Improvement of neuronal differentiation by carbon monoxide: Role of pentose phosphate pathway.

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: Increase of Neuronal Differentiation Yield by Preventing Cell Death.

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 improves neuronal differentiation and yield by increasing the functioning and number of mitochondria.

The process of cell differentiation goes hand-in-hand with metabolic adaptations, which are needed to provide energy and new metabolites. Carbon monoxide (CO) is an endogenous cytoprotective molecule able to inhibit cell death and improve mitochondrial metabolism. Neuronal differentiation processes were studied using the NT2 cell line, which is derived from human testicular embryonic teratocarcinoma and differentiates into post-mitotic neurons upon retinoic acid treatment. CO-releasing molecule A1 (CORM-A1) was used do deliver CO into cell culture. CO treatment improved NT2 neuronal differentiation and yield, since there were more neurons and the total cell number increased following the differentiation process. CO supplementation enhanced the mitochondrial population in post-mitotic neurons derived from NT2 cells, as indicated by an increase in mitochondrial DNA. CO treatment during neuronal differentiation increased the extent of the classical metabolic change that occurs during neuronal differentiation, from glycolytic to more oxidative metabolism, by decreasing the ratio of lactate production and glucose consumption. The expression of pyruvate and lactate dehydrogenases was higher, indicating an augmented oxidative metabolism. Moreover, these findings were corroborated by an increased percentage of (13) C incorporation from [U-(13) C]glucose into the tricarboxylic acid cycle metabolites malate and citrate, and also glutamate and aspartate in CO-treated cells. Finally, under low levels of oxygen (5%), which enhances glycolytic metabolism, some of the enhancing effects of CO on mitochondria were not observed. In conclusion, our data show that CO improves neuronal and mitochondrial yield by stimulation of tricarboxylic acid cycle activity, and thus oxidative metabolism of NT2 cells during the process of neuronal differentiation. The process of cell differentiation is coupled with metabolic adaptations. Carbon monoxide (CO) is an endogenous cytoprotective gasotransmitter able to prevent cell death and improve mitochondrial metabolism. Herein CO supplementation improved neuronal differentiation yield, by enhancing mitochondrial population and promoting the classical metabolic change that occurs during neuronal differentiation, from glycolytic to oxidative metabolism.

Neuroprotective effects of digested polyphenols from wild blackberry species.

PURPOSE: Blackberry ingestion has been demonstrated to attenuate brain degenerative processes with the benefits ascribed to the (poly)phenolic components. The aim of this work was to evaluate the neuroprotective potential of two wild blackberry species in a neurodegeneration cell model and compare them with a commercial variety. METHODS: This work encompasses chemical characterization before and after an in vitro digestion and the assessment of neuroprotection by digested metabolites. Some studies targeting redox/cell death systems were also performed to assess possible neuroprotective molecular mechanisms. RESULTS: The three blackberry extracts presented some quantitative differences in polyphenol composition that could be responsible for the different responses in the neurodegeneration cell model. Commercial blackberry extracts were ineffective but both wild blackberries, Rubus brigantinus and Rubus vagabundus, presented neuroprotective effects. It was verified that a diminishment of intracellular ROS levels, modulation of glutathione levels and activation of caspases occurred during treatment. The last effect suggests a preconditioning effect since caspase activation was not accompanied by diminution in cell death and loss of functionality. CONCLUSIONS: This is the first time that metabolites obtained from an in vitro digested food matrix, and tested at levels approaching the concentrations found in human plasma, have been described as inducing an adaptative response.

Classic Bartter syndrome: a rare cause of failure to thrive in a child.

Bartter syndrome is a group of rare autosomal-recessive disorders caused by a defect in distal tubule transport of sodium and chloride. Blood gases and plasma electrolytes raise suspicion of this diagnosis and the definitive diagnosis is made by genetic study. Early treatment improves prognosis. The authors present the case of an 11-month-old child with early failure to thrive and severe regurgitation. Blood gases revealed hypochloraemic metabolic alkalosis, hyponatraemia and hypokalaemia. Blood pressure was normal and polyuria was documented. She began therapy with potassium chloride supplementation and indomethacin. There was clinical improvement and plasma potassium and bicarbonate normalised. The molecular study confirmed it was the classic form of Bartter syndrome. Despite being rare in clinical practice, which may lead to unnecessary medical investigation and diagnosis delay, in a child with failure to thrive, hypochloraemic metabolic alkalosis and hypokalaemia, this diagnosis must be considered.