Ceramide is a Mediator of Apoptosis in Retina Photoreceptors

PURPOSE. The precise mechanisms involved in photoreceptor apoptosis are still unclear. We here investigated the role of ceramide, a sphingolipid precursor that induces apoptosis upon cellular stress, in activating this death in photoreceptors. METHODS. Rat retina neuronal cultures, with or without docosahexaenoic acid (DHA), were treated with the ceramide analog 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 to controls, while 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 protective effect. CONCLUSIONS. Our results suggest that oxidative stress stimulated an increase in ceramide levels, which induced photoreceptor apoptosis. DHA prevented oxidative stress and ceramide damage by up regulating 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 might provide a therapeutic tool for preventing photoreceptor death in neurodegenerative diseases.Fil: German, Olga Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Miranda, Gisela Edit. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Abrahan, Carolina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin

Sphingosine-1-Phosphate Is a Key Regulator of Proliferation and Differentiation in Retina Photoreceptors

PURPOSE. Identifying the cues required for survival and development of photoreceptors is essential for treating retina neurodegenerations. We previously established that glial derived neurotrophic factor (GDNF) stimulates proliferation and that docosahexaenoic acid (DHA) promotes photoreceptor survival and differentiation. Our later finding that ceramide triggers photoreceptor apoptosis suggested sphingolipids might also control photoreceptor development. We now investigated whether sphingosine-1-phophate (S1P), which promotes survival and differentiation in several cell types, regulates photoreceptor proliferation and differentiation and whether it is a mediator in GDNF and DHA effects. METHODS. Rat retina neuronal cultures were supplemented at day 0 or 1 with S1P, GDNF or DHA and treated with DL-threo-dihydrosphingosine (DHS) to inhibit S1P synthesis or with Brefeldin A (BFA) to block intracellular trafficking. Proliferation was quantified determining bromodeoxyuridine uptake and number of mitotic figures. Opsin, peripherin and sphingosine kinase (SphK), the enzyme required for S1P synthesis, were quantified by immunocytochemistry and Western blot. RESULTS. S1P increased proliferation of photoreceptor progenitors. It also stimulated formation of apical processes, enhanced opsin and peripherin expression and promoted their localization in these processes; DHA had similar effects. BFA prevented S1P and DHA enhancement of apical process formation, without affecting opsin expression. While GDNF and DHA enhanced SphK expression in photoreceptors, inhibiting S1P synthesis blocked GDNF mitogenic effect and DHA effects on differentiation. CONCLUSIONS. We propose S1P as a key regulator in photoreceptor development. GDNF and DHA might upregulate SphK levels to promote S1P synthesis, which would initially promote proliferation and then advance photoreceptor differentiation.Fil: Miranda, Gisela Edit. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Abrahan, Carolina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Politi, Luis Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin

Ceramide-1-phosphate: a new mediator of survival and development in retina photoreceptors

Purpose. Simple sphingolipids control crucial cellular processes in several cell types. We demonstrated that sphingolipids such as ceramide, sphingosine and sphingosine-1-phosphate are key mediators in the regulation of survival, differentiation and proliferation of retina photoreceptors. Ceramide-1-phosphate (C1P) regulates growth and survival in several cell types; however, little is known concerning its functions in the retina. We here explored whether C1P also participated in controlling photoreceptor development. Methods. Rat retina neuronal cultures were supplemented with 1-10 µM C1P. Proliferation was determined by evaluating 5-bromo-2-deoxyuridine (BrdU) uptake and number of mitotic figures and differentiation by establishing opsin and peripherin expression by immunocytochemistry and Western blot. Apoptosis was inhibited with the caspase pan-inhibitor ZVADFMK and evaluated by TUNEL assay, propidium iodide/annexin V and DAPI labeling. Preservation of mitochondrial membrane potential was evaluated. Results. C1P enhanced BrdU uptake and increased mitosis in retinal progenitors. C1P addition advanced photoreceptor differentiation, enhancing opsin and peripherin expression and stimulating development of apical processes, in which these proteins were concentrated. In the absence of their trophic factors, photoreceptors degenerate after 4 days in vitro and at day 6 almost 50% of photoreceptors were apoptotic; C1P decreased photoreceptor apoptosis, reducing this percentage by half. Inhibiting caspase activity reduced photoreceptor apoptosis in controls but did not increase opsin expression, implying C1P has separate effects on differentiation and survival. Conclusions. These results suggest for the first time that C1P is a novel mediator that plays multiple functions in photoreceptors, initially regulating their proliferation and then promoting their survival and differentiation.Fil: Miranda, Gisela Edit. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: Abrahan, Carolina Elizabeth. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: Agnolazza, Daniela Luciana. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: Politi, Luis Enrique. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; Argentin

Oxidative stress promotes proliferation and dedifferentiation of retina glial cells in vitro

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 h2o2. 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.Fil: Abrahan, Carolina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Insua, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Politi, Luis Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: German, Olga Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin

Müller glial cells induce stem cell properties in retinal progenitors in vitro and promote their further differentiation into photoreceptors

Using stem cells to replace lost neurons is a promising strategy for treating retinal neurodegenerative diseases. Among their multiple functions, Müller glial cells are retina stem cells, with a robust regenerative potential in lower vertebrates, which is much more restricted in mammals. In rodents, most retina progenitors exit the cell cycle immediately after birth, differentiate as neurons, and then cannot reenter the cell cycle. Here we demonstrate that, in mixed cultures with Müller glial cells, rat retina progenitor cells expressed stem cell properties, maintained their proliferative potential, and were able to preserve these properties and remain mitotically active after several consecutive passages. Notably, these progenitors retained the capacity to differentiate as photoreceptors, even after successive reseedings. Müller glial cells markedly stimulated differentiation of retina progenitors; these cells initially expressed Crx and then developed as mature photoreceptors that expressed characteristic markers, such as opsin and peripherin. Moreover, they were light responsive, insofar as they decreased their cGMP levels when exposed to light, and they also showed high-affinity glutamate uptake, a characteristic of mature photoreceptors. Our present findings indicate that, in addition to giving rise to new photoreceptors, Muller glial cells might instruct a pool of undifferentiated cells to develop and preserve stem cell characteristics, even after successive reseedings, and then stimulate their differentiation as functional photoreceptors. This complementary mechanism might contribute to enlarge the limited regenerative capacity of mammalian Müller cells.Fil: Simon, Maria Victoria. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: de Genaro, Pablo Adrian. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: Abrahan, Carolina Elizabeth. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: de Los Santos, Elisa Beatriz. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: Politi, Luis Enrique. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; Argentin

Antropologías hechas en Ecuador. El quehacer antropológico (Volumen IV)

Al igual que en otros países, en Ecuador la antropología no es solo una disciplina, son varias genealogías que obedecen a temas diversos con enfoques interdisciplinarios y que cambian de acuerdo al contexto social, económico y político; pero a diferencia de la región, registra pocas escuelas de antropología y centros de formación de profesionales en el área. Esta recopilación de textos muestra la diversidad y las múltiples facetas de las antropologías ecuatorianas. La antropología ecuatoriana no se agota en estas historiografías y resalta aquellas genealogías del pensamiento ecuatoriano, nutrido por reflexiones desde las escuelas clásicas de la antropología, que dialogan fuertemente con el contexto nacional y que, particularmente, tienen la capacidad de recrearse a la luz de las necesidades reales de la gente con quienes se co-construye el conocimiento