Abstract
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.
Highlights
3). [14 C]-ascorbic acid (AA) uptake analysis demonstrated the functionality of the expressed transporter as the cultures of neurons transduced with hSVCT2wt-EYFP lentivirus captured twice the amount of AA (236.74 ± 20.58 pmol × 106 cells) as compared to nontransduced neurons (127.13 ± 13.75 pmol × 106 cells) or those transduced with the EGFP lentivirus
After 72 h of Sodium-dependent vitamin C transporter2 (SVCT2) expression, neurites were clearly observed in transduced cells (E). (G–N) SVCT2 overexpression in human subventricular tumor C3 (HSVT-C3) cells. (G) Uptake of 100 μM AA was analyzed in the presence of NaCl or choline at 37 ◦ C in nontransduced and EGFP- or hSVCT2wt-EYFP-overexpressing HSVT-C3 cells. (H) Consecutive z-planes of C3 cells overexpressing SVCT2
The overexpressed protein appeared to be transported in vesicle-like structures that fused to the cell membrane and reached the thin filopodia processes (I and inset). (J) The intracellular vesicles had partial colocalization with the RER marker (J and inset); the filopodia and bleb-like and ruffes-like structures were positive for phalloidin (K). (L–M) RER (RE-RFP+) and SVCT2-EYFP
Summary
It has been postulated that vitamin C has a functional role in neuronal and progenitor cell differentiation [1,2,3,4,5,6,7]. 2 (SVCT2) has been detected in neurons of the cerebral cortex, cerebellum, hippocampus, entorhinal cortex and hypothalamus [1,3,8,9,10,11]. SVCT2 has been detected in glial cells, such as marginal astrocytes, microglia, choroid plexus epithelial cells and tanycytes [9,11,12,13,14]. SVCT2 was detected in radial glia cells with apical polarization; low-to-absent expression was observed in Antioxidants 2021, 10, 1413.
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