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Projeto de investigação
CARBON MONOXIDE MODULATION OF NEURONAL DIFFERENTIATION – NOVEL CELL THERAPY STRATEGIES FOR NEUROLOGICAL DISORDERS
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Carbon monoxide and mitochondria-modulation of cell metabolism, redox response and cell death
Publication . Almeida, Ana S.; Pereira, Cláudia Figueiredo; Vieira, Helena L. A.; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); Centro de Estudos de Doenças Crónicas (CEDOC); Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Frontiers
Improvement of neuronal differentiation by carbon monoxide
Publication . Almeida, Ana S.; Soares, Nuno L.; Sequeira, Catarina O.; Pereira, Sofia A.; SA, Pereira; Sonnewald, Ursula; Vieira, Helena L.A.; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); Centro de Estudos de Doenças Crónicas (CEDOC); Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Elsevier
Over the last decades, the silent-killer carbon monoxide (CO) has been shown to also be an endogenous cytoprotective molecule able to inhibit cell death and modulate mitochondrial metabolism. Neuronal metabolism is mostly oxidative and neurons also use glucose for maintaining their anti-oxidant status by generation of reduced glutathione (GSH) via the pentose-phosphate pathway (PPP). It is established that neuronal differentiation depends on reactive oxygen species (ROS) generation and signalling, however there is a lack of information about modulation of the PPP during adult neurogenesis. Thus, the main goal of this study was to unravel the role of CO on cell metabolism during neuronal differentiation, particularly by targeting PPP flux and GSH levels as anti-oxidant system. A human neuroblastoma SH-S5Y5 cell line was used, which differentiates into post-mitotic neurons by treatment with retinoic acid (RA), supplemented or not with CO-releasing molecule-A1 (CORM-A1). SH-SY5Y cell differentiation supplemented with CORM-A1 prompted an increase in neuronal yield production. It did, however, not alter glycolytic metabolism, but increased the PPP. In fact, CORM-A1 treatment stimulated (i) mRNA expression of 6-phosphogluconate dehydrogenase (PGDH) and transketolase (TKT), which are enzymes for oxidative and non-oxidative phases of the PPP, respectively and (ii) protein expression and activity of glucose 6-phosphate dehydrogenase (G6PD) the rate-limiting enzyme of the PPP. Likewise, whenever G6PD was knocked-down CO-induced improvement on neuronal differentiation was reverted, while pharmacological inhibition of GSH synthesis did not change CO's effect on the improvement of neuronal differentiation. Both results indicate the key role of PPP in CO-modulation of neuronal differentiation. Furthermore, at the end of SH-SY5Y neuronal differentiation process, CORM-A1 supplementation increased the ratio of reduced and oxidized glutathione (GSH/GSSG) without alteration of GSH metabolism. These data corroborate with PPP stimulation. In conclusion, CO improves neuronal differentiation of SH-S5Y5 cells by stimulating the PPP and modulating the GSH system.
Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis
Publication . Almeida, Ana S; Soares, Nuno L; Vieira, Melissa; Gramsbergen, Jan Bert; Vieira, Helena L A; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); Centro de Estudos de Doenças Crónicas (CEDOC); Instituto de Tecnologia Química e Biológica António Xavier (ITQB); PLOS - Public Library of Science
Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO's improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO's increasing number of differentiated neurons in OHSC. In conclusion, CO's increasing number of differentiated neurons is a novel biological role disclosed herein. CO improves neuronal yield due to its capacity to reduce cell death, promoting an increase in proliferative population. However, one cannot disregard a direct CO's effect on specific cellular processes of neuronal differentiation. Further studies are needed to evaluate how CO can potentially modulate cell mechanisms involved in neuronal differentiation. In summary, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies.
Carbon Monoxide modulation of neuronal differentiation
Publication . Almeida, Ana Sofia Cabral e Sousa de; Vieira, Helena Luísa Araújo
Several evidences support carbon monoxide (CO) for modulating cellular differentiation, in particular for neuronal cells. First, there are some reported studies documenting the biogenesis of mitochondria during spontaneous cell differentiation and it was demonstrated that CO promotes mitochondrial biogenesis. Secondly, ROS are signalling molecules in CO-induced pathways and are also key-players in neuronal differentiation. Third, CO has been described as anti-proliferative molecule in different cell types, namely smooth muscle, cancer and T cells, which can be involved in the balance between differentiation and proliferation that occurs during neurogenesis. Therefore, CO presents a strong potential for modulating neuronal differentiation, opening windows for the development of novel cell therapy strategies for neurological disorders.(...)
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Fundação para a Ciência e a Tecnologia
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SFRH/BD/78440/2011