aA-crystallin was associated with a decreased expression of GSH [28]. It was also reported that chaperone function of a-crystallin was compromised in diabetic rats and humans [125Ð127]. In L929 cells, overexpression of another family member, Hsp25, increases intracellular GSH and confers resistance to oxidative stress [128], whereas its down regulation decreased GSH and increased oxidative stress in vivo [129]. GSH is particularly active in the mitochondria where most of the ROS generated are scavenged by GSH dependent redox systems [1]. In this context, it is of interest that overexpression of aB-crystallin protected murine L929 cells against cell death induced by tumor necrosis factor, H2O2, or menadione [122]. Overexpression of aB-crystallin increased GSH levels which can scavenge ROS easily without inducing an imbalance in the thiol status [122]. We have shown that in a-crystallin overexpressed RPE cells, the levels of the mitochondrial pool of GSH increased with oxidative stress, possibly protecting cells from undergoing apoptosis [13]. The important link between GSH and aB-crystallin was well illustrated in a recent study showing that autosomal dominant mutation in the human aB-crystallin gene (hR120GCryAB) resulted in increased recycling of oxidized to reduced GSH due to increased expression of glucose-6-phosphate dehydrogenase, glutathione reductase, and glutathione peroxidase [130]. The mechanism(s) involved in the ability of a-crystallins to increase the intracellular GSH is not known and additional studies are required to explore the mechanism. Further evaluation is required to determine whether a-crystallin exerts its action on one or all of the processes of GSH metabolism in the retina, namely, biosynthesis, transport, or secretion.

9.5Future Perspectives

The importance of a-crystallins and GSH in different pathologies is documented in several recent reports, so the use of compounds or drugs that modulate the activity or functions of these antioxidants will prove to be an area of fruitful investigation. Although different forms of a-crystallins and GSH are used individually in therapy for multiple disorders, a combination of the two would be very beneÞcial, given their known specialized properties (Fig. 9.2). How the modulation of GSH metabolism through its biosynthesis and or transport would inßuence a-crystallin availability and function is not fully investigated. In this context, given the presence of GSH and a-crystallins, manipulation of mitochondrial pools of GSH and a-crystallin in retinal cell types to provide protection from injury will be a valuable approach. Further, whether GSH has a direct or indirect effect on the recently discovered aB-crystallin secretion would be critical, particularly with respect to neuroprotection. Additionally, small peptides of 18Ð20 amino acids isolated from aA- and aB-crystallin were shown to have similar chaperone as well as antiapoptotic functions as their parent crystallins [131Ð133]. The chaperone minipeptides offer an efÞcient modality for delivery via different vehicles including nanoencapsulation. Thus, it is believed that further understanding of the interaction between a-crystallin and sHsps in general with the cellÕs most abundant endogenous antioxidant GSH would offer valuable therapeutic beneÞts.

Fig. 9.2 Schematic representation of the protective function of a-crystallins and glutathione (GSH) in the cytosolic and mitochondrial compartments of chronically stressed cells from normal human RPE (left) and RPE overexpressing a-crystallin (right). Cells under conditions of oxidative stress produce reactive oxygen species (ROS), release cytochrome c (cyt c), and activate effector caspases. ROS oxidizes or unfolds polypeptides and causes protein aggregation which results in cell death. In a-crystallin overexpressing cells, ROS formation is signiÞcantly inhibited and the subsequent pathways in cell death are inhibited. a-crystallins as chaperones refold unfolded proteins and prevent protein accumulation. Mitochondrial GSH (mGSH) is elevated in a-crystallin overexpressing cells due to an increased synthesis of cytosolic GSH and translocation to the mitochondria. Participation of MRP1 in the efßux of GSH (and GSSG) is also shown

Acknowledgments The Arnold and Mabel Beckman Foundation, National Institutes of Health Grants (EY01545, EY03040), and a grant to the Department of Ophthalmology by Research to Prevent Blindness, Inc are acknowledged. We thank Christine Spee for expert technical help in the preparation of human polarized RPE cells and Susan Clarke for editorial assistance and Eric Barron for help with the Þgures.

References

1. Mar’ M, Morales A, Colell A, Garc’a-Ruiz C, Fern‡ndez-Checa JC (2009) Mitochondrial glutathione, a key survival antioxidant. Antioxid Redox Signal 11:2685Ð2700

2. Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL (2009) Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem 390:191Ð214 3. Moreno MC, Campanelli J, Sande P, S‡nez DA, Keller Sarmiento MI, Rosenstein RE (2004) Retinal oxidative stress induced by high intraocular pressure. Free Radic Biol Med

37:803Ð812

194 P.G. Sreekumar et al.

4. Pau H, Graf P, Sies H (1982) Glutathione content of the lens in various forms of cataract. Graefes Arch Clin Exp Ophthalmol 219:140Ð142

5. Pau H, Graf P, Sies H (1990) Glutathione levels in human lens: regional distribution in different forms of cataract. Exp Eye Res 50:17Ð20

6. Sternberg P Jr, Davidson PC, Jones DP, Hagen TM, Reed RL, Drews-Botsch C (1993) Protection of retinal pigment epithelium from oxidative injury by glutathione and precursors. Invest Ophthalmol Vis Sci 34:3661Ð3668

7. Horwitz J (1992) Alpha-crystallin can function as a molecular chaperone. Proc Natl Acad Sci U S A 89:10449Ð10453

8. Klemenz R, Fršhli E, Steiger RH, SchŠfer R, Aoyama A (1991) Alpha B-crystallin is a small heat shock protein. Proc Natl Acad Sci U S A 88:3652Ð3656

9. Kamradt MC, Chen F, Cryns VL (2001) The small heat shock protein alpha B-crystallin negatively regulates cytochrome c- and caspase-8-dependent activation of caspase-3 by inhibiting its autoproteolytic maturation. J Biol Chem 276:16059Ð16063

10. Kamradt MC, Chen F, Sam S, Cryns VL (2002) The small heat shock protein alpha B-crystallin negatively regulates apoptosis during myogenic differentiation by inhibiting caspase-3 activation. J Biol Chem 277:38731Ð38736

11. Yaung J, Jin M, Barron E, Spee C, Wawrousek EF, Kannan R, Hinton DR (2007) alphaCrystallin distribution in retinal pigment epithelium and effect of gene knockouts on sensitivity to oxidative stress. Mol Vis 13:566Ð577

12. Sun Y, MacRae TH (2005) Small heat shock proteins: molecular structure and chaperone function. Cell Mol Life Sci 62:2460Ð2476

13. Parameswaran S, Spee C, Ryan SJ, Kannan R, Hinton DR (2009) Decreased cellular glutathione (GSH) and increased GSH efßux in {alpha}-crystallin knockout retina. Invest Ophthalmol Vis Sci 50:E-Abstract 1831

14. De S, Rabin DM, Salero E, Lederman PL, Temple S, Stern JH (2007) Human retinal pigment epithelium cell changes and expression of alphaB-crystallin: a biomarker for retinal pigment epithelium cell change in age-related macular degeneration. Arch Ophthalmol 125:641Ð645 15. Masilamoni JG, Vignesh S, Kirubagaran R, Jesudason EP, Jayakumar R (2005) The neuroprotective efÞcacy of alpha-crystallin against acute inßammation in mice. Brain Res Bull

67:235Ð241

16. Ousman SS, Tomooka BH, van Noort JM, Wawrousek EF, OÕConnor KC, Haßer DA, Sobel RA, Robinson WH, Steinman L (2007) Protective and therapeutic role for alphaB-crystallin in autoimmune demyelination. Nature 448:474Ð479

17. Cooper AJ, Kristal BS (1997) Multiple roles of glutathione in the central nervous system. Biol Chem 378(793Ð802):20

18. Jones DP (2008) Radical-free biology of oxidative stress. Am J Physiol Cell Physiol 295:C849ÐC868

19. Townsend DM, Tew KD, Tapiero H (2003) The importance of glutathione in human disease. Biomed Pharmacother 57:145Ð155

20. Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52:711Ð760

21. Dalton TP, Chen Y, Schneider SN, Nebert DW, Shertzer HG (2004) Genetically altered mice to evaluate glutathione homeostasis in health and disease. Free Radic Biol Med 37:1511Ð1526

22. Lu SC (2000) Regulation of glutathione synthesis. Curr Top Cell Regul 36:95Ð116

23. Dalton TP, Dieter MZ, Yang Y, Shertzer HG, Nebert DW (2000) Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deÞciency when heterozygous. Biochem Biophys Res Commun 279:324Ð329

24. McConnachie LA, Mohar I, Hudson FN, Ware CB, Ladiges WC, Fernandez C, ChattertonKirchmeier S, White CC, Pierce RH, Kavanagh TJ (2007) Glutamate cysteine ligase modiÞer subunit deÞciency and gender as determinants of acetaminophen-induced hepatotoxicity in mice. Toxicol Sci 99:628Ð636

25. Njalsson R, Norgren S, Larsson A, Huang CS, Anderson ME, Luo JL (2001) Cooperative binding of gamma-glutamyl substrate to human glutathione synthetase. Biochem Biophys Res Commun 289:80Ð84

26. Jaiswal AK (2004) Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic Biol Med 36:1199Ð1207

27. Jin M, Yaung J, Kannan R, He S, Ryan SJ, Hinton DR (2005) Hepatocyte growth factor protects RPE cells from apoptosis induced by glutathione depletion. Invest Ophthalmol Vis Sci 46:4311Ð4319

28. Kannan R, Ouyang B, Wawrousek E, Kaplowitz N, Andley UP (2001) Regulation of GSH in alphaA-expressing human lens epithelial cell lines and in alphaA knockout mouse lenses. Invest Ophthalmol Vis Sci 42:409Ð416

29. Lee JM, Johnson JA (2004) An important role of Nrf2-ARE pathway in the cellular defense mechanism. J Biochem Mol Biol 37:139Ð143

30. Pallard— FV, Markovic J, Garc’a JL, Vi–a J (2009) Role of nuclear glutathione as a key regulator of cell proliferation. Mol Aspects Med 30:77Ð85

31. Hwang C, Sinskey AJ, Lodish HF (1992) Oxidized redox state of glutathione in the endoplasmic reticulum. Science 257:1496Ð1502

32.GrifÞth OW, Meister A (1985) Origin and turnover of mitochondrial glutathione. Proc Natl Acad Sci U S A 82(4668Ð4672):21

33. Kaplowitz N, Aw TY, Ookhtens M (1985) The regulation of hepatic glutathione. Annu Rev Pharmacol Toxicol 25:715Ð744

34. Ikeda Y, Taniguchi N (2005) Gene expression of gamma-glutamyltranspeptidase. Methods Enzymol 401:408Ð425

35. Ballatori N, Hammond CL, Cunningham JB, Krance SM, Marchan R (2005) Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/ SLC21A families of membrane proteins. Toxicol Appl Pharmacol 204:238Ð255

36. Briz O, Romero MR, Martinez-Becerra P, Macias RI, Perez MJ, Jimenez F, San Martin FG, Marin JJ (2006) OATP8/1B3-mediated cotransport of bile acids and glutathione: an export pathway for organic anions from hepatocytes? J Biol Chem 281:30326Ð30335

37. Li L, Lee TK, Meier PJ, Ballatori N (1998) IdentiÞcation of glutathione as a driving force and leukotriene C4 as a substrate for oatp1, the hepatic sinusoidal organic solute transporter. J Biol Chem 273:16184Ð16191

38. Hammond CL, Marchan R, Krance SM, Ballatori N (2007) Glutathione export during apoptosis requires functional multidrug resistance-associated proteins. J Biol Chem 282:14337Ð14347

39. Minich T, Riemer J, Schulz JB, Wielinga P, Wijnholds J, Dringen R (2006) The multidrug resistance protein 1 (Mrp1), but not Mrp5, mediates export of glutathione and glutathione disulÞde from brain astrocytes. J Neurochem 97:373Ð384

40. Kannan R, Sreekumar PG, Ryan SJ, Hinton DR (2009) Role of multidrug resistant protein (MRP1) in oxidant stress-induced glutathione efßux in RPE. Invest Ophthalmol Vis Sci 50:E-Abstract 1830

41. Chang TS, Cho CS, Park S, Yu S, Kang SW, Rhee SG (2004) Peroxiredoxin III, a mitochon- drion-speciÞc peroxidase, regulates apoptotic signaling by mitochondria. J Biol Chem 279:41975Ð41984

42. Reed DJ (1990) Glutathione: toxicological implications. Annu Rev Pharmacol Toxicol 30:603Ð631

43. Yap LP, Garcia JV, Han D, Cadenas E (2009) The energyÐredox axis in aging and age-related neurodegeneration. Adv Drug Deliv Rev 61:1283Ð1298

44. Fern‡ndez-Checa JC, Kaplowitz N, Garc’a-Ruiz C, Colell A (1998) Mitochondrial glutathione: importance and transport. Semin Liver Dis 18:389Ð401

45. Fern‡ndez-Checa JC, Kaplowitz N, Garc’a-Ruiz C, Colell A, Miranda M, Mar’ M, Ardite E, Morales A (1997) GSH transport in mitochondria: defense against TNF-induced oxidative stress and alcohol-induced defect. Am J Physiol 273(1 Pt 1):G7ÐG17

46. Palmieri F (1994) Mitochondrial carrier proteins. FEBS Lett 346:48Ð54

47. Han D, Canali R, Rettori D, Kaplowitz N (2003) Effect of glutathione depletion on sites and topology of superoxide and hydrogen peroxide production in mitochondria. Mol Pharmacol 64:1136Ð1144

48. Lash LH, Visarius TM, Sall JM, Qian W, Tokarz JJ (1998) Cellular and subcellular heterogeneity of glutathione metabolism and transport in rat kidney cells. Toxicology 130:1Ð15

49. MŒrtensson J, Jain A, Meister A (1990) Glutathione is required for intestinal function. Proc Natl Acad Sci U S A 87:1715Ð1719

50. Cazanave S, Berson A, Haouzi D, Vadrot N, Fau D, Grodet A, LettŽron P, Feldmann G, El-Benna J, Fromenty B, Robin MA, Pessayre D (2007) High hepatic glutathione stores alleviate Fas-induced apoptosis in mice. J Hepatol 46:858Ð868

51. Friesen C, Kiess Y, Debatin KM (2004) A critical role of glutathione in determining apoptosis sensitivity and resistance in leukemia cells. Cell Death Differ 11(Suppl 1):S73ÐS85

52. Armstrong JS, Steinauer KK, Hornung B, Irish JM, Lecane P, Birrell GW, Peehl DM, Knox SJ (2002) Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line. Cell Death Differ 9:252Ð263

53. Will Y, Kaetzel RS, Brown MK, Fraley TS, Reed DJ (2002) In vivo reversal of glutathione deÞciency and susceptibility to in vivo dexamethasone-induced apoptosis by N-acetylcysteine and L-2-oxothiazolidine-4-carboxylic acid, but not ascorbic acid, in thymocytes from gamma- glutamyltranspeptidase-deÞcient knockout mice. Arch Biochem Biophys 397:399Ð406

54. Garcia-Ruiz C, Fernandez-Checa JC (2006) Mitochondrial glutathione: hepatocellular sur- vival-death switch. J Gastroenterol Hepatol 21(Suppl 3):S3ÐS6

55. Lash LH (2006) Mitochondrial glutathione transport: physiological, pathological and toxicological implications. Chem Biol Interact 163:54Ð67

56. Marchetti P, Decaudin D, Macho A, Zamzami N, Hirsch T, Susin SA, Kroemer G (1997) Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function. Eur J Immunol 27:289Ð296

57. Colell A, Garc’a-Ruiz C, Miranda M, Ardite E, Mar’ M, Morales A, Corrales F, Kaplowitz N, Fern‡ndez-Checa JC (1998) Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor. Gastroenterology 115:1541Ð1551

58. Colell A, Coll O, Garc’a-Ruiz C, Par’s R, Tiribelli C, Kaplowitz N, Fern‡ndez-Checa JC (2001) Tauroursodeoxycholic acid protects hepatocytes from ethanol-fed rats against tumor necrosis factor-induced cell death by replenishing mitochondrial glutathione. Hepatology 34:964Ð971

59. Fern‡ndez-Checa JC, Garc’a-Ruiz C, Ookhtens M, Kaplowitz N (1991) Impaired uptake of glutathione by hepatic mitochondria from chronic ethanol-fed rats. Tracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress. J Clin Invest 87:397Ð405

60. Franco R, Schoneveld OJ, Pappa A, Panayiotidis MI (2007) The central role of glutathione in the pathophysiology of human diseases. Arch Physiol Biochem 113:234Ð258

61. Ayer A, Tan SX, Grant CM, Meyer AJ, Dawes IW, Perrone GG (2010) The critical role of glutathione in maintenance of the mitochondrial genome. Free Radic Biol Med 49:1956Ð1968

62. Will O, Mahler HC, Arrigo AP, Epe B (1999) Inßuence of glutathione levels and heat-shock on the steady-state levels of oxidative DNA base modiÞcations in mammalian cells. Carcinogenesis 20:333Ð337

63. Pow DV, Crook DK (1995) Immunocytochemical evidence for the presence of high levels of reduced glutathione in radial glial cells and horizontal cells in the rabbit retina. Neurosci Lett 193:25Ð28

64. Carter-Dawson L, Shen FF, Harwerth RS, Crawford ML, Smith EL 3rd, Whitetree A (2004) Glutathione content is altered in MŸller cells of monkey eyes with experimental glaucoma. Neurosci Lett 364:7Ð10

65. Huster D, Hjelle OP, Haug FM, Nagelhus EA, Reichelt W, Ottersen OP (1998) Subcellular compartmentation of glutathione and glutathione precursors. A high resolution immunogold analysis of the outer retina of guinea pig. Anat Embryol (Berl) 198:277Ð287

66. Marc RE, Cameron D (2001) A molecular phenotype atlas of the zebraÞsh retina. J Neurocytol 30:593Ð654

67. SchŸtte M, Werner P (1998) Redistribution of glutathione in the ischemic rat retina. Neurosci Lett 246:53Ð56

68. Winkler BS (2008) An hypothesis to account for the renewal of outer segments in rod and cone photoreceptor cells: renewal as a surrogate antioxidant. Invest Ophthalmol Vis Sci 49:3259Ð3261

69. Bertram KM, Baglole CJ, Phipps RP, Libby RT (2009) Molecular regulation of cigarette smoke induced-oxidative stress in human retinal pigment epithelial cells: implications for age-related macular degeneration. Am J Physiol Cell Physiol 297:C1200ÐC1210

70. Roh YJ, Moon C, Kim SY, Park MH, Bae YC, Chun MH, Moon JI (2007) Glutathione depletion induces differential apoptosis in cells of mouse retina, in vivo. Neurosci Lett 417:266Ð270

71. Nordgaard CL, Karunadharma PP, Feng X, Olsen TW, Ferrington DA (2008) Mitochondrial proteomics of the retinal pigment epithelium at progressive stages of age-related macular degeneration. Invest Ophthalmol Vis Sci 49:2848Ð2855

72. Liang FQ, Godley BF (2003) Oxidative stress-induced mitochondrial DNA damage in human retinal pigment epithelial cells: a possible mechanism for RPE aging and age-related macular degeneration. Exp Eye Res 76:397Ð403

73. Wang AL, Lukas TJ, Yuan M, Neufeld AH (2008) Increased mitochondrial DNA damage and down-regulation of DNA repair enzymes in aged rodent retinal pigment epithelium and choroid. Mol Vis 14:644Ð651

74. He Y, Ge J, Tombran-Tink J (2008) Mitochondrial defects and dysfunction in calcium regulation in glaucomatous trabecular meshwork cells. Invest Ophthalmol Vis Sci 49: 4912Ð4922

75. Bhat SP (2003) Crystallins, genes and cataract. Prog Drug Res 60:205Ð262

76. Sreekumar PG, Kannan R, Hinton DR (2009) There are three major families of crystallins: misnaming of alphaB crystallin. Acta Physiol (Oxf) 195:503

77. Horwitz J (2003) Alpha-crystallin. Exp Eye Res 76:145Ð153

78. Andley UP (2007) Crystallins in the eye: function and pathology. Prog Retin Eye Res 26:78Ð98

79. de Jong WW, Caspers GJ, Leunissen JA (1998) Genealogy of the alpha-crystallinÐsmall heatshock protein superfamily. Int J Biol Macromol 22:151Ð162

80. Ingolia TD, Craig EA (1982) Four small Drosophila heat shock proteins are related to each other and to mammalian alpha-crystallin. Proc Natl Acad Sci U S A 79:2360Ð2364

81. Bhat SP, Nagineni CN (1989) alpha B subunit of lens-speciÞc protein alpha-crystallin is present in other ocular and non-ocular tissues. Biochem Biophys Res Commun 158:319Ð325

82. Dubin RA, Wawrousek EF, Piatigorsky J (1989) Expression of the murine alpha B-crystallin gene is not restricted to the lens. Mol Cell Biol 9:1083Ð1091

83. Kato K, Shinohara H, Kurobe N, Goto S, Inaguma Y, Ohshima K (1991) Immunoreactive alpha A crystallin in rat non-lenticular tissues detected with a sensitive immunoassay method. Biochim Biophys Acta 1080:173Ð180

84. Sax CM, Piatigorsky J (1994) Expression of the alpha-crystallin/small heat-shock protein/ molecular chaperone genes in the lens and other tissues. Adv Enzymol Relat Areas Mol Biol 69:155Ð201

85. Srinivasan AN, Nagineni CN, Bhat SP (1992) alpha A-crystallin is expressed in non-ocular tissues. J Biol Chem 267:23337Ð23341

86. Xi J, Farjo R, Yoshida S, Kern TS, Swaroop A, Andley UP (2003) A comprehensive analysis of the expression of crystallins in mouse retina. Mol Vis 9(410Ð419):28

87. van Rijk AF, Bloemendal H (2000) Alpha-B-crystallin in neuropathology. Ophthalmologica 214:7Ð12

88. Brady JP, Garland DL, Green DE, Tamm ER, Giblin FJ, Wawrousek EF (2001) AlphaBcrystallin in lens development and muscle integrity: a gene knockout approach. Invest Ophthalmol Vis Sci 42:2924Ð2934

89. Arai H, Atomi Y (1997) Chaperone activity of alpha B-crystallin suppresses tubulin aggregation through complex formation. Cell Struct Funct 22:539Ð544

90. Longoni S, James P, Chiesi M (1990) Cardiac alpha-crystallin I. Isolation and identiÞcation. I. Isolation and identiÞcation. Mol Cell Biochem 99:113Ð120

91. Gangalum RK, Schibler MJ, Bhat SP (2004) Small heat shock protein alphaB-crystallin is part of cell cycle-dependent Golgi reorganization. J Biol Chem 279:43374Ð43377

92.Nakata K, Crabb JW, HollyÞeld JG (2005) Crystallin distribution in BruchÕs membranechoroid complex from AMD and age-matched donor eyes. Exp Eye Res 80:821Ð826

93. Yuan X, Gu X, Crabb JS, Yue X, Shadrach K, HollyÞeld JG, Crabb JW (2010) Quantitative proteomics: comparison of the macular BruchÕs membrane/choroid complex from agerelated macular degeneration and normal eyes. Mol Cell Proteomics 9:1031Ð1046

94. Hauck SM, Schoeffmann S, Deeg CA, Gloeckner CJ, Swiatek-de Lange M, UefÞng M (2005) Proteomic analysis of the porcine interphotoreceptor matrix. Proteomics 5:3623Ð3636

95. Steiner-Champliaud MF, Sahel J, Hicks D (2006) Retinoschisin forms a mutli-molecular matrix and cytoplasmic proteins: interactions with beta2 laminin and alphaB-crystallin. Mol Vis 12:892Ð901

96. Arrigo AP, Simon S (2010) Expression and functions of heat shock proteins in the normal and pathological mammalian eye. Curr Mol Med 10:776Ð793

97. Litt M, Kramer P, LaMorticella DM, Murphey W, Lovrien EW, Weleber RG (1998) Autosomal dominant congenital cataract associated with a missense mutation in the human alpha crystallin gene CRYAA. Hum Mol Genet 7:471Ð474

98. Koteiche HA, Mchaourab HS (2006) Mechanism of a hereditary cataract phenotype. Mutations in alphaA-crystallin activate substrate binding. J Biol Chem 281(14273Ð14279):23

99. Kumar LV, Ramakrishna T, Rao CM (1999) Structural and functional consequences of the mutation of a conserved arginine residue in alphaA and alphaB crystallins. J Biol Chem 274:24137Ð24141

100.Brown Z, Ponce A, Lampi K, Hancock L, Takemoto L (2007) Differential binding of mutant (R116C) and wildtype alphaA crystallin to actin. Curr Eye Res 32:1051Ð1054

101.Graw J (2009) Genetics of crystallins: cataract and beyond. Exp Eye Res 88:173Ð189

102.Reilich P, Schoser B, Schramm N, Krause S, Schessl J, Kress W, MŸller-Hšcker J, Walter MC, Lochmuller H (2010) The p.G154S mutation of the alpha-B crystallin gene (CRYAB) causes late-onset distal myopathy. Neuromuscul Disord 20:255Ð259

103.Maloyan A, Sanbe A, Osinska H, Westfall M, Robinson D, Imahashi K, Murphy E, Robbins J (2005) Mitochondrial dysfunction and apoptosis underlie the pathogenic 24 process in alpha-B-crystallin desmin-related cardiomyopathy. Circulation 112:3451Ð3461

104.Pilotto A, Marziliano N, Pasotti M, Grasso M, Costante AM, Arbustini E (2006) alphaBcrystallin mutation in dilated cardiomyopathies: low prevalence in a consecutive series of 200 unrelated probands. Biochem Biophys Res Commun 346:1115Ð1117

105.Chen Q, Ma J, Yan M, Mothobi ME, Liu Y, Zheng F (2009) A novel mutation in CRYAB associated with autosomal dominant congenital nuclear cataract in a Chinese family. Mol Vis 15:1359Ð1365

106.Brady JP, Garland D, Duglas-Tabor Y, Robison WG Jr, Groome A, Wawrousek EF (1997) Targeted disruption of the mouse alpha A-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein alpha B-crystallin. Proc Natl Acad Sci U S A 94:884Ð889

107.Boyle DL, Takemoto L, Brady JP, Wawrousek EF (2003) Morphological characterization of the alpha A- and alpha B-crystallin double knockout mouse lens. BMC Ophthalmol 3:3

108.Morozov V, Wawrousek EF (2006) Caspase-dependent secondary lens Þber cell disintegration in alphaA-/alphaB-crystallin double-knockout mice. Development 133:813Ð821

109.Arrigo AP, Simon S, Gibert B, Kretz-Remy C, Nivon M, Czekalla A, Guillet D, Moulin M, Diaz-Latoud C, Vicart P (2007) Hsp27 (HspB1) and alphaB-crystallin (HspB5) as therapeutic targets. FEBS Lett 581:3665Ð3674

110.Hogan JM, Pitteri SJ, McLuckey SA (2003) Phosphorylation site identiÞcation via ion trap tandem mass spectrometry of whole protein and peptide ions: bovine alpha-crystallin A chain. Anal Chem 75:6509Ð6916

111.Schaefer H, Marcus K, Sickmann A, Herrmann M, Klose J, Meyer HE (2003) IdentiÞcation of phosphorylation and acetylation sites in alphaA-crystallin of the eye lens (mus musculus) after two-dimensional gel electrophoresis. Anal Bioanal Chem 376:966Ð972

112.van den IJssel PR, Overkamp P, Knauf U, Gaestel M, de Jong WW (1994) Alpha A-crystallin confers cellular thermoresistance. FEBS Lett 355:54Ð56

113.Mehlen P, Schulze-Osthoff K, Arrigo AP (1996) Small stress proteins as novel regulators of apoptosis. Heat shock protein 27 blocks Fas/APO-1- and staurosporine-induced cell death. J Biol Chem 271:16510Ð16514

114.Merck KB, Groenen PJ, Voorter CE, de Haard-Hoekman WA, Horwitz J, Bloemendal H, de Jong WW (1993) Structural and functional similarities of bovine alpha-crystallin and mouse small heat-shock protein. A family of chaperones. J Biol Chem 268:1046Ð1052

115.Nicholl ID, Quinlan RA (1994) Chaperone activity of alpha-crystallins modulates intermediate Þlament assembly. EMBO J 13(945Ð953):25

116.Stege GJ, Renkawek K, Overkamp PS, Verschuure P, van Rijk AF, Reijnen-Aalbers A, Boelens WC, Bosman GJ, de Jong WW (1999) The molecular chaperone alphaB-27 crystallin enhances amyloid beta neurotoxicity. Biochem Biophys Res Commun 262:152Ð156

117.Narberhaus F (2002) Alpha-crystallin-type heat shock proteins: socializing minichaperones in the context of a multichaperone network. Microbiol Mol Biol Rev 66:64Ð93

118.Sreekumar PG, Kannan R, Kitamura M, Spee C, Barron E, Ryan SJ, Hinton DR (2010) aB- crystallin is apically secreted within exosomes by polarized human retinal pigment epithelium and provides neuroprotection to adjacent cells. PLoS One 5:e12578

119.Yaung J, Kannan R, Wawrousek EF, Spee C, Sreekumar PG, Hinton DR (2008) Exacerbation of retinal degeneration in the absence of alpha crystallins in an in vivo model of chemically induced hypoxia. Exp Eye Res 86:355Ð365

120.Kase S, He S, Sonoda S, Kitamura M, Spee C, Wawrousek E, Ryan SJ, Kannan R, Hinton DR (2010) alphaB-crystallin regulation of angiogenesis by modulation of VEGF. Blood 115:3398Ð3406

121.Kerr BA, Byzova TV (2010) alphaB-crystallin: a novel VEGF chaperone. Blood 115:3181Ð3183

122.Mehlen P, Kretz-Remy C, PrŽville X, Arrigo AP (1996) Human hsp27, Drosophila hsp27 and human alphaB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFalpha-induced cell death. EMBO J 15:2695Ð2706

123.Cherian M, Smith JB, Jiang XY, Abraham EC (1997) Inßuence of protein-glutathione mixed disulÞde on the chaperone-like function of alpha-crystallin. J Biol Chem 272:29099Ð29103

124.Pal J, Bera S, Ghosh SK (1998) The effect of glutathione upon chaperone activity of alphacrystallin is probably mediated through target modulation. Ophthalmic Res 30:271Ð279

125.Cherian M, Abraham EC (1995) Diabetes affects a-crystallin chaperone function. Biochem Biophys Res Commun 212:184Ð189

126.Kumar PA, Suryanarayana P, Reddy PY, Reddy GB (2005) Modulation of alpha-crystallin chaperone activity in diabetic rat lens by curcumin. Mol Vis 11:561Ð568

127.Thampi P, Zarina S, Abraham EC (2002) alpha-Crystallin chaperone function in diabetic rat and human lenses. Mol Cell Biochem 229:113Ð118

128.Baek SH, Min JN, Park EM, Han MY, Lee YS, Lee YJ, Park YM (2000) Role of small heat shock protein HSP25 in radioresistance and glutathione-redox cycle. J Cell Physiol 183:100Ð107

129.Yan LJ, Christians ES, Liu L, Xiao X, Sohal RS, Benjamin IJ (2002) Mouse heat shock transcription factor 1 deÞciency alters cardiac redox homeostasis and increases mitochondrial oxidative damage. EMBO J 21:5164Ð5172

130.Rajasekaran NS, Connell P, Christians ES, Yan LJ, Taylor RP, Orosz A, Zhang XQ, Stevenson TJ, Peshock RM, Leopold JA, Barry WH, Loscalzo J, Odelberg SJ, Benjamin IJ (2007) Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice. Cell 130:427Ð439

131.Bhattacharyya J, Padmanabha Udupa EG, Wang J, Sharma KK (2006) Mini-alphaB- crystallin: a functional element of alphaB-crystallin with chaperone-like activity. Biochemistry 45:3069Ð3076

132.Kannan R, Sreekumar PG, Kannan N, Ryan SJ, Kompella UB, Sharma K, Hinton DR (2010) Alpha crystallin derived peptide chaperone protects human RPE cells from oxidative injury. Invest Ophthalmol Vis Sci 51:E-Abstract 1441

133.Santhoshkumar P, Sharma KK (2004) Inhibition of amyloid Þbrillogenesis and toxicity by a peptide chaperone. Mol Cell Biochem 267:147Ð155

134.Gu M, Owen AD, Toffa SE, Cooper JM, Dexter DT, Jenner P, Marsden CD, Schapira AH (1998) Mitochondrial function, GSH and iron in neurodegeneration and Lewy body diseases. J Neurol Sci 158:24Ð29

135.Liu H, Wang H, Shenvi S, Hagen TM, Liu RM (2004) Glutathione metabolism during aging and in Alzheimer disease. Ann N Y Acad Sci 1019:346Ð349

136.Gironi M, Bianchi A, Russo A, Alberoni M, Ceresa L, Angelini A, Cursano C, Mariani E, Nemni R, Kullmann C, Farina E, Martinelli Boneschi F (2011) Oxidative imbalance in different neurodegenerative diseases with memory impairment. Neurodegener Dis 8(3):129Ð37

137.Sian J, Dexter DT, Lees AJ, Daniel S, Jenner P, Marsden CD (1994) Glutathione-related enzymes in brain in ParkinsonÕs disease. Ann Neurol 36:356Ð361

138.Baillet A, Chanteperdrix V, TrocmŽ C, Casez P, Garrel C, Besson G (2010) The role of oxidative stress in amyotrophic lateral sclerosis and ParkinsonÕs disease. Neurochem Res 35:1530Ð1537

139.Jiang H, Talaska AE, Schacht J, Sha SH (2007) Oxidative imbalance in the aging inner ear. Neurobiol Aging 28(1605Ð1612):22

140.Estrela JM, Ortega A, Obrador E (2006) Glutathione in cancer biology and therapy. Crit Rev Clin Lab Sci 43:143Ð181

141.Ortega A, Carretero J, Obrador E, Estrela JM (2008) Tumoricidal activity of endotheliumderived NO and the survival of metastatic cells with high GSH and Bcl-2 levels. Nitric Oxide 19:107Ð114

142.Leopold JA, Loscalzo J (2005) Oxidative enzymopathies and vascular disease. Arterioscler Thromb Vasc Biol 25:1332Ð1340

143.Li S, Zheng MQ, Rozanski GJ (2009) Glutathione homeostasis in ventricular myocytes from rat hearts with chronic myocardial infarction. Exp Physiol 94:815Ð824

144.Kobayashi T, Watanabe Y, Saito Y, Fujioka D, Nakamura T, Obata JE, Kitta Y, Yano T, Kawabata K, Watanabe K, Mishina H, Ito S, Kugiyama K (2010) Mice lacking the gluta- mate-cysteine ligase modiÞer subunit are susceptible to myocardial ischaemia-reperfusion injury. Cardiovasc Res 85:785Ð795

145.Malhotra D, Thimmulappa R, Navas-Acien A, Sandford A, Elliott M, Singh A, Chen L, Zhuang X, Hogg J, Pare P, Tuder RM, Biswal S (2008) Decline in NRF2-regulated antioxidants in chronic obstructive pulmonary disease lungs due to loss of its positive regulator, DJ-1. Am J Respir Crit Care Med 178:592Ð604

146.van der Toorn M, Smit-de Vries MP, Slebos DJ, de Bruin HG, Abello N, van Oosterhout AJ, Bischoff R, Kauffman HF (2007) Cigarette smoke irreversibly modiÞes glutathione in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 293:L1156ÐL1162

147.Roum JH, Buhl R, McElvaney NG, Borok Z, Crystal RG (1993) Systemic deÞciency of glutathione in cystic Þbrosis. J Appl Physiol 75:2419Ð2424

148.Hassan MQ, Hadi RA, Al-Rawi ZS, Padron VA, Stohs SJ (2001) The glutathione defense system in the pathogenesis of rheumatoid arthritis. J Appl Toxicol 21:69Ð73

149.Staro A, Makosa G, Koter-Michalak M (2010 Nov 17) Oxidative stress in erythrocytes from patients with rheumatoid arthritis. Rheumatol Int. [Epub ahead of print]

150.Sido B, Hack V, Hochlehnert A, Lipps H, Herfarth C, Dršge W (1998) Impairment of intestinal glutathione synthesis in patients with inßammatory bowel disease. Gut 42:485Ð492

151.Biagioni C, Favilli F, Catarzi S, Marcucci T, Fazi M, Tonelli F, Vincenzini MT, Iantomasi T (2006) Redox state and O2*- production in neutrophils of CrohnÕs disease patients. Exp Biol Med (Maywood) 231:186Ð195

152.Zargari M, Allameh A, Sanati MH, Tiraihi T, Lavasani S, Emadyan O (2007) Relationship between the clinical scoring and demyelination in central nervous system with total antioxidant capacity of plasma during experimental autoimmune encephalomyelitis development in mice. Neurosci Lett 412:24Ð28

153.Choi IY, Lee SP, Denney D, Lynch S (2011) Lower levels of glutathione in the brains of secondary progressive multiple sclerosis patients measured by 1H magnetic resonance chemical shift imaging at 3 T. Mult Scler 17(3):289Ð96

154.Kšk•am I, Naziro lu M (1999) Antioxidants and lipid peroxidation status in the blood of patients with psoriasis. Clin Chim Acta 289:23Ð31

155.Herzenberg LA, De Rosa SC, Dubs JG, Roederer M, Anderson MT, Ela SW, Deresinski SC, Herzenberg LA (1997) Glutathione deÞciency is associated with impaired survival in HIV disease. Proc Natl Acad Sci U S A 94:1967Ð1972

156.Sharma A, Kharb S, Chugh SN, Kakkar R, Singh GP (2000) Effect of glycemic control and vitamin E supplementation on total glutathione content in non-insulin-dependent diabetes mellitus. Ann Nutr Metab 44:11Ð13

157.Maher P, Hanneken A (2005) Flavonoids protect retinal ganglion cells from oxidative stressinduced death. Invest Ophthalmol Vis Sci 46:4796Ð4803

158.Coral K, Raman R, Rathi S, Rajesh M, Sulochana KN, Angayarkanni N, Paul PG, Ramakrishnan S (2006) Plasma homocysteine and total thiol content in patients with exudative age-related macular degeneration. Eye (Lond) 20:203Ð207

159.Javadzadeh A, Ghorbanihaghjo A, Bahreini E, Rashtchizadeh N, Argani H, Alizadeh S (2010) Plasma oxidized LDL and thiol-containing molecules in patients with exudative agerelated macular degeneration. Mol Vis 16:2578Ð2584

160.Iwaki T, Kume-Iwaki A, Liem RK, Goldman JE (1989) Alpha B-crystallin is expressed in non-lenticular tissues and accumulates in AlexanderÕs disease brain. Cell 57:71Ð78

161.Renkawek K, de Jong WW, Merck KB, Frenken CW, van Workum FP, Bosman GJ (1992) alpha B-crystallin is present in reactive glia in Creutzfeldt-Jakob disease. Acta Neuropathol 83:324Ð327

162.Rekas A, Adda CG, Andrew Aquilina J, Barnham KJ, Sunde M, Galatis D, Williamson NA, Masters CL, Anders RF, Robinson CV, Cappai R, Carver JA (2004) Interaction of 26 the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid Þbril formation and chaperone activity. J Mol Biol 340:1167Ð1183

163.Renkawek K, Voorter CE, Bosman GJ, van Workum FP, de Jong WW (1994) Expression of alpha B-crystallin in AlzheimerÕs disease. Acta Neuropathol 87:155Ð160

164.Waudby CA, Knowles TP, Devlin GL, Skepper JN, Ecroyd H, Carver JA, Welland ME, Christodoulou J, Dobson CM, Meehan S (2010) The interaction of alphaB-crystallin with mature alpha-synuclein amyloid Þbrils inhibits their elongation. Biophys J 98:843Ð851

165.Vicart P, Caron A, Guicheney P, Li Z, PrŽvost MC, Faure A, Chateau D, Chapon F, TomŽ F, Dupret JM, Paulin D, Fardeau M (1998) A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nat Genet 20:92Ð95

166.Sinclair C, Mirakhur M, Kirk J, Farrell M, McQuaid S (2005) Up-regulation of osteopontin and alphaBeta-crystallin in the normal-appearing white matter of multiple sclerosis: an immunohistochemical study utilizing tissue microarrays. Neuropathol Appl Neurobiol 31:292Ð303

167.Rao NA, Saraswathy S, Wu GS, Katselis GS, Wawrousek EF, Bhat S (2008) Elevated retinaspeciÞc expression of the small heat shock protein, alphaA-crystallin, is associated with photoreceptor protection in experimental uveitis. Invest Ophthalmol Vis Sci 49:1161Ð1171

168.Kase S, Parikh JG, Rao NA (2009) Expression of alpha-crystallin in retinoblastoma. Arch Ophthalmol 127:187Ð192

169.Jones SE, Jomary C, Grist J, Thomas MR, Neal MJ (1998) Expression of alphaB-crystallin in a mouse model of inherited retinal degeneration. Neuroreport 9:4161Ð4165

170.Mackay DS, Andley UP, Shiels A (2003) Cell death triggered by a novel mutation in the alphaA-crystallin gene underlies autosomal dominant cataract linked to chromosome 21q. Eur J Hum Genet 11:784Ð793

171.Liu M, Ke T, Wang Z, Yang Q, Chang W, Jiang F, Tang Z, Li H, Ren X, Wang X, Wang T, Li Q, Yang J, Liu J, Wang QK (2006) IdentiÞcation of a CRYAB mutation associated with autosomal dominant posterior polar cataract in a Chinese family. Invest Ophthalmol Vis Sci 47:3461Ð3466

Chapter 10

The Role of Mitochondrial Oxidative Stress

in Retinal Dysfunction

Stuart G. Jarrett, Alfred S. Lewin, and Michael E. Boulton

Abstract In the eye, the retina and surrounding tissues are exposed to one of the most highly oxidizing microenvironments in the entire human body. This is due, in part, to constant visible light exposure, elevated oxygen partial pressure and phagocytosis of the polyunsaturated fatty acid-loaded photoreceptor outer segments. Accordingly, numerous retinal degenerations including age-related macular degeneration (AMD), diabetic retinopathy, and glaucoma are associated with oxidative stress. Oxidative damage and mitochondrial dysfunction are considered to be signiÞcant factors underlying the initiation and progression of cellular changes during aging and disease. This chapter discusses the high vulnerability of mitochondria and outlines current evidence implicating this organelle as a weak link in the retina. In particular, mitochondrial DNA (mtDNA) damage and defects in the mtDNA repair system may be particularly important to the pathogenesis retinal degenerations. We also consider the importance of mitochondrial biogenesis as well as removal of damaged mitochondria via autophagy as cellular strategies to minimize the effect of mitochondrial damage on cellular function. The speciÞc targeting of mitochondria (e.g., biogenesis, removal, antioxidants, and DNA repair) with pharmacological agents able to protect against retinal damage may offer novel alternatives for the treatment of retinal degenerations.

S.G. Jarrett

Department of Molecular and Biomedical Pharmacology, College of Medicine, University of Kentucky, Lexington, KY, USA

A.S. Lewin

Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, FL, USA

M.E. Boulton (*)

Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, FL, USA

e-mail: meboulton@uß.edu

in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-61779-606-7_10, © Springer Science+Business Media, LLC 2012