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.

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.

In this study we show that insulin-like growth factor (IGF)-I selectively promotes survival and differentiation of amacrine neurons. In cultures lacking this factor, an initial degeneration pathway, selectively affecting amacrine neurons, led to no lamellipodia development and little axon outgrowth. Cell lysis initially affected 50% of amacrine neurons; those remaining underwent apoptosis leading to the death of approximately 95% of them by day 10. Apoptosis was preceded by a marked increase in c-Jun expression. Addition of IGF-I or high concentrations (over 1 mM) of either insulin or IGF-II to the cultures prevented the degeneration of amacrine neurons, stimulated their neurite outgrowth, increased phospho-Akt expression and decreased c-Jun expression. The high insulin and IGF-II concentrations required to protect amacrine cells suggest that these neurons depend on IGF-I for their survival, IGF-II and insulin probably acting through IGF-I receptors to mimic IGF-I effects. Inhibition of phosphatidylinositol-3 kinase (PI 3-kinase) with wortmannin blocked insulin-mediated survival. Wortmannin addition had similar effects to IGF-I deprivation: it prevented neurite outgrowth, increased c-Jun expression and induced apoptosis. These results suggest that IGF-I is essential for the survival and differentiation of amacrine neurons, and activation of PI 3-kinase is involved in the intracellular signaling pathways mediating these effects.

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.