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Age-related macular degeneration (AMD) is among the main pathologies leading to blindness in adults and has currently no cure or effective treatment. Selective apoptosis of retina pigment epithelial (RPE) cells results in the progressive loss of photoreceptor neurons, with the consequent gradual vision loss. Oxidative stress plays an important role in this process. We have previously determined that activation of RXRs protects rat photoreceptor neurons from oxidative stress-induced apoptosis. In this study we investigated whether RXR ligands prevented apoptosis in an RPE cell line, D407 cells, exposed to hydrogen peroxide (H2O2). H2O2 induced apoptosis of D407 cells, promoting p65NFκB nuclear translocation, increasing Bax mRNA expression, activating caspase-3 and altering cell morphology. We show, for the first time, that HX630, a RXR pan-agonist, protected D407 cells from H2O2-induced apoptosis, preventing p65NFκB nuclear translocation, increasing Bclxl and PPARγ mRNA levels and simultaneously decreasing Bax mRNA levels and caspase-3 activation. Pretreatment with a RXR antagonist blocked HX630 protection. LG100754, which binds RXRs but only activates heterodimers and is an antagonist of RXR homodimers, also had a protective effect. In addition, only agonists known to bind to RXR/PPARγ were protective. As a whole, our results suggest that RXR activation protects RPE cells from oxidative stress-induced apoptosis and this protection might involve signaling through a heterodimeric receptor, such as RXR/PPARγ. These data also imply that RXR agonists might provide potential pharmacological tools for treating retina degenerative diseases., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Apoptosis, a form of programmed cell death, is a process fundamental to normal growth and development, immune response, tissue remodeling after injury or insult, and homeostasis of the intestinal epithelium. Recently, it has become apparent that apoptosis is a crucial process in skeletal development and homeostasis, and that signaling by the parathyroid hormone (PTH) receptor can either promote or suppress apoptosis depending on the cellular context. In this study, we evaluated the role of PTH in intestinal apoptosis using human colonic Caco-2 cells. To that end, Caco-2 cells were exposed to PTH (10-8 mol/L) for 1-5 days. Evaluation of cell survival by use of resazurin staining, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) staining, and crystal violet staining revealed that PTH treatment diminishes the number of viable cells. Assessment of cells after PTH treatment by use of propidium iodide showed that the hormone increased the number of red-stained (dead) cells (178%, 5 days). Moreover, we found that the hormone induced disruption of actin filaments with changes to cellular shape, alteration of cell-to-cell junctions, externalization of membrane phosphatidylserine, chromatin condensation, and DNA fragmentation of the nucleus, which are morphological features consistent with apoptosis. In addition, PTH treatment revealed a cytosolic staining pattern of 14-3-3. However, the significance of such differential localization for 14-3-3 function remains unknown. Taken together, the present study suggests that PTH promotes apoptosis in Caco-2 intestinal cells.

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.

Purpose: The precise mechanisms involved in photoreceptor apoptosis are still unclear. In the present study, the role of ceramide, a sphingolipid precursor that induces apoptosis on cellular stress, was investigated in relation to the activation of cell death in photoreceptors., Methods: Rat retina neuronal cultures, with or without docosahexaenoic acid (DHA), were treated with the ceramide analogue acetylsphingosine (C2-ceramide), and with a glucosylceramide synthase inhibitor. Ceramide synthesis in cultures treated with the oxidant paraquat was evaluated with [3H]palmitate. The effect of inhibitors of ceramide de novo synthesis, fumonisin B1 and cycloserine, on photoreceptor apoptosis was investigated. Apoptosis, mitochondrial membrane potential, and Bcl-2 expression were determined., Results: Addition of C2-ceramide induced photoreceptor apoptosis. Paraquat increased formation of [3H]ceramide in photoreceptors, compared with the control, whereas inhibition of ceramide synthesis, immediately before paraquat treatment, prevented paraquat-induced photoreceptor apoptosis. Fumonisin also reduced photoreceptor apoptosis during early development in vitro. DHA, the retina major polyunsaturated fatty acid, which protects photoreceptors from oxidative stress-induced apoptosis, completely blocked C2-ceramide-induced photoreceptor death, simultaneously increasing Bcl-2 expression. Inhibiting glucosylceramide synthase, which catalyzes ceramide glucosylation, before ceramide or paraquat treatment blocked DHA's protective effect., Conclusions: 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.

Pigment epithelium‐derived factor (PEDF) is a cytoprotective protein for the retina. We hypothesize that this protein acts on neuronal survival and differentiation of photoreceptor cells in culture. The purpose of the present study was to evaluate the neurotrophic effects of PEDF and its fragments in an in vitro model of cultured primary retinal neurons that die spontaneously in the absence of trophic factors. We used Wistar albino rats. Cell death was assayed by immunofluorescence and flow cytometry through TUNEL assay, propidium iodide, mitotracker, and annexin V. Immunofluorescence of cells for visualizing rhodopsin, CRX, and antisyntaxin under confocal microscopy was performed. Neurite outgrowth was also quantified. Results show that PEDF protected photoreceptor precursors from apoptosis, preserved mitochondrial function and promoted polarization of opsin enhancing their developmental process, as well as induced neurite outgrowth in amacrine neurons. These effects were abolished by an inhibitor of the PEDF receptor or receptor‐derived peptides that block ligand/receptor interactions. While all the activities were specifically conferred by short peptide fragments (17 amino acid residues) derived from the PEDF neurotrophic domain, no effects were triggered by peptides from the PEDF antiangiogenic region. The observed effects on retinal neurons imply a specific activation of the PEDF receptor by a small neurotrophic region of PEDF. Our findings support the neurotrophic PEDF peptides as neuronal guardians for the retina, highlighting their potential as promoters of retinal differentiation, and inhibitors of retinal cell death and its blinding consequences. Cover Image for this issue:https://doi.org/10.1111/jnc.15089 [ABSTRACT FROM AUTHOR]

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 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. [ABSTRACT FROM AUTHOR]