Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated In Vitro

Abstract

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.

SVCT2 Overexpression and Ascorbic Acid Uptake Increase Cortical Neuron Differentiation, Which Is Dependent on Vitamin C Recycling between Neurons and Astrocytes

“…As has been characterized in detail in Espinoza et al [30], neurospheres grown in vitro and treated with AA, in the presence of U87 cells (DHA recycling cells), generated neurites efficiently (Figure 5A,B). Thus, similar cultures of neurospheres were co-cultured with U87 cells and treated with 5 µM WZB117 at 24 and 48 h of AA treatment.…”

“…We propose that the main function of vitamin C and SVCT2 is fundamentally to enhance neurite differentiation and inhibit ROS production in brain postnatal states [30,39]. In previous studies, we observed increased SVCT2 expression in pyramidal neurons of the cerebral and cerebellar cortex on postnatal days [1,8].…”

“…Before conducting experiments that allow us to analyze the effect of DHA recycling in the cerebral cortex of postnatal rats and in neurites growth, we conducted studies in 3D cultures of neural precursors that form neurospheres. Recently, we showed that neurospheres expressed high levels of SVCT2 and, when treated with AA, actively generated neurites [30]. This approach seemed robust and complementary to SVCT2 overexpression using primary culture of neurons transduced with lentiviruses.…”

“…This approach seemed robust and complementary to SVCT2 overexpression using primary culture of neurons transduced with lentiviruses. The neurospheres treated with AA also produced DHA, which must be recycled to prevent inhibition of neurite growth and fragmentation [30] In this way, we analyzed the in vitro effects of the GLUT1 inhibitor, WZB117, on DHA recycling and neurite genesis, in cocultures of neurospheres and U87 cells (recycling cells), as well as the cell viability of U87, neurons and astrocytes.…”

Role of Natural Antioxidants on Neuroprotection and Neuroinflammation

“…Furthermore, in a recent article, Dr. Espinoza and collaborators demonstrated that vitamin C treatment improved neuronal differentiation. In their work, the authors described how vitamin C recycling is an important mechanism in regulating neuronal morphology during neuronal differentiation and maturation [ 10 ]. Several studies have shown that other nutraceutical compounds, such as carotenoids, polyphenols and omega-3s, when consumed daily, reduce the risks of chronic neurodegenerative diseases.…”