viability [57Ð59]. Thus, strategies to restore mitochondrial GSH from depletion or to prevent the impairment of GSH mitochondrial transport may be of therapeutic signiÞcance in the treatment of several pathologies such as hypoxia and reperfusion injury, liver diseases, neurological disorders, diabetes, and aging [1].

9.2.3GSH as a ROS Scavenger

Among the cellular antioxidants, GSH reacts directly with ROS. It is a cofactor for the H2O2-removing enzyme glutathione peroxidase and for dehydroascorbate dehydrogenase, and it is thereby directly or indirectly involved in many ROS-detoxifying reactions [52]. Usually, generation of ROS oxidizes GSH to GSSG, ultimately reducing the total GSH level. However, studies have also shown that a reduction in the intracellular GSH is necessary for the formation of ROS [60]. GSH depletion has been shown to directly modulate both the loss of mitochondrial membrane potential and the activation of executioner caspases [52, 60]. GSH homeostasis plays a vital role in the maintenance of mitochondrial DNA and respiratory competency of cells [61]. In various types of untreated cultured mammalian cells, the levels of total GSH were found to be inversely correlated with the levels of DNA base modiÞcations [62]. Since mitochondria lack catalase, the metabolism of H2O2 is mainly accomplished by GSH, with the involvement of either GSH peroxidase or peroxiredoxin [1].

9.2.4GSH Distribution in the Retina and RPE in Health and Disease

The retina is one of the most vascularized tissues in the body and has one of the highest oxidative metabolic rates per tissue weight. The GSH system is one of the most important antioxidant systems involved in retinal protection. In early immunological studies of the rabbit retina, GSH was found to be mainly distributed in the Muller cells, horizontal cells, RPE, and the choroid [63]. The outer segments of the rod and cone photoreceptor cells were negative for GSH staining. Subsequent studies also supported these Þndings in Zebra Þsh, monkey, and guinea pig [64Ð67]. However, Winkler postulated that the deÞciency of GSH will not damage the outer segments under unstressed conditions because periodic renewal of outer segments replaces products of oxidation damage before they increase to the toxic level [68]. Under pathological situations, this process alone is not capable of protecting photoreceptors, and accordingly, photoreceptors are believed to be highly vulnerable to cell death or degeneration [68]. Oxidative stress caused by exposure of RPE cells to cigarette smoke extract sensitizes cells to apoptosis by altering mitochondrial functions and decreasing intracellular GSH [69]. GSH depletion by systemic injection

amyotrophic lateral sclerosis, Alexander disease, and AMD [87] (see Table 9.1). Studies by Brady et al. highlighted the importance of aB-crystallin, not only in the lens but also in affecting muscle integrity[88]. In various tissues, aB-crystallin is associated with different cytoskeletal elements, such as tubulin, actin, and desmin

[89, 90]. It is believed that aB-crystallin is involved in cell growth and differentiation and that it also helps in Golgi reorganization during cell division [78, 91]. BruchÕs membrane, drusen and the subjacent choroidal connective tissue from AMD tissues showed greater immunoreactivity for aA- and aB-crystallins, suggesting that it may represent a stress response to protect RPE in AMD [92]. Recent quantitative proteomic analysis of the macular BruchÕs membrane/choroid complex provides strong evidence of the upregulation of a-crystallins in AMD samples [93]. Interestingly, aB-crystallin is also found in extracellular drusen deposits and has been reported as a component of the interphotoreceptor matrix, suggesting the possibility that it may be secreted [94, 95].

The major disorder caused by mutations in the aA-crystallin gene is cataract (of different forms), sometimes associated with microcornea or microphthalmia. Besides the loss in chaperone activity, the pathology may arise from the altered interaction with the lens cytoskeleton proteins such as actin, tubulin, or intermediate Þlaments [78, 96]. The Þrst dominant cataract mutation affecting the human aA- crystallin gene described is the Arg116Cys mutation [97]. Subsequently, several other independent studies also reported mutation in the same position, suggesting this position as a mutational hotspot in the aA-crystallin gene. Among the two mechanisms proposed to explain enhanced protein aggregation due to a-crystallin mutations, the Þrst suggests an alteration in the structure of the chaperone protein and the second a loss of regulation of the oligomeric structure of a-crystallin, resulting in higher coaggregation of mutant protein and substrates [98]. Since the Arg116Cys mutation was the Þrst human aA-crystallin mutation reported, a broad variety of experimental approaches have been undertaken to Þnd out the functional consequences of this mutation. The mutant protein shows reduced chaperone activity [99] and interaction with actin [100]. In addition to the nine aA-crystallin mutations in human, there are also four aA-crystallin mutants reported in mouse. Although aA-crystallin is expressed outside the lens, only cataracts are reported to be caused by mutations in the aA-crystallin encoding gene [101].

In contrast to the clear relationship of aA-crystallin mutations and the formation of cataracts, the role of aB-crystallin is more heterogeneous. In humans, nine mutations are reported affecting the aB-crystallin gene [101]. A few of them are associated with dominant cataracts only, but some are also suggested to be causative for desmin-related myopathy or dilated cardiomyopathy [96]. The missense mutation p.Gly154Ser in aB-crystallin gene is associated with a late-onset distal vacuolar myopathy with protein aggregates [102]. The R120G mutation in aB-crystallin (CryABR120G) causes desmin-related myopathy characterized by early mitochondrial dysfunction and activation of intrinsic (mitochondrial-based) apoptotic signaling [103]. The missense mutation p.Gly154Ser in exon 3 of Cryab gene, previously described in isolated cardiomyopathy [104], is also causative for late-onset progressive distal myopathy without cardiac involvement and without signiÞcant cataracts [102]. A mutation at nucleotide 32 in the Þrst exon of aB-crystallin resulting in an amino acid change from arginine to histidine at codon 11 (R11H) was responsible for the autosomal dominant nuclear congenital cataract [105]. However, when compared with aA-crystallin gene mutations, mutations in aB-crystallin genes caused more

serious cardiac problems than retinal disorders. Posttranslational modiÞcations of aA- and aB-crystallin, including truncation, deamidation, oxidation, glycation, phosphorylation, and racemization/isomerization, promote cataract formation in aging organisms through modiÞcation of chaperone activity and solubility [12].

Generation of mice lacking aA- and aB-crystallin has provided valuable insights into the functional roles of these proteins in the lens. Targeted disruption of aA-crystallin gene in mice induces cataract and dense cytoplasmic inclusion bodies in lens Þber cells [106]. The presence of dense aB-crystallin inclusion bodies was also observed in the central lens Þber cells of aA-crystallin knockout (aA−/−) mice, suggesting the possible role of aA-crystallin for maintaining the solubility of other crystallins in the lens [106]. It was also found that the absence of aA-crystallin increases cell death during the mitotic phase. On the other hand, disruption of aB-crystallin gene (HSPB2, an adjacent gene which was also deleted) did not produce cataracts or abnormal retinal phenotype, but aB-crystallin knockout (aB−/−) mice showed skeletal muscle degeneration, spine curvature, and a life span one half that of wild type mice [88]. Further, lens cells from aB−/− mice exhibited a greater tendency for hyper-proliferation and genomic instability [78]. Combined deletion of aA- and aB-crystallin genes leads to gross abnormalities of the Þber cell structure, consistent with their important role in lens Þber development [107]. aA−/−/aB−/−-crystallin double knockout mouse lenses are signiÞcantly smaller than wild-type, and Þber cell formation is severely disturbed [107] due to caspase-dependent Þber cell degeneration [108].

An intriguing property of aB-crystallin is its ability to undergo phosphorylation at multiple sites and therefore it is subject to modiÞcation by several transduction pathways. aB-crystallin has serine phosphorylated sites in the N-terminal part of the polypeptide, and close to the a-crystallin domain [109]. aB-crystallin is phosphorylated at three serine sites corresponding to residues 19, 45, and 59. At least two pathways are implicated in the aB-crystallin phosphorylation: the mitogenactivated protein kinases associated protein kinases 2 are responsible of the phosphorylation of serine 59, while p42/p44 MAP Kinase appears to control serine 45. The kinase responsible of the phosphorylation of serine 19 is still not known [109]. Multiple phosphorylation sites of aA-crystallin have been reported in the literature although all the phosphorylation sites have not been characterized and pathways of activation not studied [110, 111].

9.3.2Nonchaperone Functions of a -Crystallins

As stated earlier, a-crystallins are multifunctional proteins involved in many cellular processes including those which are not directly related to protein folding and aggregation [96]. Some of these functions include maintaining eye lens transparency [106]; thermotolerance [112]; resistance to apoptosis [11, 106, 113, 114]; cytoskeleton modulation [115]; prevention of amyloid formation [116]; various developmental processes [117]; protection against oxidative stress [11, 118, 119]; and neuroprotection [16, 118]. The interaction between aB-crystallin and a

proteosomal subunit might suggest that a-crystallins facilitate not only refolding but also selective degradation of target proteins [109]. Our laboratory has recently shown that aB-crystallin plays an important role in the regulation of vascular permeability and angiogenesis by modulating vascular endothelial growth factor in laser-induced choroidal neovascularization and in retinopathy of prematurity models [120, 121].

9.3.3Secretory Function of a B-Crystallin in RPE and Its Relevance

Until recently, it was thought that a-crystallins are intracellular proteins since they lack secretory signal sequence. We have shown in human RPE cells and in polarized human RPE monolayers that aB-crystallin is secreted by a nonclassical pathway involving exosomes [118]. In highly polarized RPE monolayers, aB-crystallin was selectively secreted toward the apical, photoreceptor-facing side under steady-state conditions. Severe oxidative stress resulted in barrier breakdown and release of aB- crystallin to the basolateral choroidal side. To support these Þndings in vivo in mouse retinal sections, we localized aB-crystallin in the interphotoreceptor matrix. Under oxidative stress, the secreted aB-crystallin is taken up to offer protection from apoptosis by inhibition of caspase 3 and PARP activation [118]. Further, we also found that aB-crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for aB-crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of aB-crystallin in extracellular subRPE deposits in the stressed microenvironment in AMD.

9.4Interlink Between GSH and a -Crystallins

Several reports have shown that GSH is the coenzyme of various redox reactions [20]. The antioxidant activity of a-crystallin was found to depend on reduced GSH [122]. Incubation of a-crystallin with oxidized GSH results in signiÞcant loss of its chaperone activity because of the formation of proteinÐGSH mixed disulÞdes [123]. An in vitro study on the effect of oxidized and reduced GSH on the chaperone activity of a-crystallin demonstrated that reduced GSH enhanced the chaperone function while oxidized GSH diminished the activity, suggesting that GSH may modulate the target protein which could inßuence the chaperone activity of a-crystallin [124]. We have shown that overexpression of a-crystallins offers protection, while deÞciency of a-crystallins renders RPE cells susceptible to oxidative stress-induced apoptosis [11, 13]. A direct positive correlation between aA-crystallin and GSH was found in lens epithelial cells overexpressing aA-crystallin, whereas absence of