These results show that oxidative damage induces apoptosis in retinal neurons during their early development in culture and suggest that the loss of mitochondrial membrane integrity is crucial in the apoptotic death of these cells. DHA activates intracellular mechanisms that prevent this loss and by modulating the levels of pro- and antiapoptotic proteins of the Bcl-2 family selectively protect photoreceptors from oxidative stress.

Identifying the trophic factors for retina photoreceptors and the intracellular pathways activated to promote cell survival is crucial for treating retina neurodegenerative diseases. Docosahexaenoic acid (DHA), the major retinal polyunsaturated fatty acid, prevents photoreceptor apoptosis during early development in vitro, and upon oxidative stress. However, the signaling mechanisms activated by DHA are still unclear. We investigated whether the extracellular signal regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) or the phosphatidylinositol-3-kinase (PI3K) pathway participated in DHA protection. 1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophynyltio) butadiene (U0126), a specific MEK inhibitor, completely blocked the DHA anti-apoptotic effect. DHA rapidly increased ERK phosphorylation in photoreceptors, whereas U0126 blocked this increase. U0126 hindered DHA prevention of mitochondrial depolarization, and blocked the DHA-induced increase in opsin expression. On the contrary, PI3K inhibitors did not diminish the DHA protective effect. DHA promoted the early expression of Bcl-2, decreased Bax expression and reduced caspase-3 activation in photoreceptors. These results suggest that DHA exclusively activates the ERK/MAPK pathway to promote photoreceptor survival during early development in vitro and upon oxidative stress. This leads to the regulation of Bcl-2 and Bax expression, thus preserving mitochondrial membrane potential and inhibiting caspase activation. Hence, DHA, a lipid trophic factor, promotes photoreceptor survival and differentiation by activating the same signaling pathways triggered by peptidic trophic factors.

The results suggest that oxidative stress stimulated an increase in ceramide levels that induced photoreceptor apoptosis. DHA prevented oxidative stress and ceramide damage by upregulating Bcl-2 expression and glucosylating ceramide, thus decreasing its intracellular concentration. This shows for the first time that ceramide is a critical mediator for triggering photoreceptor apoptosis in mammalian retina and suggests that modulating ceramide levels may provide a therapeutic tool for preventing photoreceptor death in neurodegenerative diseases.

tions as structural membrane components and energetic fuels. However, the fi ndings of the previous half century have extended these roles, shedding light on their indisputable relevance as signaling molecules controlling key aspects of cellular life and development. The identifi cation of diacylglycerol and inositol 1,4,5 triphosphate ( 1 ) as second messengers seemed at the time a peculiarity of these lipids. The subsequent fi ndings of the roles of arachidonic acid metabolites ( 2 ), platelet activating factor ( 3 ), and other minor inositol lipids ( 4 ) in cell signaling began to establish that being intracellular messengers was not an oddity but a habitual task for lipids in cells. The family of lipid signaling molecules largely expanded in the last two decades, when several sphingolipids were successively shown to regulate a multiplicity of cell functions. Sphingosine (Sph) was the fi rst sphingolipid to be established as a bioactive lipid, involved in the regulation of protein kinase (PK)C activity ( 5 ). Ceramide (Cer) and sphingosine-1-phosphate (S1P) were next shown to signal a myriad of cell functions and the number of sphingolipid molecules with bioactive properties has gone on enlarging in recent years ( 6, 7 ).Though a vast literature exists on the functions of sphingolipids in several tissues, less is known concerning its roles in the eye, and in the retina in particular. Accumulation of sphingolipids originated in defects in the lysosomal Abstract Many sphingolipids have key functions in the regulation of crucial cellular processes. Ceramide (Cer) and sphingosine (Sph) induce growth arrest and cell death in multiple situations of cellular stress. On the contrary, sphingosine-1-phosphate (S1P), the product of Sph phosphorylation, promotes proliferation, differentiation, and survival in different cell systems. This review summarizes the roles of these simple sphingolipids in different tissues and then analyzes their possible functions in the retina. Alterations in proliferation, neovascularization, differentiation, and cell death are critical in major retina diseases and collective evidence points to a role for sphingolipids in these processes. Cer induces infl ammation and apoptosis in endothelial and retinal pigmented epithelium cells, leading to several retinopathies. S1P can prevent this death but also promotes cell proliferation that might lead to neovascularization and fibrosis. Recent data support Cer and Sph as crucial mediators in the induction of photoreceptor apoptosis in diverse models of oxidative damage and neurodegeneration, and suggest that regulating their metabolism can prevent this death. New evidence proposes a central role for S1P controlling photoreceptor survival and differentiation. Finally, this review discusses the ability of trophic factors to regulate sphingolipid metabolism and transactivate S1P signaling pathways to control survival and development in retina photoreceptors. When asked about the roles of cellular lipids, the fi rst thought usually coming to our minds re...

GDNF and DHA acted as molecular cues, counterbalancing the decision of photoreceptors to remain in or exit the cell cycle. The results strongly suggest that both factors participate in determining the number of photoreceptors in vitro, regulating the cell cycle and survival at early and late stages of development, respectively. Hence, GDNF and DHA may coordinately control the histogenesis of photoreceptors in the retina by modulating both neurogenesis and apoptosis.

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Oxidative stress is involved in activating photoreceptor death in several retinal degenerations. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, protects cultured retina photoreceptors from apoptosis induced by oxidative stress and promotes photoreceptor differentiation. Here we investigated whether eicosapentaenoic acid (EPA), a metabolic precursor to DHA, had similar effects and whether retinal neurons could metabolize EPA to DHA. Adding EPA to rat retina neuronal cultures increased opsin expression and protected photoreceptors from apoptosis induced by the oxidants paraquat (PQ) and hydrogen peroxide (H2O2). Palmitic, oleic, and arachidonic acids had no protective effect, showing the specificity for DHA. We found that EPA supplementation significantly increased DHA percentage in retinal neurons, but not EPA percentage. Photoreceptors and glial cells expressed Δ6 desaturase (FADS2), which introduces the last double bond in DHA biosynthetic pathway. Pre-treatment of neuronal cultures with CP-24879 hydrochloride, a Δ5/Δ6 desaturase inhibitor, prevented EPA-induced increase in DHA percentage and completely blocked EPA protection and its effect on photoreceptor differentiation. These results suggest that EPA promoted photoreceptor differentiation and rescued photoreceptors from oxidative stress-induced apoptosis through its elongation and desaturation to DHA. Our data show, for the first time, that isolated retinal neurons can synthesize DHA in culture.

Oxidative damage is involved in triggering neuronal death in several retinal neurodegenerative diseases. The recent finding of stem cells in the retina suggests that both preventing neuronal death and replacing lost neurons might be useful strategies for treating these diseases. We have previously shown that oxidative stress induces apoptosis in cultured retinal neurons. We now investigated the response of Müller cells, proposed as retina stem cells, to this damage. Treatment of glial cell cultures prepared from rat retinas with the oxidant paraquat (PQ) did not induce glial cell apoptosis. Instead, PQ promoted their rapid dedifferentiation and proliferation. PQ decreased expression of a marker of differentiated glial cells, simultaneously increasing the expression of smooth muscle actin, shown to increase with glial dedifferentiation, the levels of cell-cycle markers, and the number of glial cells in the cultures. In addition, glial cells protected neurons in coculture from apoptosis induced by PQ and H(2)O(2). In pure neuronal cultures, PQ induced apoptosis of photoreceptors and amacrine neurons, simultaneously decreasing the percentage of neurons preserving mitochondrial membrane potential; coculturing neurons with glial cells completely prevented PQ-induced apoptosis and preserved mitochondrial potential in both neuronal types. These results demonstrate that oxidative damage activated different responses in Müller glial cells; they rapidly dedifferentiated and enhanced their proliferation, concurrently preventing neuronal apoptosis. Glial cells might not only preserve neuronal survival but also activate their cell cycle in order to provide a pool of new progenitor cells that might eventually be manipulated to preserve retinal functionality.