Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999
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4. CONCLUSIONS
Progress is being made in characterization of the RPE and its apical microvilli. The years to come will bring further definition of key proteins and pathways present in RPE microvilli as well as a better understanding of their function in vision.
5. ACKNOWLEDGMENTS
Supported by NIH grants EY06603, EY14239, EY014240 and an infrastructure grant EY015638, a Research Center grant from the Foundation Fighting Blindness, a grant from the National Glaucoma Research Program of American Health Assistance Foundation (G2004-047 to SKB), and funds from the Cleveland Clinic Foundation.
6.REFERNCES
1.K. M. Zinn, and J. V. Benjamin-Henkind., Anatomy of the human retinal pigment epithelium. in: The Retinal Pigment Epithelium, edited by K. M. Zinn, M. F. Marmor, (Harvard University Press; Cambridge, MA: 1979), pp:3-31.
2.D. Bok, The retinal pigment epithelium: a versatile partner in vision. J Cell Sci Suppl 17:189-195 (1993).
3.V. L. Bonilha, S. K. Bhattacharya, K. A. West, J. S. Crabb, J. Sun, M. E. Rayborn, M. Nawrot, J. C. Saari, and J. W. Crabb., Support for a proposed retinoid-processing protein complex in apical retinal pigment epithelium. Exp Eye Res 79:419-422 (2004).
4.T. D. Lamb, and E. N. Jr. Pugh, Dark adaptation and the retinoid cycle of vision. Prog Retin Eye Res 23:307380 (2004).
5.R. H. Steinberg, and I. Wood, The Relationship of the Retinal Pigment Epithelium to Photoreceptor Outer Segments in Human Retina. in: The Retinal Pigment Epithelium, edited by K. M. Zinn, M. F. Marmor (Harvard University Press; Cambridge, MA: 1979), pp:32-44.
6.D. Gundersen, J. Orlowski, and E. Rodriguez-Boulan, Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton. J Cell Biol 112:863-872 (1991).
7.D. Gundersen, S. K. Powell, and E. Rodriguez-Boulan, Apical polarization of N-CAM in retinal pigment epithelium is dependent on contact with the neural retina. J Cell Biol 121:335-343 (1993).
8.A. D. Marmorstein, S. C. Finnemann, V. L. Bonilha, and E. Rodriguez-Boulan, Morphogenesis of the retinal pigment epithelium: toward understanding retinal degenerative diseases. Ann N Y Acad Sci 857:1-12 (1998).
9.D. K. Vaughan, and S. K. Fisher, The distribution of F-actin in cells isolated from vertebrate retinas. Exp Eye Res 44:393-406 (1987).
10.T. Hasson, M. B. Heintzelman, J. Santos-Sacchi, D. P. Corey, and M. S. Mooseker, Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type 1B. Proc Natl Acad Sci U S A 92:9815-9819 (1995).
11.D. Hofer, and D. Drenckhahn, Molecular heterogeneity of the actin filament cytoskeleton associated with microvilli of photoreceptors, Muller’s glial cells and pigment epithelial cells of the retina. Histochemistry 99:29-35 (1993).
12.K. Owaribe, and G. Eguchi, Increase in actin contents and elongation of apical projections in retinal pigmented epithelial cells during development of the chicken eye. J Cell Biol 101:590-596 (1985).
13.V. L. Bonilha, S. C. Finnemann, and E. Rodriguez-Boulan, Ezrin promotes morphogenesis of apical microvilli and basal infoldings in retinal pigment epithelium. J Cell Biol 147:1533-1548 (1999).
14.V. L. Bonilha, and E. Rodriguez-Boulan, Polarity and developmental regulation of two PDZ proteins in the retinal pigment epithelium. Invest Ophthalmol Vis Sci 42:3274-3282 (2001).
15.M. Nawrot, K. West, J. Huang, D. E. Possin, A. Bretscher, J. W. Crabb, and J. C. Saari, Cellular retinaldehydebinding protein interacts with ERM-binding phosphoprotein 50 in retinal pigment epithelium. Invest Ophthalmol Vis Sci 45:393-401 (2004).
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16.N. G. Cooper, B. I. Tarnowski, and B. J. McLaughlin, Lectin-affinity isolation of microvillous membranes from the pigmented epithelium of rat retina. Curr Eye Res 6:969-979 (1987).
17.V. L. Bonilha, S. K. Bhattacharya, K. A. West, J. Sun, J. W. Crabb, M. E. Rayborn, and J. G. Hollyfield, Proteomic characterization of isolated retinal pigment epithelium microvilli. Mol Cell Proteomics 3:1119-1127 (2004)
18.M. F. Marmor. Mechanisms of Retinal Adhesion. in: Progress in Retinal Research, edited by N. Osborne, G. Chader (Pergamon Press, New York, NY: 1993), pp. 179-204.
19.M. A. Maw, B. Kennedy, A. Knight, R. Bridges, K. E. Roth, E. J. Mani, J. K. Mukkadan, D. Nancarrow, J. W. Crabb, and M. J. Denton, Mutation of the gene encoding cellular retinaldehyde-binding protein in autosomal recessive retinitis pigmentosa. Nat Genet 17:198-200 (1997).
20.Q. Wang, Q. Chen, K. Zhao, L. Wang, and E. I. Traboulsi, Update on the molecular genetics of retinitis pigmentosa. Ophthalmic Genet 22:133-154 (2001).
21.M. D. Farber, S. Lam, H. H. Tessler, T. J. Jennings, A. Cross, and M. M. Rusin, Reduction of macular oedema by acetazolamide in patients with chronic iridocyclitis: a randomised prospective crossover study. Br J Ophthalmol 78:4-7 (1994).
22.G. A. Fishman, L. D. Gilbert, R. G. Fiscella, A. E. Kimura, and L. M. Jampol, Acetazolamide for treatment of chronic macular edema in retinitis pigmentosa. Arch Ophthalmol 107:1445-1452 (1989).
23.J. C. Chen, F. W. Fitzke, and A. C. Bird, Long-term effect of acetazolamide in a patient with retinitis pigmentosa. Invest Ophthalmol Vis Sci 31:1914-1918 (1990).
24.M. F. Marmor, Hypothesis concerning carbonic anhydrase treatment of cystoid macular edema: example with epiretinal membrane. Arch Ophthalmol 108:1524-1525 (1990).
25.T. J. Wolfensberger. The role of carbonic anhydrase inhibitors in the management of macular edema. Doc Ophthalmol 97:387-397 (1999).
26.I. Weisse, Changes in the aging rat retina. Ophthalmic Res. 1995;27:154-163.
27.T. Hirai, S. Kojima, A. Shimada, T. Umemura, M. Sakai, and C. Itakura, Age-related changes in the olfactory system of dogs. Neuropathol Appl Neurobiol 22:531-539 (1996).
28.I. Jang, K. Jung, and J. Cho, Influence of age on duodenal brush border membrane and specific activities of brush border membrane enzymes in Wistar rats. Exp Anim 49:281-287 (2000).
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32.L. Teillet, L. Preisser, J. M. Verbavatz, and B. Corman, Kidney aging: cellular mechanisms of problems of hydration equilibrium. Therapie 4:147-154 (1999).
33.D. L. Schmucker, K. Thoreux, R. L. Owen, Aging impairs intestinal immunity. Mech Ageing Dev 122:13971411 (2001).
34.P. Hahn, T. Dentchev, Y. Qian, T. Rouault, Z. L. Harris, and J. L. Dunaief JL, Immunolocalization and regulation of iron handling proteins ferritin and ferroportin in the retina. Mol Vis. 10:598-607 (2004).
35.P. Hahn, Y. Qian, T. Dentchev, L. Chen, J. Beard J, Z. L. Harris, and J. L. Dunaief, Disruption of ceruloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration. Proc Natl Acad Sci U S A 101:13850-13855 (2004). Epub 12004 Sep 13813.
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CHAPTER 73
UPREGULATION OF TRANSGLUTAMINASE IN THE GOLDFISH RETINA DURING OPTIC NERVE REGENERATION
Kayo Sugitani, Toru Matsukawa, Ari Maeda, and Satoru Kato*
1. SUMMARY
To elucidate the molecular involvement of transglutaminase (TG) in central nervous system (CNS) regeneration, we cloned a full-length cDNA for neural TG (TGN) from axotomized goldfish retinas and produced a recombinant TGN protein from this cDNA. The levels of TGN mRNA and protein were increased at 10-30 days after optic nerve transection, and this increase in TGN was only localized in the ganglion cells in goldfish retinas. In retinal explant cultures, the recombinant TGN protein induced a drastic enhancement of neurite outgrowth, while TGN-specific RNAi significantly suppressed this neurite outgrowth. Taken together, these data strongly indicate that TGN is a key regulatory molecule for CNS regeneration.
2. INTRODUCTION
Transglutaminase (TG), a protein cross-linking enzyme, is widely distributed in mammalian cells and tissues. Neural TG (TGN), which is expressed in neural tissue, rapidly increased in rat sciatic nerves1 and superior cervical ganglia2 after nerve injury. In the central nerve system, the TGN activity of goldfish optic nerve increased, whereas that of rat optic nerve decreased after optic nerve crush.3 Fish can successfully regenerate the optic axons and eventually function after nerve injury, whereas rat cannot regenerate their optic axons. Therefore, to elucidate a functional role of TGN on CNS regeneration in genetic level, we first isolated a full-length cDNA clone for TGN from a cDNA library prepared from axotomized goldfish retinas. In addition, we produced a recombinant TGN protein and anti-TGN
* Satoru Kato, Department of Molecular Neurobiology, Graduate School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan, TEL: +81 76 265 2450; E-mail: satoru@med.kanazawa-u.ac.jp.
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antiserum. We also investigated the expression and localization of the TGN protein by immunohistochemical staining. Moreover, we made a TGN specific small interference RNA to estimate the effect of TGN on neurite outgrowth in explant culture system. In the present study, we showed a novel functional role of TGN on axonal elongation of goldfish optic nerve after injury.
3. METHODS
3.1. Animals
Adult common goldfish (Carassius auratus; body length about 6-8cm) were used throughout this study. Goldfish were anesthetized with ice-cold water. The optic nerve was sectioned 1mm away from the posterior of the eyeball with scissors. After surgery the goldfish were kept in water tanks at 22°C ± 1°C for 1-40 days.
3.2. Cloning of Goldfish Neural Transglutaminase (TGN)
A cDNA library was constructed from poly (A)+ RNA (5 mg) from goldfish retinas of which optic nerve had been transected 5 days before as described previously.4 Tissue-type transglutaminase (tTG) cDNA from red sea bream (Pagrus major) liver5 (gift from Dr. Yasueda, Ajinomoto Co.) was labeled with [32P] dCTP and 2 ¥ 105 colonies were screened with this probe. Five positive clones were subcloned into pBK-CMV phagemid and sequenced usig DNA seqencer. Two independent clones were obtained. The 5’ TGN mRNA was cloned by the RACE method.
3.3. Purification of Recombinant TGN
A full-length TGN cDNA clone was inserted into the expression vector pFLAG- CMV-1, and the constructs were transfected into HEK 293 cells using Lipofectamine. For the control, only the pFLAG-CMV-1 vector was transfected to create mock cells. All cells were maintained in Dulbecco’s MEM containing 10% fetal calf serum in a 5% CO2 humidified incubator for 48 h at 37°C. The cells were then harvested, lysed and centrifuged for 30 min at 15,000 g. The FLAG-tagged enzyme was purified using ANTI-FLAG M2 affinity gel.
3.4. Immunohistochemistry
A rabbit antiserum against TGN was obtained by subcutaneous injection of purified TGN. Tissue fixation and cryosectioning were carried out as described previously.6 Retinal sections were autoclaved at 121°C for 15 min in 10 mM citrate buffer. After washing and blocking, the sections were incubated with the rabbit polyclonal anti-TGN antibody (1:100 dilution) overnight at 4°C. Following incubation with a biotinylated secondary antibody for 2 h at room temperature, the bound antibodies were detected using horseradish peroxidaseconjugated streptavidin and 3-amino-9-ethylcarbazole.
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3.5. Retinal Explant Culture
Retinal explant culture was performed according to a previous method of Matsukawa et al.6 For siRNA synthesis, the in vitro Transcription T7 Kit for siRNA synthesis was used according to the manufacture’s instruction. Transfections of siRNA to retinal explants were carried out using Lipofectamine 2000. For each transfection sample, 2 ml of Lipofectamine 2000 diluted in 98 ml of L-15 medium was mixed with 100 pmol of siRNA diluted in 100 ml of L-15 medium, incubated for 20 min at room temperature to allow complex formation and then added to 0.8 ml of the resuspended retinal culture. The retinal explants were gently mixed with the culture medium for 3 h and then divided into two 35-mm culture dishes. After incubation at 28°C overnight, 50 ml of fetal calf serum was added to each dish and the culture was continued.
4. RESULTS
4.1. TGN Expression in the Retina after Optic Nerve Transection
The expression of TGN protein levels in the goldfish retina was investigated after optic nerve transection using the anti-TGN antiserum. Weak signals for the TGN protein could be seen in the ganglion cell layers of control retina (Fig. 73.1a).
The immunoreactivity in the ganglion cell layer started to increase at 10 days and peaked at 20-30 days (Fig. 73.1b) and then decreased by 40 days after axotomy (Fig. 73.1c). The increase in TGN immunoreactivity was only localized in the ganglion cells and nerve layers (Fig. 73.1a,b,c). The expression pattern of TGN mRNA after optic nerve injury was the same as that of TGN protein (data not shown).
4.2. Moduration of Neurite Outgrowth by a Recombinant TGN Protein and RNAi in Retinal Explant Cultures
Addition of the recombinant TGN protein induced a large number of explants with long and thick neurites after 2 days (Fig. 73.2b,d), as compared with the control culture (Fig. 73.2a,d). The neurite outgrowth of the culture containing recombinant TGN protein was evoked in 50% of the explants during 2 days of culture whereas neurite outgrowth of the
Figure 73.1. Immunohistochemical staining of goldfish retina with the anti-TGN antibody. (a) control retina,
(b) at 20 days after optic nerve transection, (c) at 40 days after optic nerve transection. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar = 40 mm.
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Figure 73.2. Explant culture of adult goldfish retinas treated with recombinant TGN protein and TGN-specific RNAi. (a) No addition. (b) Recombinant TGN protein (4 mg/ml). (c) TGN-specific siRNA (100 pmol/ml). (d) Graphical representation of the neurite outgrowth for 2 days of culture. Note a suppression of neurite outgrowth by TGN- specific siRNA (**p < 0.01) and an enhancement of neurite outgrowth by recombinant TGN (*p < 0.01) compared with control cultures. The values represent the mean ± SD in five independent experiments. Scale bar = 200 mm.
control culture was evoked in 30% of the explants (Fig. 73.2d). 21-bp siRNAs for TGN- specific siRNA and random siRNA were chemically synthesized and transfected into retinal explants in culture using Lipofectamine 2000. Figure 73.2c shows the neurite outgrowth from retinal explants transfected with the TGN-specific siRNA (100 pmol/ml) was clearly inhibited and only short neurites could be seen (Figure 73.2c,d). In contrast, retinal explants transfected with the random siRNA (100 pmol/ml) had no difference of neurite outgrowth of the control retina (data not shown).
5. DISCUSSION
5.1. Changes in TGN Expression after Optic Nerve Transection
Eitan and Schwaltz reported that a crude neural TG (TGN) enzyme preparation from injured goldfish optic nerves could partially regenerate injured rat optic nerves in vivo.7 Hence, we used goldfish retinas for characterization of the TGN in this study. First, to inves-
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tigate the functional role of TGN in CNS regeneration at a genetic level, we identified a fulllength of TGN cDNA clone using a retinal cDNA library from axotomized goldfish retinas.4 The cDNA clone for TGN encoded 678 amino acid residues with a molecular mass of 76 kDa. Levels of TGN mRNA and protein started to increase in the goldfish retina at 10 days and peaked at 20-30 days after axotomy. Furthermore, we clearly showed that the increases in TGN mRNA and protein was only localized in the retinal ganglion cells (RGCs) and nerve fiber layers. The period of 10-30 days for the upregulation of TGN corresponds to a stage of axonal elongation in the goldfish visual system.8 In our immunohistochemical study, TGN protein in the rat RGCs rapidly decreased at 3 days after optic nerve injury (data not shown). These contrastive results of TGN expression in the rat and goldfish retinas suggest that induction of TGN is an important event for optic nerve regeneration in goldfish.
5.2. The Functional Role of TGN during Optic Nerve Regeneration
TG catalyzes post-translational, covalent protein cross-linking reactions in diverse processes in nervous systems.9 During development, TGN activity is highest in the early postnatal stage of the mouse CNS.10 In cerebellar granule neurons, TG inhibitors caused destabilization of neurites during the initial outgrowth period of cultured granule cells.11 In our culture study, we clearly demonstrated that recombinant TGN induced a drastic extension of long and thick neurites and that TGN-specific RNAi significantly inhibited neurite outgrowth. The culture study shows that TGN activity directly enhanced neurite outgrowth from RGCs after nerve injury via dimerization of bioactive peptides. Although the target proteins or substrates for TGN have not yet been identified, midkine and galectin-3 are known to be present in embryonic mouse cerebellar granule neurons as putative substrates.11,12 In a study on the partial regeneration of rat optic nerves mediated by goldfish TGN, the authors described that interleukin-2 (IL-2) was a substrate for TGN.7,13 However in the current study on goldfish, the retinal explants do not contain any oligodendrocytes and therefore have no myelin inhibitory factors. Hence, cytotoxic IL-2 is not the substrate for TGN in this goldfish optic nerve regeneration system.
6.REFERENCES
1.S. Shyne-Athwal, R.V. Riccio, G. Chakraborty, and N.A. Ingoglia, Protein modification by amino acid addition is increased in crushed sciatic but not optic nerves, Science 231:603-605 (1986).
2.M. Ando, S. Kunii, T. Tatematsu, and Y. Nagata, Rapid and transient alterations in transglutaminase activity in rat superior cervical ganglia following denervation or axotomy, Neurosci. Res. 17:47-52 (1993).
3.G. Chakaraborty, T. Leach, M.F. Zanakis, J.A. Sturman, and N.A. Ingoglia, Posttranslational protein modification by polyamines in intact and regenerating nerves, J. Neurochem. 48:669-675 (1987).
4.S. Eitan, A. Solomon, V. Lavie, E. Yoles, D.L. Hirschberg, M. Belkin, and M. Schwartz, Recovery of visual response of injured adult rat optic nerves treated with transglutaminase, Science 264:1764-1768 (1994).
5.H. Yasueda, K. Nakanishi, Y. Kumazawa, K. Nagase, M. Motoki, and H. Matsui, Tissue-type transglutaminase from red sea bream (Pagrus major). Sequence analysis of the cDNA and functional expression in Escherichia coli, Eur. J. Biochem. 232:411-419 (1995).
6.T. Matsukawa, K. Sugitani, K. Mawatari, Y. Koriyama, Z. Liu, M. Tanaka, and S. Kato, Role of purpurin as a retinol-binding protein in goldfish retina during the early stage of optic nerve regeneration: its priming action on neurite outgrowth, J. Neurosci. 24:8346-8353 (2004).
7.Z.W. Liu, T. Matsukawa, K. Arai, M. Devadas, H. Nakashima, M. Tanaka, K. Mawatari, and S. Kato, Na,K- ATPase alpha3 subunit in the goldfish retina during optic nerve regeneration, J. Neurochem. 80:763-770 (2002).
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8.T. Matsukawa, K. Arai, Y. Koriyama, Z. Liu, and S. Kato, Axonal regeneration of fish optic nerve after injury,
Biol. Pharm. Bull. 27:445-451 (2004).
9.M. Lesort, J. Tucholski, M.L. Miller, and G.V. Johnson, Tissue transglutaminase: a possible role in neurodegenerative diseases, Prog. Neurobiol. 61:439-463 (2000).
10.M.J. Perry and L.W. Haynes, Localization and activity of transglutaminase, a retinoid-inducible protein, in developing rat spinal cord, Int. J. Dev. Neurosci. 11:325-337 (1993).
11.S.A. Mahoney, M. Wilkinson, S. Smith, and L.W. Haynes, Stabilization of neurites in cerebellar granule cells by transglutaminase activity: identification of midkine and galectin-3 as substrates, Neuroscience 101:141-155 (2000).
12.S. Kojima, T. Inui, H. Muramatsu, Y. Suzuki, K. Kadomatsu, M. Yoshizawa, S. Hirose, T. Kimura, S. Sakakibara, and T. Muramatsu, Dimerization of midkine by tissue transglutaminase and its functional implication, J. Biol. Chem. 272:9410-9416 (1997).
13.S. Eitan and M. Schwartz, A transglutaminase that converts interleukin-2 into a factor cytotoxic to oligodendrocytes, Science 261:106-108 (1993).
CHAPTER 74
SURVIVAL SIGNALING IN RETINAL PIGMENT EPITHELIAL CELLS IN RESPONSE TO OXIDATIVE STRESS: SIGNIFICANCE IN RETINAL DEGENERATIONS
Nicolas G. Bazan*
1. SUMMARY
Photoreceptor survival depends on the integrity of retinal pigment epithelial (RPE) cells. The pathophysiology of several retinal degenerations involves oxidative stressmediated injury and RPE cell death; in some instances it has been shown that this event is mediated by A2E and its epoxides. Photoreceptor outer segments display the highest DHA content of any cell type. RPE cells are active in DHA uptake, conservation, and delivery. Delivery of DHA to photoreceptor inner segments is mediated by the interphotoreceptor matrix. DHA is necessary for photoreceptor function and at the same time is a target of oxidative stress-mediated lipid peroxidation. It has not been clear whether specific mediators generated from DHA contribute to its biological properties. Using ARPE-19 cells, we demonstrated the synthesis of 10,17S-docosatriene [neuroprotectin D1 (NPD1)]. This synthesis was enhanced by the calcium ionophore A-23187, by IL-1b, or by supplying DHA. Added NPD1 (50 nM) potently counteracted H2O2/tumor necrosis factor-a oxidative stresstriggered apoptotic DNA damage in RPE. NPD1 also up-regulated the anti-apoptotic proteins Bcl-2 and Bcl-xL and decreased pro-apoptotic Bax and Bad expression. Moreover, NPD1 (50 nM) inhibited oxidative stress-induced caspase-3 activation. NPD1 also inhibited IL-1b-stimulated expression of COX-2. Furthermore, A2E-triggered oxidative stress induction of RPE cell apoptosis was also attenuated by NPD1. Overall, NPD1 protected RPE cells from oxidative stress-induced apoptosis. In conclusion, we have demonstrated an additional function of the RPE: its capacity to synthesize NPD1. This new survival signaling is potentially of interest in the understanding of the pathophysiology of retinal degenerations and in exploration of new therapeutic modalities.
* Neuroscience Center of Excellence and Department of Ophthalmology, Louisiana State University Health Sciences Center School of Medicine in New Orleans, 2020 Gravier Street, Suite D, New Orleans, LA 70112.
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2. INTRODUCTION
Omega-3 fatty acids provided by the diet are necessary for retina, brain, and overall cellular functional integrity and human health (Simopoulos et al., 1999). Docosa-hexaenoic acid (22:6, n-3, DHA, found in fish oil and marine algae) is a quantitatively major omega- 3 fatty acid highly concentrated in photoreceptors, brain, and retinal synapses (Bazan, 1990). Either DHA or its precursor 18:3, n-3 from the diet are initially taken up by the liver and then released into blood lipoproteins for distribution. DHA is in in high demand during photoreceptor cell biogenesis and synaptogenesis (Scott and Bazan, 1989) and has been shown to be critical for brain and retina development, excitable membrane function (Salem et al., 1986; Litman et al., 2001), memory (Catalan et al., 2002; Moriguchi and Salem, 2003), photoreceptor biogenesis and function (Wheeler et al., 1975; Stinson et al., 1991; Organisciak et al., 1996; Anderson et al., 2001; Bicknell et al., 2002; Anderson et al., 2002), and neuroprotection (Kim et al., 2000; Rotstein et al., 2003).
3. PHOTORECEPTOR RENEWAL AND THE SIGNIFICANCE OF THE RPE IN DHA CONSERVATION
In the outer segments of photoreceptors, rhodopsin is immersed in phospholipids endowed with the highest content of DHA of any cell type (Bazan, 1990; Choe and Anderson, 1990; Anderson et al., 2002). The RPE cells, which are in close contact with the photoreceptor tips, are the most active phagocytes of the body, and phagocytize the distal tips of photoreceptor outer segments in a daily process of rod outer segment renewal (Hu and Bok, 2001) that is completed by addition of new membrane to the base of the outer segments. DHA is conserved in photoreceptors by its retrieval through the interphotoreceptor matrix, which supplies the fatty acid for outer segment biogenesis (Bazan et al., 1985; Stinson et al., 1991; Gordon et al., 1992). This renewal is tightly regulated to maintain photoreceptor length and chemical composition, including that of their phospholipids. Most of the DHA in photoreceptor phospholipids is esterified in carbon-2 of the glycerol backbone, but DHA-containing molecular species of phospholipids also occupy both C1 and C2 positions of the glycerol backbone (Aveldano de Caldironi and Bazan, 1977; Wiegand and Anderson, 1983; Choe and Anderson, 1990). Retina and brain tenaciously retain DHA, even during very prolonged dietary deprivation of essential fatty acids of the omega-3 family. Dietary deprivation for more than one generation has been necessary to effectively reduce the content of DHA in retina and brain in rodents and even in non-human primates (Neuringer et al., 1986; Weisinger et al., 2002), conditions under which impairments of retinal function occur (e.g., Wheeler et al., 1975; Neuringer et al., 1984).
RPE cells also participate in transport and reisomerization of bleached visual pigments, and contribute to the integrity of the blood-outer retinal barrier. Injury to the RPE, including retinal detachment or trauma, triggers cellular dysfunctions that lead to the onset and development of proliferative vitreoretinopathy.
Oxidative stress-mediated injury and cell death in RPE can in turn trigger photoreceptor death and impair vision, particularly when the macular RPE cells are affected. Oxidative stress leading to apoptosis of RPE cells is key in the pathophysiology of many retinal degenerations, such as age-related macular degenerations, including Stargardt’s disease (Sieving et al., 2001; Radu et al., 2003; Sparrow et al., 2003).