mitochondrion, where superoxide (O2−2) is generated from complexes I and III and quickly dismutated to H2O2 and oxygen by superoxide dismutase [23]. It follows, therefore, that mitochondrial function that is compromised is a central component of the impairments seen in degenerative disease [11, 24, 25]. One of the most prevalent ROS in cells is H2O2; its biological function is characterized by its reactivity, redox potential,relativestabilityincells,anditsabilitytotraversemembranes[19,20,26,27]. Furthermore, H2O2 is a second messenger modulating the homeostatic redox state [28] and the actions of platelet-derived growth factor (PDGF) [29, 30], epidermal growth factor (EGF) [31], and endocrine regulation. H2O2 detoxiÞcation is accomplished by enzymatic (catalase, glutathione peroxidases, peroxiredoxins) and nonenzymatic (glutathione, vitamins A, C, and E, and bilirubin) mechanisms.

In retinal degeneration, clinical evidence reveals that photoreceptor cell death takes place over the course of several years. The constant rebuilding of photoreceptor outer segments requires molecular building blocks, energy, and an environment suitable for promoting cellular integrity. Photoreceptors shed outer segment tips which are then phagocytized daily, in an intermittent, circadian fashion by mammalian RPE cells [32Ð34]. The length of the outer segments remains constant due to the highly regulated biogenesis of outer segment membrane components in inner segments, and the phagocytosis of these shed tips at an equal, compensatory rate. During photoreceptor outer segment renewal, proteins turn over and are continually replaced [35]. Therefore, it is critical to identify early prosurvival, antiinßammatory signaling mechanisms essential for maintaining photoreceptor cell integrity as this may lead to novel therapeutic interventions for halting or slowing disease progression.

7.2DHA in Photoreceptor Cells

The lipid milieu of the outer segments membranes in which rhodopsin and other proteins perform their functions is distinguished by phospholipids rich in DHA and in omega-3 fatty acid derivatives longer than C22. A salient characteristic of photoreceptor outer segments is the very high concentration of omega-3 fatty acid family members of which all are essentially esteriÞed in phospholipids [1, 3, 36]. In some phospholipids, there are two omega-3 fatty acids esteriÞed at both the sn-1 and sn-2 positions of the same glycerol backbone yielding the supraenoic or supraene molecular species [37Ð40]. In addition, identiÞcation of di-docosahexaenoyl diglycerides in the amphibian retina [41, 42] and the observation that DHA is enriched in phosphatidic acid of the retina [43, 44] suggests that the composition, metabolism, and function of supraenoic molecular species of phospholipids are important in photoreceptor organization and function. Supraenoic molecular species of phospholipids represent 31% of phosphatidylcholine, 52% of phosphatidylserine, and 20% of phosphatidylethanolamine in photoreceptor discs [37]. The supraenoic phosphatidylcholines that contain DHA at position sn-2 and the 24:6Ð36:6 elongation products of the omega-3 fatty acid family series at position sn-1 are tightly bound to

rhodopsin [45]. These very long-chain fatty acids at sn-1 may ÒcurlÓ and restrict rhodopsin motion, likely forming a disc membrane domain that does not favor the classical bilayer membrane organization [45]. In fact, phospholipids containing DHA provide a favorable environment within which G-protein-coupled events can occur [45]. Another point of interest is the physiologically selective enrichment of DHA in phosphatidylserine of neural cells resulting in the positive modulation of Akt survival signaling [46, 47]. These aspects of DHA content are not mutually exclusive and might indicate speciÞc functions for membrane DHA.

Under normal conditions, DHA is retained and protected from peroxidation. However, in experimental models of retinal degeneration [48], when lipid peroxidation takes place, perturbations of photoreceptor function, damage, and cell death occur. In UsherÕs syndrome [49, 50] and several forms of RP [49, 51Ð55], a decrease in the blood content of DHA has been found. This implies that retinal impairment may be a consequence of decreased DHA supply and a decreased availability of DHA to photoreceptors. However, the relationship between decreased DHA in the blood supply, disease initiation and progression remains unclear. Indeed, rats overexpressing rhodopsin mutations homologous to human RP display decreased amounts of DHA in photoreceptors [56]. This Þnding might represent a retinal response to metabolic stress, whereby decreasing the amount of the major target of lipid peroxidation DHA, contributes to photoreceptor protection [56]. Moreover, constant-light- mediated retinal degeneration causes a loss of DHA from photoreceptors. Still, rats reared in bright cyclic light are protected from both photoreceptor loss and degeneration, thus suggesting the potential for an adaptive, plastic response [57].

Is the shortage of DHA in the blood of RP [55, 58, 59] and UsherÕs syndrome patients [49, 50] reßected in the relationship between very-long-chain, DHA-derived acyl groups and rhodopsin? Or, as shown in experimental retinal degeneration, is the peroxidation of DHA, which is closely associated with rhodopsin, impairing the function of this protein? Although these questions have not yet been answered, we do know that DHA promotes survival [60] and inhibits apoptosis [61] of photoreceptors. Similarly, in an AlzheimerÕs disease (AD) mouse model, DHA exerts neural protection [62] and several studies have shown neuroprotective properties of DHA [1, 3, 46, 60, 61].

7.3Neuroprotectin D1 Synthesis is an Early Response to Oxidative Stress in RPE Cells

RPE cells, the most active phagocytes of the body, are derived from the neuroectoderm and support photoreceptor cells by participating in the daily shedding, internalization, and degradation (phagocytosis) of the tips of the photoreceptor outer segments (membrane discs). In mammals, circadian shedding and phagocytosis of one entire rod outer segment (ROS) has been calculated to be complete after 10 days [34, 35]. In rhesus monkeys, every RPE cell makes contact with 20Ð45 photoreceptor tips [63], whereas each contacts 23 photoreceptors in the human macula [64]. An RPE cell interacts

Fig. 7.1 Biosynthesis of NPD1. A membrane phospholipid containing a docosahexaenoyl chain in sn-2 is hydrolyzed by a phospholipase A2, generating a free (unesteriÞed) DHA. The carbons of DHA are numbered and the omega-3 (n-3) tail highlighted. Lipoxygenation is then followed by epoxidation and hydrolysis to generate NPD1. (Republished from Bazan [150], with permission. The Association for Research in Vision and Ophthalmology is the copyright holder)

through its apical side with the ROS and phagocytizes about 10% of the photoreceptor outer segments daily. This immense task is comparable to engulÞng and degrading (phagolysosomal processing) the equivalent of 5Ð10 red blood cell membranes per day. Outer segment phagocytosis is an intricate process that engages RPE cell-surface receptors to recognize, bind to, and internalize photoreceptor tips [32, 65, 66]. The cells undergo cytoskeletal rearrangements, genes are induced [67], lysosomeÐphagosome fusion takes place, and recycling of retinol and DHA is initiated [3, 68]. Consequently, ROS renewal occurs, resulting in an outer segment that is unmodiÞed in length because, as the discs at the tips are phagocytized, membrane biogenesis from the inner segment precisely replaces the amount of membrane removed. RPE cells perform other roles such as transport and reisomerization of bleached visual pigments, synthesis and secretion of neurotrophic factors, and contribution to the integrity of the barrier between choroidal blood and the photoreceptors. In this way, RPE cells display complex, pleiotropic behavior capable of resembling a variety of cell types that range from macrophages to classic cuboidal epithelial cells and glial cells.

One response of the RPE cell to oxidative stress is induction of NPD1 synthesis [69] (Fig. 7.1). The name ÒNPD1Ó is based upon its neuroprotective bioactivity and

attenuating the expression of those that challenge cell survival (e.g., Bax, Bad, Bid, and Bik). An NPD1-mediated, coordinated regulation of the availability of Bcl-2 proteins for subsequent downstream signaling might be crucial for cell survival [69, 82]. Translational or post-translational events may also integrate a response to counteract oxidative stress. Bcl-xL is a major antiapoptotic Bcl-2 protein required for cell survival; however, phosphorylation at residue Ser-62 renders this protein proapoptotic. The serine/threonine protein phosphatase 2A (PP2A) is a key regulator of Bcl-xL phosphorylation at residue Ser-62 in the ARPE-19 cell. Bcl-xL phosphorylation is increased under oxidative stress with the application of okadaic acid, a PP2A inhibitor, or the depletion of the catalytic subunit of PP2A (PP2A/C) by small interfering RNA. PP2A/C colocalizes and interacts with S62Bcl-xL in cells undergoing OS. Disruption of PP2A/C exacerbates OSÐinduced apoptosis. NPD1 downregulates OS -induced phosphorylation of Bcl-xL by increasing protein phosphatase activity and increasing the association of PP2A/C with S62Bcl-xL and total Bcl-xL [83]. It also attenuates apoptosis induced by OS and PP2A/C knockdown. NPD1 enhances the heterodimerization of Bcl-xL with the proapoptotic protein Bax and modulates the activation of Bcl-xl through dephosphorylation by PP2A. This suggests a coordinated, NPD1-mediated regulation of cell survival in response to OS [83].

The Bcl-2 family of proteins regulates apoptotic signaling at the level of mitochondria and the endoplasmic reticulum. Caspase-3, a downstream effector of proapoptotic and antiapoptotic Bcl-2 proteins, is activated as a consequence of mitochondrial cytochrome c release into the cytoplasm and activation of the apoptosome [84]. In RPE cells, cleavage of endogenous substrates by caspase-3 is enhanced by oxidative stress, as indicated by increased accumulation of poly(ADP-ribose) polymerases (PARPs). NPD1 inhibits caspase-3 activation when added at the onset of oxidative stress [69], this effect is interpreted as a downstream consequence of NPD1 modulation of the premitochondrial Bcl-2 proteins. It is signiÞcant that DHA itself inhibits apoptosis in parallel with a time-dependent formation of NPD1. Interestingly, the potency of DHA for cytoprotection is much higher than that of added NPD1 [69] suggesting that NPD1 might exert its action near the subcellular site of its synthesis. It is important to note that these actions of DHA cannot be mimicked by other PUFAs (e.g., 20:4,n-6). Other NPD-like mediators might potentially participate in promoting RPE cell survival in an attempt to cope with the multiplicity of signaling pathways that are impacted by RPE cells or neurons confronted by oxidative stress. DNA array-based human genome expression proÞling has revealed that NPD1 turns off several proinßammatory and proapoptotic genes and induces antiapoptotic genes in human neural progenitor cells [82]. Remarkably, DHA, and even more so NPD1, mediate opposite changes from those elicited by the amyloid b peptide Ab42 which enhances expression of genes encoding cytokine exodus protein-1 (CEX-1), IL-1b, tumor necrosis factor alpha (TNF-a), and cyclooxygenase-2 (COX-2), in addition to the TNF-a-inducible proinßammatory element B94 [82]. These observations further suggest that NPD1 induces a gene expression program in both RPE and neural cells that is neuroprotective