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

<jats:title>Abstract</jats:title><jats:sec><jats:label /><jats:p>The process of cell differentiation goes hand‐in‐hand with metabolic adaptations, which are needed to provide energy and new metabolites. Carbon monoxide (<jats:styled-content style="fixed-case">CO</jats:styled-content>) is an endogenous cytoprotective molecule able to inhibit cell death and improve mitochondrial metabolism. Neuronal differentiation processes were studied using the <jats:styled-content style="fixed-case">NT</jats:styled-content>2 cell line, which is derived from human testicular embryonic teratocarcinoma and differentiates into post‐mitotic neurons upon retinoic acid treatment. <jats:styled-content style="fixed-case">CO</jats:styled-content>‐releasing molecule A1 (<jats:styled-content style="fixed-case">CORM</jats:styled-content>‐A1) was used do deliver <jats:styled-content style="fixed-case">CO</jats:styled-content> into cell culture. <jats:styled-content style="fixed-case">CO</jats:styled-content> treatment improved <jats:styled-content style="fixed-case">NT</jats:styled-content>2 neuronal differentiation and yield, since there were more neurons and the total cell number increased following the differentiation process. <jats:styled-content style="fixed-case">CO</jats:styled-content> supplementation enhanced the mitochondrial population in post‐mitotic neurons derived from <jats:styled-content style="fixed-case">NT</jats:styled-content>2 cells, as indicated by an increase in mitochondrial <jats:styled-content style="fixed-case">DNA</jats:styled-content>. <jats:styled-content style="fixed-case">CO</jats:styled-content> 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 <jats:sup>13</jats:sup>C incorporation from [U‐<jats:sup>13</jats:sup>C]glucose into the tricarboxylic acid cycle metabolites malate and citrate, and also glutamate and aspartate in <jats:styled-content style="fixed-case">CO</jats:styled-content>‐treated cells. Finally, under low levels of oxygen (5%), which enhances glycolytic metabolism, some of the enhancing effects of <jats:styled-content style="fixed-case">CO</jats:styled-content> on mitochondria were not observed. In conclusion, our data show that <jats:styled-content style="fixed-case">CO</jats:styled-content> improves neuronal and mitochondrial yield by stimulation of tricarboxylic acid cycle activity, and thus oxidative metabolism of <jats:styled-content style="fixed-case">NT</jats:styled-content>2 cells during the process of neuronal differentiation.</jats:p></jats:sec><jats:sec><jats:label /><jats:p> <jats:boxed-text content-type="graphic" position="anchor"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" mimetype="image/png" position="anchor" specific-use="enlarged-web-image" xlink:href="graphic/jnc13653-fig-0007-m.png"><jats:alt-text>image</jats:alt-text></jats:graphic></jats:boxed-text> 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.</jats:p></jats:sec>