-crystallin marked in red. The diseases associated with the various functions are shown in green. DR, diabetic retinopathy; CNV, choroidal neovascularization; ROP, retinopathy of prematurity; AMD, age-related macular degeneration; PVR, proliferative vitreretinopathy; EAE, autoimmune encephalomyelitis; CMA autophagy, chaperone-mediated autophagy.

Another area of research that is certain to attract a lot of attention is the extracellular localization of αB-crystallin and its functional significance. While intracellular properties of αB-crystallin have been widely studied, its extracellular localization has only been recently identified. Further the functional role in extracellular space and on the surface of adjacent cells that take up αB-crystallin will be an area of active investigation. For example, the mechanisms of the anti-inflammatory actions of extracellular αB-crystallin are only now being elucidated. Recent findings that αB-crystallin is secreted via exosomes and is neuroprotective is important in this regard. Further research on how this secretion is controlled, what other components exist in exosomes and how αB-crystallin interacts with other exosomal proteins will prove to be of value. For example, it is known that several microRNAs which are responsible for regulating a number of cellular processes are secreted in exosomes (Mathivanan et al., 2010, 2012; Yuan et al 2009). Which of these microRNAs regulate αB-crystallin or vice versa in RPE may shed some light on modulating their extracellular therapeutic action. Identification of multiple short-chain peptides that have similar or even higher chaperone activity than the parent α-crystallins have provided researchers with a platform for evaluating the beneficial roles of these peptides in preventing ocular disease. These αB-crystallin-derived minichaperone peptides are antiapoptotic in addition to being efficient chaperones. Further research will be needed to find out their roles in other areas such as autophagy, inflammation, and angiogenesis. Generation of novel nanoparticle containing minichaperones of α-crystallins for efficient delivery to the retina would greatly advance the utility of these peptides as therapeutic agents.

One pathophysiological feature of AMD is the accumulation of toxic compounds in drusen among which β-amyloid peptide is an important component. αB-Crystallin was shown to inhibit β-amyloid fibril formation and thus reduce β-amyloid toxicity. However this interpretation gets complicated because of a very recent finding that while αB-crystallin can prevent fibrillogenesis, it can by itself form a toxic β-amyloid oligomer (Laganowsky et al., 2012). This important area calls for further work to address the controversy particularly with respect to precise conditions of β-amyloidogenesis in RPE, identify the actual αB-crystallin peptide sequence(s) that affect β-amyloid formation and toxicity and other primary or secondary factors that may play a part. It would also be valuable to perform such toxicity studies with both individual pro-and anti-chaperone peptides that have been discovered recently.

Recent finding of αB-crystallin expression by a subset of progenitor cells in the human fetal telencephalon suggests its importance in the developing human brain (Kida et al., 2010). Whether αB-crystallin plays a similar significant role in retinal development remains to be studied using retinal progenitor cells.

Similarly intriguing are the effects of αB-crystallin on angiogenesis. While deficiency of αB-crystallin strongly inhibits the angiogenic response, at least in part through loss of binding of VEGF, treatment with αB-crystallin could be either pro- or anti-angiogenic by promoting endothelial cell survival and VEGF expression or inhibiting pro-angiogenic inflammatory responses.

While αA- and αB-crystallins are differentially expressed in various retinal cell types, much needs to be learned about the mechanisms controlling the genetic and epigenetic regulation of crystallin gene expression and post-translational modifications in each of these cell types in normal retina and in disease. It is believed that as more and more functions of α-crystallins are unraveled, these versatile sHSPs are likely to serve as important therapeutic targets in ocular disease.