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... more
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...
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In their transit from the caput to the cauda segments of the epididymis, rat spermatozoa undergo significant modifications in lipid content and composition. The amount of lipid phosphorus per cell decreases, and most lipid classes show... more
In their transit from the caput to the cauda segments of the epididymis, rat spermatozoa undergo significant modifications in lipid content and composition. The amount of lipid phosphorus per cell decreases, and most lipid classes show specific changes in their constituent fatty acids. A depletion of phosphatidylcholine and phosphatidylethanolamine, concomitant with a virtually unchanged amount of the corresponding plasmalogens, are the major alterations, plasmenylcholine thereby becoming the major phospholipid. Diphosphatidylglycerol, sphingomyelin and the phosphoinositides decrease to a lesser extent or do not change at all, also resulting in relative increases with sperm maturation. Concerning the fatty acids, the proportions of oleate (C18:1, n-9) and linoleate (C18:2, n-6) in most lipids decrease on movement of sperm from caput to cauda, augmenting in turn the proportions of longer-chain (C20 to C24) and more unsaturated fatty acids. Docosapentaenoate (C22:5, n-6) is a major ac...
Research Interests: Biochemistry, Biology, Phospholipids, Medicine, Biological Sciences, and 15 moreSperm, Animals, Male, Sphingomyelin, Biochemical, Lipid metabolism, CHEMICAL SCIENCES, Rats, Spermatozoa, Epididymis, Sexual Maturation, Phosphatidylcholine, plasmalogens, Medical and Health Sciences, and In Vitro Techniques
The synthesis of very long chain (C24 to C36) polyunsaturated (four, five and six double bonds) fatty acids (VLCPUFA) is investigated in bovine retina using [14C]acetate. Saturates on the one hand (mainly palmitate), and polyenes on the... more
The synthesis of very long chain (C24 to C36) polyunsaturated (four, five and six double bonds) fatty acids (VLCPUFA) is investigated in bovine retina using [14C]acetate. Saturates on the one hand (mainly palmitate), and polyenes on the other (mainly VLCPUFA), incorporate most of the label found in lipids. Phosphatidylcholine (PC) is the most highly labelled lipid class, since both types of 14C-labelled fatty acids, but especially this novel series of VLCPUFA, are concentrated in this phospholipid. Radioactivity from [14C]acetate is found in very long chain tetra, penta and hexaenoic fatty acids of PC. The labelling of 20:4(n − 6), 20:5(n − 3), 22:5(n − 6) and 22:6(n − 3) is much lower than that of longer polyenes of each of these series, indicating that VLCPUFA are synthesized in situ by successive elongations of the above polyenes, pre-existing in retina lipids. In various subcellular fractions isolated from retinas after incubations with [14C]acetate (including cytosol, microsome...
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DHA, released from membrane phospholipids upon oxidative damage, activates retinoid X receptors (RXR), thus triggering multiple mechanisms, including upregulation or activation of pro-survival proteins, antioxidant defense mechanisms and... more
DHA, released from membrane phospholipids upon oxidative damage, activates retinoid X receptors (RXR), thus triggering multiple mechanisms, including upregulation or activation of pro-survival proteins, antioxidant defense mechanisms and the ERK/MAPK pathway to promote photoreceptor survival.
Research Interests: Biology, Cell Biology, Oxidative Stress, Medicine, Lipids, and 15 moreHumans, Animals, Cell Death, Retina, Light, Retinal Degeneration, Retinal, Ciencias Biológicas, Oxidative Damage, Cell Survival, Docosahexaenoic Acid, Biochemistry and cell biology, Photoreceptor, Photoreceptor cells, and Lipid peroxides
In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on... more
In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [(3)H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of ...
Research Interests: Biology, Cell Cycle, Cell Biology, Mitochondria, Apoptosis, and 15 moreMedicine, Cell Culture, Cell Division, Biological Sciences, DNA, Cell Differentiation, Animals, Ciencias Biológicas, Apoptose, Neurotrophic Factors, Cell Survival, Docosahexaenoic Acid, Drug synergism, Medical and Health Sciences, and Drug combinations
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Research Interests: Biochemistry, Chromatography, Biology, Phospholipids, Mitochondria, and 15 moreMedicine, Biological Sciences, Animals, Physical sciences, Endoplasmic Reticulum, Membrane Lipids, Esterification, Membrane, Cattle, Phosphatidylinositol, Ethanolamine, Phosphatidylcholine, Phosphatidylserine, Arachidonic Acid, and microsomes
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Dietary deficiency of essential fatty acids of the n-3 and n-6 series is known to promote a compensatory increase in polyenoic fatty acids of the n-9 series in the lipids of mammalian tissues. In the present study long-chain n-9 polyenes... more
Dietary deficiency of essential fatty acids of the n-3 and n-6 series is known to promote a compensatory increase in polyenoic fatty acids of the n-9 series in the lipids of mammalian tissues. In the present study long-chain n-9 polyenes were found to be normal components of the epididymis and especially of sperm isolated from that tissue, in healthy, well-fed, fertile rats maintained on essential fatty acid-sufficient diets. The n-9 polyenes occurred in large concentrations in the choline glycerophospholipids (CGP), the major phospholipid class of spermatozoa in epididymal cauda, and were highly concentrated in plasmenylcholine, the major subclass of CGP. The uncommon polyene 22:4n-9 was found in the highest proportion, followed in order of relative abundance by 22:3n-9, 20:3n-9 and 24:4n-9. These polyenes were probably derived from oleate (18:1n-9) in much the same way as long-chain polyenes of the n-6 and n-3 series are derived from linoleate (18:2n-6) and linolenate (18:3n-3), respectively.
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Unless docosahexaenoic acid (DHA, 22:6n-3) is added, retina photoreceptor cells in culture undergo a degeneration process that leads to their massive death in 2 wk (1). We recently showed that this occurs by the activation of an apoptotic... more
Unless docosahexaenoic acid (DHA, 22:6n-3) is added, retina photoreceptor cells in culture undergo a degeneration process that leads to their massive death in 2 wk (1). We recently showed that this occurs by the activation of an apoptotic pathway and that DHA provides against such event, postponing the onset of apoptosis and slowing down its progression (2). Our objective in this project is to investigate the factors associated with this action of DHA. Here we present the effect of diverse fatty acids including DHA on lipid composition and metabolism and on the apoptosis and differentiation of photoreceptors. Rat retina neuronal cultures prepared from 1-2-d-old rats (1,3) were incubated in a chemically defined serum-free medium, in the absence and in the presence of DHA and other fatty acids (20:4, 18:1, 16:0), unlabeled and radioactive. Cells were identified by morphological and immunocytochemical techniques, and apoptosis was determined using nuclear markers (2). In cells incubated with DHA, its proportion in lipids increased fourto sixfold, in contrast to other fatty acids whose addition did not affect the cell lipid composition. However, all labeled fatty acids were actively incorporated and esterified in neuronal lipids, particularly in phosphatidylcholine, showing that efficient mechanisms exist for the uptake of each. Apart from this common feature the distribution of radioactivity among lipids varied, showing a characteristic profile for each fatty acid. The addition of unlabeled DHA to cells did not affect the incorporation of labeled 16:0 or 18:1 in phosphatidylcholine but significantly decreased that of 20:4. Retinal neurons thus have specific mechanisms for handling fatty acids of different length and unsaturation, the incorporation of DHA taking priority in this phase of development. This may contribute to the high proportion of DHA-enriched phospholipids found in retinal neurons. Furthermore, neurons may have specific mechanisms for handling DHA. While there was a net uptake of all fatty acids tested when added as radioactive substrates, only DHA was able to modify the lipid acyl chain composition when supplied as unla-
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The insulin receptor (IR) and IR signaling proteins are widely distributed throughout the CNS. IR signaling provides a trophic signal for transformed retinal neurons in culture and we recently reported that deletion of IR in rod... more
The insulin receptor (IR) and IR signaling proteins are widely distributed throughout the CNS. IR signaling provides a trophic signal for transformed retinal neurons in culture and we recently reported that deletion of IR in rod photoreceptors by Cre/lox system resulted in stress‐induced photoreceptor degeneration. These studies suggest a neuroprotective role of IR in rod photoreceptor cell function. However, there are no studies available on the role of insulin‐induced IR signaling in the development of normal photoreceptors. To examine the role of insulin‐induced IR signaling, we analyzed cultured neuronal cells isolated from newborn rodent retinas. In insulin‐lacking cultures, photoreceptors from wild‐type rat retinas exhibited an abnormal morphology with a wide axon cone and disorganization of the actin and tubulin cytoskeleton. Photoreceptors from IR knockout mouse retinas also exhibited a similar abnormal morphology. A novel finding in this study was that addition of docosahex...
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Research Interests: Chemistry, Biology, Oxidative Stress, Apoptosis, Medicine, and 15 moreAnimals, Retina, Ciencias Biológicas, Rats, Oxidative Damage, Receptor, Wistar Rats, Lipid, Retinoid, Structure activity Relationship, Cell Survival, Docosahexaenoic Acid, Biochemistry and cell biology, Photoreceptors, and Medical biochemistry and metabolomics
Research Interests: Fluorescence Microscopy, Biology, Cell Cycle, Enzyme Inhibitors, Cell Biology, and 15 moreConfocal Microscopy, Apoptosis, Medicine, Biological Sciences, Cell Differentiation, Animals, Phosphorylation, Ciencias Biológicas, Rats, Pyridines, Imidazoles, Cell Survival, Antineoplastic Agents, Photoreceptors, and Medical and Health Sciences
Research Interests: Neurogenesis, Biology, Cell Cycle, Cell Biology, Medicine, and 15 moreBiological Sciences, Cell Differentiation, Animals, Retina, Neurons, Ciencias Biológicas, Rats, Cell Cycle regulation, Cell Cycle Proteins, Neurotrophic Factors, Cell Survival, Docosahexaenoic Acid, Coordinated Control, fluorescent dyes, and Medical and Health Sciences
Research Interests: Chemistry, Biology, Immunohistochemistry, Cell Biology, Medicine, and 15 moreBiological Sciences, Cell Differentiation, Pregnancy, Female, Animals, Cellular differentiation, Differentiation, Ciencias Biológicas, CRX, Pax, Axons, Docosahexaenoic Acid, Photoreceptor, Medical and Health Sciences, and Cell culture techniques
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Research Interests: Ophthalmology, Biology, Oxidative Stress, Mitochondria, Apoptosis, and 15 moreMedicine, Biological Sciences, Cell Differentiation, Animals, Cell Death, Cytoprotection, Paraquat, Ciencias Biológicas, Hydrogen Peroxide, Apoptose, Carotenoid, Lutein, Cell Survival, Docosahexaenoic Acid, and Medical and Health Sciences
Research Interests: Biology, Cell Biology, Oxidative Stress, Mitochondria, Apoptosis, and 15 moreMedicine, Cell Culture, Investigative Ophthalmology, Biological Sciences, Animals, Cell Death, Cytoprotection, Neurons, Herbicides, Apoptose, Cell Survival, Docosahexaenoic Acid, Mitochondrion, Medical and Health Sciences, and Cell culture techniques
Research Interests: Biology, Immunohistochemistry, Enzyme Inhibitors, Cell Biology, Oxidative Stress, and 15 moreMitochondria, Apoptosis, Medicine, Biological Sciences, Animals, Ceramide, Paraquat, Ciencias Biológicas, Rats, Fumonisins, Ceramides, Docosahexaenoic Acid, Sphingosine, Photoreceptor, and Medical and Health Sciences
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Research Interests: Ophthalmology, Biology, Immunohistochemistry, Enzyme Inhibitors, Cell Biology, and 15 moreMedicine, Western blotting, Biological Sciences, Cell Differentiation, Animals, Proliferation, Differentiation, Ciencias Biológicas, Opsins, Rats, Cell Proliferation, Cell Survival, Sphingosine, Photoreceptors, and Medical and Health Sciences
The formation of 14C-labelled long-chain and very-long-chain (n-3) pentaenoic and hexaenoic fatty acids was studied in bovine retina by following the metabolism of [14C]docosapentaenoate [C22:5, n-3 fatty acid (22:5 n-3)],... more
The formation of 14C-labelled long-chain and very-long-chain (n-3) pentaenoic and hexaenoic fatty acids was studied in bovine retina by following the metabolism of [14C]docosapentaenoate [C22:5, n-3 fatty acid (22:5 n-3)], [14C]docosahexaenoate (22:6 n-3), and [14C]acetate. With similar amounts of 22:5 n-3 and 22:6 n-3 as substrates, the former was actively transformed into 24:5 n-3, whereas the latter was virtually unmodified. Labelled 24:5, 26:5, 24:6 and 22:6 were formed from [1-14C]22:5 n-3, showing that pentaenoic fatty acids including 24:5 n-3 can be elongated and desaturated within the retina. When retinal microsomes were incubated with [1-14C]22:5 n-3, 24:5 n-3 was the only fatty acid formed. In retinas incubated with [14C]acetate, 24:5 n-3 was the most highly labelled fatty acid among the polyenes synthesized, 24:6 n-3 being a minor product. Such selectivity in the elongation of two fatty acids identical in length, 22:5 n-3 and 22:6 n-3, despite the fact that 22:5 is a mino...
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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... more
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. 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 antiangi...