Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008
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SUMMARY AND PROSPECTS
Proteomic analyses have identified a number of proteins from the TM and optic nerve of POAG and normal tissue donors. Two of these proteins, namely cochlin and PAD2, were confirmed to be uniquely present in the glaucomatous tissues and appear to contribute to glaucoma pathology. Other proteins detected only in glaucomatous tissues may also be associated with or critical for transforming normal physiological process into pathophysiology, and their glaucoma-related significance remains open to further research. The lack of detection by LC MS/MS does not necessarily mean absence of protein expression; therefore, the unique presence of proteins in glaucomatous tissues must be verified. Since most protein and mRNA abundance levels in tissues are not proportional (64), direct validation of protein expression levels is important, for example by immunoblot and immunohistochemistry. While proteomic analyses provide an unbiased comparison of diseased and normal tissues, overexpressed proteins are more readily detected than low abundancy components. Proteomic detection of down-regulated and low abundance proteins can be enhanced by multidimensional fractionation of both tissue extracts and peptide mixtures prior to mass spectrometric analysis (50,65,66). Emerging quantitative mass spectrometric technologies (67–70) also offer significant promise for further insights regarding molecular mechanisms of ocular pathologies like glaucoma.
Cochlin, discovered in glaucomatous TM by proteomic analyses, is mechanistically implicated in late onset, progressive diseases involving altered fluid flow through extracellular matrices. In the inner ear, cochlin is a major component of the cochlear extracellular matrix (ECM) and overexpression as well as presence of mutant cochlin impacts cochlear fluid flow and has been implicated in the progressive hearing loss disorder DFNA9. In the anterior eye, Cochlin forms co-deposits with mucopolysaccharide around Schlemm’s canal, which may impact aqueous humor outflow through the TM and contribute to elevated IOP. Additional studies are needed to determine whether cochlin deposition in the TM causes increased IOP and whether treatment strategies to lower cochlin expression offer therapeutic potential for glaucoma.
PAD2, discovered in POAG optic nerve by proteomic analyses, may be mechanistically associated with glaucomatous optic neuropathy due of its deiminase activity resulting in the formation of citrulline from peptidyl arginine. Other PAD isoforms and/or citrullination have been implicated in demyelinating diseases including multiple sclerosis (39,58), secondary progressive multiple sclerosis (71), and encephalomyelitis (60) as well as rheumatoid arthritis (44,46) and amyotrophic lateral sclerosis (48). PAD2 has previously been associated with neurodegeneration in rat brain (34,49,72) and now with increased citrullination in POAG optic nerve (12). In POAG optic nerve, several myelin associated proteins appear to be citrullinated, suggesting that glaucomatous optic neuropathy may involve structural disruption of the myelin sheath. Further research is required to corroborate current in vitro and in vivo results implicating elevated IOP with concomitant increased intracellular calcium as stimulators of PAD2 expression. Additional studies are also needed to better understand the normal physiological function and regulation of citrullination and whether treatment strategies to lower PAD2 expression offer therapeutic potential for glaucoma.
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ACKNOWLEDGMENTS
This research was supported in part by the National Glaucoma Research Program of American Health Assistance Foundation, NIH grants EY015266, EY16112, EY6603, EY14239, EY015638, a Research Center Grant from The Foundation Fighting Blindness, a Research to Prevent Blindness (RPB) challenge grant, a RPB Career Development Award (SKB), RPB senior investigator award (JWC) and funds from University of Miami and funds from the Cleveland Clinic Foundation. We are grateful to all our colleagues who participated in the original studies.
REFERENCES
1.Coleman AL. Epidemiology of Glaucoma. In: Morrison JC, Pollack IP, eds. Glaucoma Science and Practice. New York: Thieme Medical Publishers Inc.; 2003:2–11.
2.Quigley HA. Number of people with glaucoma worldwide. Br J Ophthalmol 1996;80(5): 389–93.
3.Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol 2006;90(3):262–7.
4.Hollows FC, Graham PA. Intra-ocular pressure, glaucoma, and glaucoma suspects in a defined population. Br J Ophthalmol 1966;50(10):570–86.
5.Stone EM, Fingert JH, Alward WL, et al. Identification of a gene that causes primary open angle glaucoma. Science 1997;275(5300):668–70.
6.Rezaie T, Child A, Hitchings R, et al. Adult-onset primary open-angle glaucoma caused by mutations in optineurin. Science 2002;295(5557):1077–9.
7.Wirtz MK, Samples JR. Glaucoma Genetics. New York: Thieme; 2003.
8.Wiggs, JL. Genetic etiologies of glaucoma. Arch Ophthalmol 2007;125(1):30–37.
9.Bhattacharya SK, Rockwood EJ, Smith SD, et al. Proteomics reveals cochlin deposits associated with glaucomatous trabecular meshwork. J Biol Chem 2005;280(7):6080–4.
10.Bhattacharya SK, Peachey NS, Crabb JW. Cochlin and glaucoma: a mini-review. Vis Neurosci 2005;22(5):605–13.
11.Bhattacharya SK, Annangudi SP, Salomon RG, Kuchtey RW, Peachey NS, Crabb JW. Cochlin deposits in the trabecular meshwork of the glaucomatous DBA/2J mouse. Exp Eye Res 2005;80(5):741–4.
12.Bhattacharya SK, Crabb JS, Bonilha VL, Gu X, Takahara H, Crabb JW. Proteomics implicates peptidyl arginine deiminase 2 and optic nerve citrullination in glaucoma pathogenesis.
Invest Ophthalmol Vis Sci 2006;47:2508–14.
13.Robertson NG, Hamaker SA, Patriub V, Aster JC, Morton CC. Subcellular localisation, secretion, and post-translational processing of normal cochlin, and of mutants causing the sensorineural deafness and vestibular disorder, DFNA9. J Med Genet 2003;40(7):479–86.
14.Robertson NG, Khetarpal U, Gutierrez-Espeleta GA, Bieber FR, Morton CC. Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening. Genomics 1994;23(1):42–50.
15.Kamarinos M, McGill J, Lynch M, Dahl H. Identification of a novel COCH mutation, I109N, highlights the similar clinical features observed in DFNA9 families. Hum Mutat 2001;17(4):351.
16.Ikezono T, Shindo S, Li L, et al. Identification of a novel Cochlin isoform in the perilymph: insights to Cochlin function and the pathogenesis of DFNA9. Biochem Biophys Res Commun 2004;314(2):440–6.
17. Kaplan F, Ledoux P, Kassamali FQ, et al. A novel developmentally regulated gene in lung mesenchyme: homology to a tumor-derived trypsin inhibitor. Am J Physiol 1999;276(6 Pt 1): L1027–36.
456 |
Bhattacharya and Crabb |
18.Liepinsh E, Trexler M, Kaikkonen A, et al. NMR structure of the LCCL domain and implications for DFNA9 deafness disorder. EMBO J 2001;20(19):5347–53.
19.Tuckwell D. Evolution of von Willebrand factor A (VWA) domains. Biochem Soc Trans 1999;27(6):835–40.
20.John SW, Smith RS, Savinova OV, et al. Essential iris atrophy, pigment dispersion, and glaucoma in DBA/2J mice. Invest Ophthalmol Vis Sci 1998;39(6):951–62.
21.Chang B, Smith RS, Hawes NL, et al. Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice. Nat Genet 1999;21(4):405–9.
22.Bayer AU, Neuhardt T, May AC, et al. Retinal morphology and ERG response in the DBA/2NNia mouse model of angle-closure glaucoma. Invest Ophthalmol Vis Sci 2001;42(6):1258–65.
23.Savinova OV, Sugiyama F, Martin JE, et al. Intraocular pressure in genetically distinct mice: an update and strain survey. BMC Genet 2001;2(1):12.
24.Ueda J, Wentz-Hunter K, Yue BY. Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human eyes. Invest Ophthalmol Vis Sci 2002;43(4):1068–76.
25.Lutjen-Drecoll E, Shimizu T, Rohrbach M, Rohen JW. Quantitative analysis of ‘plaque material’ in the innerand outer wall of Schlemm’s canal in normaland glaucomatous eyes. Exp Eye Res 1986;42(5):443–55.
26.Masliah E, Hashimoto M. Development of new treatments for Parkinson’s disease in transgenic animal models: a role for beta-synuclein. Neurotoxicology 2002;23(4–5):461–8.
27.McKinnon SJ. Glaucoma: ocular Alzheimer’s disease? Front Biosci 2003;8:s1140–56.
28.Pareti FI, Niiya K, McPherson JM, Ruggeri ZM. Isolation and characterization of two domains of human von Willebrand factor that interact with fibrillar collagen types I and III. J Biol Chem 1987;262(28):13835–41.
29.Marchant JK, Hahn RA, Linsenmayer TF, Birk DE. Reduction of type V collagen using a dominant-negative strategy alters the regulation of fibrillogenesis and results in the loss of corneal-specific fibril morphology. J Cell Biol 1996;135(5):1415–26.
30.Anderson MG, Smith RS, Savinova OV, et al. Genetic modification of glaucoma associated phenotypes between AKXD-28/Ty and DBA/2J mice. BMC Genet 2001;2(1):1.
31.Mabuchi F, Lindsey JD, Aihara M, Mackey MR, Weinreb RN. Optic nerve damage in mice with a targeted type I collagen mutation. Invest Ophthalmol Vis Sci 2004;45(6):1841–5.
32.Libby RT, Smith RS, Savinova OV, et al. Modification of ocular defects in mouse developmental glaucoma models by tyrosinase. Science 2003;299(5612):1578–81.
33.Rodriguez CI, Cheng JG, Liu L, Stewart CL. Cochlin, a secreted von Willebrand factor type a domain-containing factor, is regulated by leukemia inhibitory factor in the uterus at the time of embryo implantation. Endocrinology 2004;145(3):1410–8.
34.Asaga H, Ishigami A. Protein deimination in the rat brain after kainate administration: citrulline-containing proteins as a novel marker of neurodegeneration. Neurosci Lett 2001;299(1–2):5–8.
35.Bhattacharya SK, Bhat MB, Takahara H. Modulation of peptidyl arginine deiminase 2 and implication for neurodegeneration. Curr Eye Res 2006;31(12):1063–71.
36.Nakashima K, Hagiwara T, Yamada M. Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes. J Biol Chem 2002;277(51):49562–8.
37.Cuthbert GL, Daujat S, Snowden AW, et al. Histone deimination antagonizes arginine methylation. Cell 2004;118(5):545–53.
38.Wang Y, Wysocka J, Sayegh J, et al. Human PAD4 regulates histone arginine methylation levels via demethylimination. Science 2004;306(5694):279–83.
39.Moscarello MA, Pritzker L, Mastronardi FG, Wood DD. Peptidylarginine deiminase: a candidate factor in demyelinating disease. J Neurochem 2002;81(2):335–43.
Proteomic Advances in Glaucoma |
457 |
40.Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays 2003;25(11): 1106–18.
41.Kubilus J, Baden HP. Purification and properties of a brain enzyme which deiminates proteins. Biochim Biophys Acta 1983;745(3):285–91.
42.Arai T, Kusubata M, Kohsaka T, Shiraiwa M, Sugawara K, Takahara H. Mouse uterus peptidylarginine deiminase is expressed in decidual cells during pregnancy. J Cell Biochem 1995;58(3):269–78.
43.Terakawa H, Takahara H, Sugawara K. Three types of mouse peptidylarginine deiminase: characterization and tissue distribution. J Biochem (Tokyo) 1991;110(4):661–6.
44.Scofield RH. Autoantibodies as predictors of disease. Lancet 2004;363(9420):1544–6.
45.Gyorgy B, Toth E, Tarcsa E, Falus A, Buzas EI. Citrullination: A posttranslational modifi-
cation in health and disease. Int J Biochem Cell Biol 2006;38(10):1662–77.
46. van Gaalen F, Ioan-Facsinay A, Huizinga TW, Toes RE. The devil in the details: the emerging role of anticitrulline autoimmunity in rheumatoid arthritis. J Immunol 2005;175(9):5575–80.
47.Kim JK, Mastronardi FG, Wood DD, Lubman DM, Zand R, Moscarello MA. Multiple sclerosis: an important role for post-translational modifications of myelin basic protein in pathogenesis. Mol Cell Proteomics 2003;2(7):453–62.
48.Chou SM, Wang HS, Taniguchi A. Role of SOD-1 and nitric oxide/cyclic GMP cascade on neurofilament aggregation in ALS/MND. J Neurol Sci 1996;139 Suppl:16–26.
49.Asaga H, Akiyama K, Ohsawa T, Ishigami A. Increased and type II-specific expression of peptidylarginine deiminase in activated microglia but not hyperplastic astrocytes following kainic acid-evoked neurodegeneration in the rat brain. Neurosci Lett 2002;326(2):129–32.
50.Bhattacharya SK, Crabb JS, West KA, et al. Optic nerve fractionation for proteomics. In: Smejkal GB, Lazarev A, eds. Separation Methods in Proteomics. Boca Raton: CRC Press; 2006:135–55.
51.Osborne SL, Herreros J, Bastiaens PI, Schiavo G. Calcium-dependent oligomerization of synaptotagmins I and II. Synaptotagmins I and II are localized on the same synaptic vesicle and heterodimerize in the presence of calcium. J Biol Chem 1999;274(1):59–66.
52.Kursula P, Tikkanen G, Lehto VP, Nishikimi M, Heape AM. Calcium-dependent interaction between the large myelin-associated glycoprotein and S100beta. J Neurochem 1999;73(4):1724–32.
53.Pritzker LB, Joshi S, Gowan JJ, Harauz G, Moscarello MA. Deimination of myelin basic protein. 1. Effect of deimination of arginyl residues of myelin basic protein on its structure and susceptibility to digestion by cathepsin D. Biochemistry 2000;39(18):5374–81.
54.Boggs JM, Yip PM, Rangaraj G, Jo E. Effect of posttranslational modifications to myelin basic protein on its ability to aggregate acidic lipid vesicles. Biochemistry 1997;36(16):5065–71.
55.Pritzker LB, Joshi S, Harauz G, Moscarello MA. Deimination of myelin basic protein. 2. Effect of methylation of MBP on its deimination by peptidylarginine deiminase. Biochemistry 2000;39(18):5382–8.
56.D’Souza CA, Wood DD, She YM, Moscarello MA. Autocatalytic cleavage of myelin basic
protein: an alternative to molecular mimicry. Biochemistry 2005;44(38):12905–13.
57. D’Souza CA, Moscarello MA. Differences in susceptibility of MBP charge isomers to digestion by stromelysin-1 (MMP-3) and release of an immunodominant epitope. Neurochem Res 2006;31(8):1045–54.
58.Mastronardi FG, Moscarello MA. Molecules affecting myelin stability: a novel hypothesis regarding the pathogenesis of multiple sclerosis. J Neurosci Res 2005;80(3):301–8.
59.Harauz G, Musse AA. A tale of two citrullines-structural and functional aspects of myelin basic protein deimination in health and disease. Neurochem Res 2007;32(2):137–58.
458 |
Bhattacharya and Crabb |
60.Nicholas AP, Sambandam T, Echols JD, Barnum SR. Expression of citrullinated proteins in murine experimental autoimmune encephalomyelitis. J Comp Neurol 2005;486(3):254–66.
61.Raijmakers R, Vogelzangs J, Croxford JL, Wesseling P, van Venrooij WJ, Pruijn GJ. Citrullination of central nervous system proteins during the development of experimental autoimmune encephalomyelitis. J Comp Neurol 2005;486(3):243–53.
62.Gong H, Zolzer F, von Recklinghausen G, Havers W, Schweigerer L. Arginine deiminase inhibits proliferation of human leukemia cells more potently than asparaginase by inducing cell cycle arrest and apoptosis. Leukemia 2000;14(5):826–9.
63.Gong H, Zolzer F, von Recklinghausen G, et al. Arginine deiminase inhibits cell proliferation by arresting cell cycle and inducing apoptosis. Biochem Biophys Res Commun 1999;261(1):10–4.
64.Gygi SP, Rochon Y, Franza BR, Aebersold R. Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 1999;19(3):1720–30.
65.Washburn MP, Wolters D, Yates JR, 3rd. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 2001;19(3):242–7.
66.Bhattacharya SK, West KA, Gu X, et al. Fractionation of Retina for proteomic analyses. In: Smejkal GB, Lazarev A, eds. Separation Methods in Proteomics. Boca Raton: CRC Press; 2006:157–85.
67.Ross PL, Huang YN, Marchese JN, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 2004;3(12):1154–69.
68.Qian WJ, Monroe ME, Liu T, et al. Quantitative proteome analysis of human plasma following in vivo lipopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach. Mol Cell Proteomics 2005;4(5):700–9.
69.Silva JC, Denny R, Dorschel C, et al. Simultaneous qualitative and quantitative analysis of the Escherichia coli proteome: a sweet tale. Mol Cell Proteomics 2006;5(4):589–607.
70.Hajkova D, Rao KC, Miyagi M. pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase and trypsin. J Proteome Res 2006;5(7):1667–73.
71.Nicholas AP, Sambandam T, Echols JD, Tourtellotte WW. Increased citrullinated glial fibrillary acidic protein in secondary progressive multiple sclerosis. J Comp Neurol 2004;473(1):128–36.
72.Asaga H, Yamada M, Senshu T. Selective deimination of vimentin in calcium ionophoreinduced apoptosis of mouse peritoneal macrophages. Biochem Biophys Res Commun 1998;243(3):641–6.
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Molecular and Cellular Responses in the Eye to Glaucoma
Ocular Gene Expression in Experimental Glaucoma
Tomonari Ojima, md, phd, and Nagahisa Yoshimura, md, phd
CONTENTS
Animal Models for Glaucoma Research
Analysis of Gene Expression Using the Microarray System References
ANIMAL MODELS FOR GLAUCOMA RESEARCH
Glaucoma is a group of optic neuropathies characterized by the death of retinal ganglion cells (RGCs) and their axons. To better understand the mechanism of RGC damage, various experimental models have been used, including both in vitro and in vivo models. In vitro culturing of cells from postmortem tissues allows the study of how cells isolated from other cell types respond to various stimuli. Many cell types can be extracted from eye tissues and cultured, including RGCs (1), trabecular meshwork cells (2), ciliary muscle cells (3), and lamina cribrosa cells (4). Although the simplicity of this system makes it very attractive, conditions may differ greatly from those of the in vivo ocular environment. The organ culture system involves the culturing of pieces of whole tissue, such as retina (5), ciliary body, or trabecular meshwork (6), and allows the study of cell responses in association with neighboring cells or extracellular matrix. Although the cellular responses in organ culture are more like those encountered in human glaucoma than are those of the more simple cell-culturing system, the experimental responses elicited, including tissue reorganization and cellular differentiation, may differ from human glaucomatous cell responses. Although postmortem human eyes are used to study the histopathology of glaucoma, their availability is limited and the eyes obtained are heterogeneous and have no controls, making difficult the investigation of gene expression changes related to glaucoma development and progression.
From: Ophthalmology Research: Mechanisms of the Glaucomas
Edited by: J. Tombran-Tink, C. J. Barnstable, and M. B. Shields © Humana Press, Totowa, NJ
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In contrast to the above-mentioned systems, animal models of glaucoma allow for more physiologic conditions; so, cells in the retina and optic nerve more closely mimic the ocular conditions in human glaucoma. Furthermore, in contrast to in vitro systems, the response in animal models to certain stimuli, for example, to a new drug, is actually the combined responses of many cell types, which is similar to the response of the human ocular system in glaucoma. To generate glaucoma models, the monkey, rat, and mouse are generally used, and as each animal model has advantages and disadvantages, the choice of animal species is important. The rat model is the most commonly used because the eye is moderate in size and handling of the animal is comparatively easy. The monkey ocular system is closer to the human ocular system than is that of the rat, but the cost is high, availability is somewhat limited, and handling can be difficult. The mouse has a smaller eye than the rat, and measurement of intraocular pressure (IOP) is more difficult than in other models, but the mouse has the advantage of transgenic and mutant strains, which are scarcely available in other species.
Rat Glaucoma Model
Axons of the rat optic nerve are unmyelinated and are supported by astrocytes at the scleral level, similar to primate eyes, but the lamina cribrosa of the rat eye is relatively
Fig. 1. Rat glaucoma model induced by episcleral vein ligation. (Top) Schema of episcleral vein ligation. (Bottom) Photographs of episcleral vein before (left) and after (right) ligation.
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sparse and thin, suggesting that the rat optic nerve is more susceptible to high IOP than are human eyes. There are several methods to induce high IOP in rat eyes, and these can be divided into two categories: the first is based on obstruction of aqueous humor outflow, whereas the second involves uveal vascular congestion. The first type of method includes injection of hypertonic saline solution into the episcleral vein to induce scarring of aqueous humor outflow pathways (7), and laser photocoagulation of trabecular meshwork after injection of India ink into the anterior chamber to improve energy uptake (8). The second category of method includes cauterization (9) or ligation (10) of the episcleral veins or the vortex veins (see Fig. 1). Chronic elevation of IOP induced by these methods is known to result in RGC loss and in remodeling of the optic nerve head, similar to the changes seen in human eyes with glaucoma (7). Because the experimental rat model is of relatively low cost and is easy to handle, it is suitable for studying the cellular mechanisms of glaucomatous damage, thereby connecting in vitro cellular studies with what takes place in human glaucoma.
Monkey Glaucoma Model
Monkey eyes have a structure that is almost identical to that of human eyes, and the monkey glaucoma model thus most closely resembles human glaucoma. In this glaucoma model, high IOP is induced primarily by one of two methods: repeated laser photocoagulation of the trabecular meshwork (11,12), or injection of latex microspheres (13) or cross-linked polyacrylamide microgels (14) into the anterior chamber. In the latter model, the IOP elevation is less consistent than in the photocoagulation model. In both models, however, the elevated IOP results in optic disc findings similar to those of human glaucoma, including optic disc cupping, excavation, and neuroretinal rim thinning (12), although the time-course in the monkey eye is relatively accelerated (see Fig. 2). The characteristic feature of the monkey model is that it is possible to correlate pathology with function. It has even been reported that trained monkeys with glaucoma demonstrate perimetric visual field loss in a pattern similar to that seen in human glaucoma patients (15).
Mouse Glaucoma Model
Despite the much smaller size, mouse eyes have well-developed anterior chamber components, including trabecular meshwork, Schlemm’s canal, and ciliary body (16), similar to those of the human eye. Mouse eyes are also known to have a uveoscleral aqueous humor outflow pathway (17) and aqueous humor dynamics that are physiologically similar to human and monkey eyes (18). Nevertheless, it is difficult to accurately measure the IOP in mouse eyes because of their small size. Recently, however, new methods have been developed that now make possible the measurement of mouse IOP. The direct measurement using a glass-tipped micropipette inserted into the anterior chamber and connected to a pressure transducer can provide accurate results, but this method requires anesthesia, and repeated measurement over a short period of time is difficult (19). Measurement using the Tono-Pen is non-invasive, but accuracy is inferior to that of the direct method and repeated measurements by a skilled operator are necessary in order to obtain useful results (20). In mice, an elevated IOP occurs spontaneously in certain strains or can be induced in other strains by various experimental procedures (21). Despite the anatomical difference between the mouse eye and
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the human eye (i.e., the mouse eye lacks a lamina cribrosa) (22), optic nerve damage is reported to correlate with the duration and extent of IOP elevation, suggesting a mechanism similar to that of human glaucoma (23). The advantage of experimental glaucoma using the mouse model is the opportunity to induce genetic alterations in transgenic mice or in a targeted mutation model. The inbred DBA/2J mouse has two gene mutations, Tyrp 1 and Gpnmb; these cause an immune response to the iris, and result in iris atrophy and pigment dispersion, which, in turn, leads to an elevated IOP that increases in an age-dependent manner (24). The RGC loss and the optic nerve head cupping seen in this DBA/2J mouse is known to be dependent on the duration of the IOP elevation (25).
Gene Expression in Experimental Glaucoma
To better understand the mechanism of neuronal and axonal damage observed in glaucoma, studies have been performed using animal models of glaucoma, such as those mentioned above. Chronic elevation of IOP is reported to change the expression levels and distribution of selected mRNAs and proteins in the retina and optic nerve head. These investigations utilized molecular biological methods, including northern blot analysis, western blot analysis, immunostaining, and reverse transcriptase-polymerase chain reaction (RT-PCR). Many of the changes in molecular expression seen in glaucoma are associated with glial activation, apoptosis, and RGC loss.
Fig. 2. Optic disc configuration changes in monkey glaucoma model. Longitudinal sections (left) and photographs (right) of optic discs in control eye (top) and glaucoma eye (bottom) (Courtesy of Teruyoshi Miyahara, MD, PhD).
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Astrocytes and Muller cells are major glial cells in the retina, and astrocyte and oligodendrocytes are major glial cells in the optic nerve head. These glial cells play important roles in homeostasis of the retina and optic nerve head. In response to IOP elevation, these glial cells are activated, and glial fibrillary acidic protein (GFAP), a glia-specific intermediate filament, is up-regulated in both Muller cells and astrocytes (26). Activated glial cells play important roles in the protection of neural cells and in promotion of tissue repair, and are associated with increasing synthesis of heat shock proteins (HSP60 and HSP27), in both the retina and the optic nerve head (27). At the same time, activated glial cells may have cytotoxic effects on neural cells by expressing tumor necrosis factor (TNF)-alpha on glial cells and TNF-alpha receptor-1 on RGCs (28).
Microglia belong to the mononuclear phagocyte group and play an important role in development of the central nervous system; in the developing nervous system, they are responsible for the removal of cell debris that results from naturally occurring cell death (29). In experimental glaucoma, microglia activation is reported to coincide with RGC degeneration, and the expression of OX42 protein, which is a marker of activated microglia, is up-regulated (30,31).
RGCs are known to die by apoptosis in glaucoma eyes of both human and experimental animal models, as well as in optic nerve transection models (32). Apoptosis in glaucoma is indicated by characteristic findings, including TdT-mediated dUTP nick end labeling (TUNEL) of RGCs and DNA laddering in electrophoretic analysis of the retina (10). Chronically elevated IOP interferes with axonal transport of RGCs, especially with retrograde delivery of various neurotrophins that are essential for RGC survival. In experimental glaucoma, neurotrophins are reported to be depleted in the RGC layer of the retina. These depleted neutrophins include basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glial-derived neurotrophic factor (GDNF), and, subsequently, apoptosis of the RGCs occurs (10). RGC death may also be induced by glutamate excitotoxicity (33), possibly resulting from down-regulation of retinal glutamate transporters, which has been reported to occur in an experimental glaucoma model (34). In apoptotic RGC death, members of the caspase family are known to play central roles in glaucoma as well as in other neurodegenerative diseases, including Alzheimer’s disease (35). In a rat experimental model, mRNA of caspase-3 and caspase-8 is reported to be upregulated (36), suggesting activation of the caspase cascade by exposure to elevated IOP. In addition to the caspase cascade, a prosurvival gene IAP-1, a caspase inhibitor, is also reported to be up-regulated in experimental rat glaucoma (37), which may suggest the simultaneous activation of proapoptotic mechanism and intrinsic neuroprotective mechanism in glaucoma. In damaged RGCs, the expression of Thy1 mRNA, a surface glycoprotein uniquely expressed by RGCs, is decreased independent of RGC cell number, suggesting that this molecule as an early marker of RGC stress (38).
ANALYSIS OF GENE EXPRESSION USING
THE MICROARRAY SYSTEM
In the 1990s, DNA microarrays were developed that enabled researchers to examine simultaneously the expression of many genes. DNA microarrays are glass slides studded with a large number of oligonucleotides, each of which is a probe for a specific gene.