During brain development, sodium–vitamin C transporter (SVCT2) has been detected primarily in radial glial cells in situ, with low-to-absent expression in cerebral cortex neuroblasts. However, strong SVCT2 expression is observed during the first postnatal days, resulting in increased intracellular concentration of vitamin C. Hippocampal neurons isolated from SVCT2 knockout mice showed shorter neurites and low clustering of glutamate receptors. Other studies have shown that vitamin C-deprived guinea pigs have reduced spatial memory, suggesting that ascorbic acid (AA) and SVCT2 have important roles in postnatal neuronal differentiation and neurite formation. In this study, SVCT2 lentiviral overexpression induced branching and increased synaptic proteins expression in primary cultures of cortical neurons. Analysis in neuroblastoma 2a (Neuro2a) and human subventricular tumor C3 (HSVT-C3) cells showed similar branching results. SVCT2 was mainly observed in the cell membrane and endoplasmic reticulum; however, it was not detected in the mitochondria. Cellular branching in neuronal cells and in a previously standardized neurosphere assay is dependent on the recycling of vitamin C or reduction in dehydroascorbic acid (DHA, produced by neurons) by glial cells. The effect of WZB117, a selective glucose/DHA transporter 1 (GLUT1) inhibitor expressed in glial cells, was also studied. By inhibiting GLUT1 glial cells, a loss of branching is observed in vitro, which is reproduced in the cerebral cortex in situ. We concluded that vitamin C recycling between neurons and astrocyte-like cells is fundamental to maintain neuronal differentiation in vitro and in vivo. The recycling activity begins at the cerebral postnatal cortex when neurons increase SVCT2 expression and concomitantly, GLUT1 is expressed in glial cells.

The reduced form of vitamin C, ascorbic acid (AA), has been related with gene expression and cell differentiation in the cerebral cortex. In neurons, AA is mainly oxidized to dehydroascorbic acid (DHA); however, DHA cannot accumulate intracellularly because it induces metabolic changes and cell death. In this context, it has been proposed that vitamin C recycling via neuron–astrocyte coupling maintains AA levels and prevents DHA parenchymal accumulation. To date, the role of this mechanism during the outgrowth of neurites is unknown. To stimulate neuronal differentiation, adhered neurospheres treated with AA and retinoic acid (RA) were used. Neuritic growth was analyzed by confocal microscopy, and the effect of vitamin C recycling (bystander effect) in vitro was studied using different cells. AA stimulates neuritic growth more efficiently than RA. However, AA is oxidized to DHA in long incubation periods, generating a loss in the formation of neurites. Surprisingly, neurite growth is maintained over time following co-incubation of neurospheres with cells that efficiently capture DHA. In this sense, astrocytes have high capacity to recycle DHA and stimulate the maintenance of neurites. We demonstrated that vitamin C recycling in vitro regulates the morphology of immature neurons during the differentiation and maturation processes.