Abstract

Vitamin C is found in two states: reduced ascorbic acid (AA) and oxidized dehydroascorbic acid (DHA). In pathophysiological conditions, such as cerebral ischemia and reperfusion (IR), AA is oxidized to DHA. Interestingly, IR induces a particular type of cell death called necroptosis, which is characterized by activation of RIPK1, RIPK3 and MLKL. DHA alters neuronal metabolism and induces cell death; however, the pathway is unknown. Here, we propose that vitamin C oxidation and intracellular DHA production induces neuronal death by activation of necroptosis in vitro. The neural lineage cells, N2a and HN33.11, were supplemented with 200µM AA for accumulation of vitamin C. “Ischemic-like” oxidative stress was induced by deprivation of glucose and 0.5mM H2O2 for 30 min. A “reperfusion-like” condition was induced by leaving cells in complete medium for 3h. Intracellular AA concentration was measured by FRASC method, cell viability by XTT and confocal real-time live-cell microscopy. In addition, 3D reconstructions were performed in Imaris software. Morphology was analyzed by elliptical parameters: oblate, prolate, spherical. Cellular size was analyzed by Bounding-Box tool. Necroptosis was evaluated using necrostain-1 (RIPK1 inhibitor), compound-1 (MLKL inhibitor) and zVAD.FMK (apoptosis control). Characteristics of cell disintegration were analyzed by 4D real-time live-cell confocal-spectral microscopy, using the following fluorescent probes: mitotracker CMXRos (mitochondrial activity), Cellmask (plasma membrane stain and morphology), Hoechst 33342 (integrity of nuclei) and Phalloindin-alexa488 (integrity of plasma membrane). DHA production induces 50% neuronal death and necroptotic disintegration. Necroptosis inhibition with necrostatin-1 and compound-1 prevents neuronal death. However, apoptosis inhibition with zVAD does not. During necroptosis, live cell imaging shows bubbles formation prior to loss of plasma membrane integrity and cytoplasm shedding. In conclusion we propose that DHA could regulates activation of necroptosis in neuronal cells in vitro.