Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

Retinal DHA is a target of oxidative stress-mediated lipid peroxidation (Organisciak et al., 1996), which generates neuroprostanes from DHA through an enzyme-independent reaction (Roberts et al., 1998). In an intriguing contrast, some studies demonstrated DHA-mediated neuroprotection in photoreceptors (Politi et al., 2001; Rotstein et al., 2003) and brain (Kim et al., 2000). Does this neuroprotection result from the replenishment of DHA into membranes, or is there a selective neuroprotective signaling by a DHA-derived mediator?

4. IDENTIFICATION AND CHARACTERIZATION OF NPD1

We recently reported the isolation and structural characterization of 10,17S- docosatriene in ARPE-19 cells using tandem LC-PDA-ESI-MS-MS-based lipidomic analysis and ARPE-19 cells (Mukherjee et al., 2004). We termed the newly isolated dihydroxy-containing DHA derivative “neuroprotectin D1” (NPD1) (1) because of its neuroprotective properties in brain ischemia-reperfusion (Marcheselli et al., 2003) and in oxidative stress-challenged RPE cells (Mukherjee et al., 2004); (2) because of its potent ability to in-activate pro-apoptotic signaling (Mukherjee et al., 2004); and (3) because it is the first identified neuroprotective mediator of DHA. NPD1 synthesized by ARPE-19 cells is the same as that of the docosatriene found in human blood, glial cells, mouse brain (Hong et al., 2003), and during brain ischemia-reperfusion (Marcheselli et al., 2003). The biological activity of NPD1 seems to be exerted through potent inhibition of oxidative stressinduced apoptosis and of cytokine-triggered pro-inflammatory COX-2 gene-promoter induction (Figure 74.1).

5. PHOSPHOLIPASE A2 AND NPD1 SYNTHESIS

NPD1 is formed through enzyme-mediated steps involving a phospholipase A2 followed by a 15-lipoxygenase-like enzyme (Figure 74.1). Retina synthesizes mono-, di-, and trihydroxy derivatives of DHA, and certain lipoxygenase inhibitors block this synthesis, which suggests a lipoxygenase enzymatic process (Bazan et al., 1984). The availability of unesterified DHA is tightly regulated in RPE cells, as in brain, which we demonstrated in ARPE19 cells, and which is supported by the observation that DHA pool size in retina and brain is negligible under basal, unstimulated conditions (Bazan, 1970; Aveldano and Bazan, 1974; Aveldano and Bazan, 1975). Therefore, the regulation of the phospholipase A2 that releases free DHA is important in the pathway leading to the formation of NPD1. Ischemia or seizures elicit rapid activation of free DHA release in brain as well (Bazan, 1970; Aveldano and Bazan, 1975). The calcium ionophore A23187, or to a lesser extent, IL-1b, activates the synthesis of NPD1 in ARPE-19 cells. Under these conditions, there is a time-dependent increase in endogenous free DHA that is approximately 3- to 4-fold higher than the amount of NPD1 being synthesized.

DHA is highly concentrated as an acyl group of phospholipids in photoreceptor outer segment disc membranes (Bazan, 1990). The RPE cell actively recycles DHA from phagocytized disc membranes back to the inner segment of the photoreceptor cell (Bazan et al., 1985). In addition, the RPE cell takes up DHA from the blood stream through the choriocapillaris, and in turn supplies the fatty acid to photoreceptors through the interphoto-

receptor matrix (Bazan et al., 1985). This uptake is very active during early postnatal development, when photoreceptor outer segment biogenesis occurs (Scott and Bazan, 1989). In addition, active docosahexaenoyl CoA synthases in the RPE and the retina channel free DHA to acyltransferases that incorporate the fatty acid into membrane phospholipids (Reddy and Bazan, 1984). The RPE cell thus is very active in the uptake, conservation, and delivery of DHA to photoreceptors (Bazan, 1990; Bazan et al., 1985). We have demonstrated an additional function of the RPE cell; i.e., its capacity to synthesize NPD1 (Mukherjee et al., 2004). The biological activity of NPD1 may be elicited through a receptor, and in turn modulate signaling including induction of NF-kB and other transcription factors, and as a consequence, down-regulate pro-inflammatory genes (Marcheselli et al., 2003; Bazan, in press). NPD1 may act in autocrine fashion and/or may diffuse through interphotoreceptor matrix proteins and act in paracrine fashion on photoreceptor cells.

6. OXIDATIVE STRESS IN THE RETINAL PIGMENT EPITHELIUM

Oxidative stress triggers multiple signaling pathways, including some that are cytoprotective and others that contribute to cell damage and eventually cell death. Among these are the Bcl-2 family proteins. In fact, expression of proand anti-apoptotic Bcl-2 family proteins is altered by oxidative stress, and these proteins represent a major factor, insofar as the outcome of the apoptotic signaling, since cell survival reflects the predominance of one set of proteins over the other (Mattson and Bazan, in press). In the RPE and retina, oxidative stress, increased by several factors including retinal light exposure or reactive oxygen species, shifts the balance of the Bcl-2 family protein expression toward those that favor cell damage (e.g., Osborne et al., 1997; Liang et al., 2000). Since our results show oxidative stress-induced changes in the expression of Bcl-2 proteins (Mukherjee et al., 2004), they imply that the early RPE response to oxidative stress includes transcriptional, translational, and/or post-translational events upstream of the mitochondrial apoptotic step. In this connection we have observed that oxidative stress-triggered ARPE-19 cell damage includes changes in the expression of Bcl-2, Bcl-xL, Bax, and Bad. It is conceivable that oxidative stress, cytokines, and other intercellular signals (certain growth factors?) may activate NPD1 formation, in an effort to counteract the injury/pro-inflammatory response and restore homeostasis. Exogenous NPD1 (50 nM) promotes a differential modification in the expression of Bcl-2 family proteins under these conditions, up-regulating the protective Bcl-2 proteins and attenuating the expression of the proteins that challenge cell survival, particularly Bax and Bad (Mukherjee et al., 2004). These observations suggest a critical coordinate regulation of the availability of Bcl-2 proteins for subsequent downstream signaling. NPD1 may act at the level of signaling that regulates promoters of the genes encoding death repressors and effectors of the Bcl-2 family of proteins we studied (Mattson and Bazan, in press). On the other hand, translational or post-translational events may also integrate a concerted responsive machinery to counteract oxidative stress. The precise molecular mechanisms remain to be defined. The exploration of these events will provide an important insight into regulatory survival signaling. Bcl-2 family proteins regulate apoptotic signaling at the level of the mitochondrion and endoplasmic reticulum. As a consequence, cytochrome c is released from mitochondria and effector caspase-3 is activated (Mattson and Bazan, in press). In agreement with this sequence, we showed that oxidative stress activates caspase- 3 in ARPE-19 cells and that the lipid mediator NPD1 (50 nM) decreases oxidative stress-

activated caspase-3 (Mukherjee et al., 2004). Moreover, apoptosis was an outcome of TNFa/H2O2-induced oxidative stress in ARPE-19 cells. Interestingly, NPD1 was very effective in counteracting oxidative stress-induced apoptosis in ARPE-19 cells. This action was not mimicked by eicosanoids such as PGE2, LTB4, or even arachidonic acid (Mukherjee et al., 2004). This supports the selectivity of the new class of mediator, NPD1. Perhaps one of the most interesting observations is that DHA itself inhibited oxidative stress-induced apoptosis. Under those conditions, a remarkable, time-dependent formation of NPD1 occurred. Significantly, the potency of DHA for cytoprotection was much higher than that of added NPD1 (Mukherjee et al., 2004). This suggests that endogenously generated NPD1 may exert its action near the subcellular site of its synthesis. Alternatively, it may imply that other NPD1-like mediators may participate in promoting RPE cell survival (Bazan, in press). It is indeed possible that related NPD mediators are formed in an attempt to cope with the multiplicity of cellular signaling that has the potential of going awry in RPE or neurons when confronted with oxidative stress. In support of this possibility, brain does make a series of other potentially bioactive DHA-oxygenated derivatives, such as those generated in the presence of aspirin during ischemia-reperfusion (Marcheselli et al., 2003).

7. DHA AND RETINAL DEGENERATION

Until now it was thought that high DHA content in photoreceptors and RPE mainly endowed photoreceptor membrane domains with physical properties that contribute to the modulation of receptors (e.g., rhodopsin), ion channels, transporters, etc. In other cells, DHA modulates G-protein-coupled receptors and ion channels. Moreover, DHA has been suggested to regulate membrane function by maintaining its concentration in phosphatidylserine (Salem et al., 2001). As a target of oxidative stress, DHA is acted upon by reactive oxygen intermediates that generate DHA-peroxidation products that in turn participate in RPE and photoreceptor cell damage.

Rhodopsin mutations in rodent retinitis pigmentosa are associated with decreased photoreceptor DHA (Anderson et al., 2002), which can be interpreted as a retinal response to metabolic stress, where the strategy of decreasing the amount of the major target of lipid peroxidation, DHA, contributes to protection (Anderson et al., 2002). We propose that the retinal DHA pool size available for synthesis of neuroprotective docosanoids is compromised due to lipid peroxidation. Retinal degeneration induced by constant light promotes DHA loss from photoreceptors, and rats reared in bright cyclic light are protected, suggesting an unidentified adaptation or plasticity that may involve endogenous molecules (Li et al., 2001). Some of these may be lipid mediators, such as NPD1.

8. NPD1 PREVENTS APOPTOSIS AFTER A2E PHOTO-OXIDATION

N-retinylidene-N-retinylethanolamine (A2E) is the major hydrophobic fluorophore that accumulates in retinal pigment epithelial cells in Stargardt’s disease and in age-related retinal degeneration (Radu et al., 2003). Blue light accelerates the production of A2E epoxides, which trigger apoptosis in RPE cells. NPD1 (50 nM) prevented A2E-induced cell death in ARPE-19 cells (Barreiro et al., 2005). We also monitored in parallel by LC-PDA-ESI- MS-MS the formation of A2E epoxides under these conditions. A2E epoxides produced

oxidative stress and apoptosis in ARPE cells. NPD1 inhibited apoptosis when the A2E was added 15 minutes after blue light exposure. A2E alone, without light, did not trigger apoptosis at lower concentrations and short exposures. The accumulation of A2E in retinal pigment epithelial cells participates in apoptotic cell death and RPE responses in retinal degenerations such as Stargardt’s disease. Moreover, blue light accelerates the oxidation of this compound into epoxides, and apoptosis can be prevented by NPD1 (Barreiro et al., 2005).

9. CONCLUSIONS AND EVOLVING QUESTIONS

Do growth factors contribute to the pro-survival actions of NPD1? For example, FGF2 induces bovine RPE cell survival in cultures through a sustained adaptive phenomenon that involves ERK2 activation by secreted FGF1 and ERK2-dependent Bcl-xL production (Bryckaert et al., 1999). Bcl-xL may play a key role in integrating and transmitting exogenous FGF2 signals for RPE cell survival. Very recently evidence has been provided that PEDF promotes NPD1 synthesis in human RPE cell primary cultures (Bazan et al., 2005). These issues present intriguing possibilities for future investigations.

RPE cell damage and apoptosis impair photoreceptor cell survival, a dominant factor in age-related macular degeneration (Hinton et al., 1998). In Stargardt’s disease (a juvenile form of macular degeneration), oxidative stress mediated by the lipofuscin fluorophore A2E damages RPE, and caspase-3 is part of the damaging cascade; whereas Bcl-2 exerts cellular protection (Sparrow and Cai, 2001).

Thus NPD1, a DHA-derived mediator endogenously synthesized by neuro-epithelium- derived RPE cells, is a modulator of signaling pathways that promote cell survival (Bazan, in press). One pathway is the regulation of Bcl-2 family protein expression, a premitochondrial apoptotic target of NPD1 under conditions of oxidative stress. Consequently, downstream signaling, including effector caspase-3 activation and DNA degradation, is attenuated (Mukherjee et al., 2004). NPD1 also potently counteracted cytokine-triggered pro-inflammatory COX-2 gene induction, another major factor in cell damage (Mukherjee et al., 2004). In ischemia-reperfusion-injured hippocampus and in neural progenitor cells stimulated by IL-1b, COX-2 expression seems to be related to NF-kB activation. NPD1 inhibits NF-kB and COX-2 induction under those conditions (Marcheselli et al., 2003). A similar regulatory mechanism may operate in RPE cells; i.e., NPD1 down-regulation of cytokine-mediated NF-kB activation. Pro-inflammatory injury of the RPE can promote pathoangiogenesis and proliferative vitreoretinopathy, hallmarks of several diseases, including diabetic retinopathy. NPD1’s neuroprotective bioactivity in brain ischemia-reperfusion includes decreased infarct size and inhibition of polymorphonuclear leukocyte infiltration (Marcheselli et al., 2003). The addition of DHA to the culture medium promoted strong cytoprotection when RPE cells were confronted with oxidative stress (Mukherjee et al., 2004). In vivo the active DHA supply to brain and retina from the liver through the blood stream is necessary for cell development and function, and may play a critical role in conditions where, due to enhanced oxidative stress, the polyunsaturated fatty acyl chains of membrane phospholipids are decreased as a consequence of lipid peroxidation, as occurs in aging, retinal degenerations, and neurodegenerations such as Alzheimer’s disease (Scott and Bazan, 1989; Nourooz-Zadeh et al., 1999). A greater understanding of the signals that modulate NPD1 synthesis may provide windows for therapeutic intervention in neurodegenera-

tive diseases. In addition, selective DHA-delivery systems to the retina and brain may promote neuroprotection. Moreover, NPD1 and its cellular activities might be manipulated by novel approaches that result in RPE cytoprotection and enhanced photoreceptor survival in retinal degenerations.

10. ACKNOWLEDGMENT

This work was supported by NIH grants EY05121 (NEI) and P20RR16816 (from the COBRE Program, NCRR), the Eye, Ear, Nose and Throat Foundation, and the American Health Assistance Foundation.

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