Abstract

Vitamin C is the principal antioxidant in central nervous system. Vitamin C is found in 2 states, reduced ascorbic acid (AA) and oxidized dehydroascorbic acid (DHA). When there is oxidative damage, AA performs its antioxidant function, oxidizing to DHA. On one side stood, AA is incorporated by neurons through the co-transporter, sodium-dependent vitamin C transporter (SVCT2). On the other side, oxidized form, (DHA), diffuses into nerve cells by glucose transporters (GLUTs). Our laboratory has showed that accumulation of DHA induces neuronal death in oxidative stress, but pathways involved in cell death are unknown. We propose study cellular and mitochondrial morphology, because is known that morphological alterations and mitochondrial fission/fusion processes are associated with cell death. Neuro2a cell line and rat cortical neurons were supplemented with AA and thereafter treated with H2O2. Cell viability was quantified by XTT method. Cellular and mitochondrial morphology, and distribution of vitamin C transporters were analyzed by spectral microscopy, super-resolution structured illumination microscopy (SIM). In addition, 3D reconstructions were performed in Imaris software. Morphology was analyzed by elliptical parameters: oblate, prolate, spherical. Mitochondrial and cellular size was analyzed by Bounding-Box tool. Oxidation of vitamin C generates 50% of neuronal death. Prior to neuronal death, cells modified GLUT1 distribution in perinuclear zone and SVCT2 in plasmatic membrane. Oxidation of vitamin C, induces mitochondria decreases in size and volume on average 0,3m and 0,2m3 respectively. Intracellular DHA generation induces neuronal death. This process involving redistribution of SVCT2/GLUT1. Mitochondrial size and volume decreases during oxidation of vitamin C. Furthermore, we note decrease of mitochondrial activity (MitoTracker, red) with 4D live cell experiments, condition finished in permeabilization of the plasmatic membrane and incorporation of phalloidin (green).