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J Neurochem. 2016 Aug;138(3):423-35. doi: 10.1111/jnc.13653. Epub 2016 Jun 18.

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

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CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciência Médicas, Universidade Nova de Lisboa, Lisboa, Portugal.
Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Oeiras, Portugal.
Instituto de Biologia Experimental e Tecnológica (iBET), Oeiras, Portugal.
Department of Clinical Neuroscience, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.


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.


NT2 cell line; TCA; carbon monoxide; metabolism; neuronal differentiation; oxidative phosphorylation

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