Mechanisms Underlying H2O2-Evoked Carbonyl Modification of Cytoskeletal Protein and Axon Injury in PC-12 Cells.

Background/Aims: To investigate the mechanism that enables oxidative stress and cytoskeleton protein carbonylation to contribute to axonal dysfunction in traumatic brain injury (TBI). Methods: We created an in vitro model of neuronal oxidative damage by exposing a neuron-like cell line (PC-12) to different concentrations (100 mu M, 200 mu M, and 300 mu M) of H202 for 24 h or 48 h. Carbonyl modification of cytoskeletal proteins (beta-actin and beta-tubulin) and its impact on beta-actin/beta-tubulin filament dynamics were determined by enzyme-linked immunosorbent assay, immunostaining, and western blotting. Depolymerization of beta-actin/beta-tubulin filaments was evaluated using the monomer/polymer ratio of each protein via western blotting. Phosphorylation of the neurofilament heavy chain (P-NFH) was used as an axonal injury marker and detected by immunostaining. Results: Our results showed that H2O2 treatment led to increased oxidative stress in PC-12 cells, as indicated by the increased generation of malondialdehyde and 8-hydroxydeoxyguanosine and decreased intracellular glutathione levels. H2O2 treatment also increased carbonyl modification of total proteins and cytoskeleton proteins beta-actin/beta-tubulin, which occurred concurrently with the suppression of proteasome activity. Moreover, H2O2 treatment increased the generation of the axonal injury marker P-NFH, and depolymerization of the beta-actin/beta-tubulin filaments was indicated by increased monomer/polymer ratios of each protein. Lastly, overexpression of the proteasome beta 5 subunit in PC-12 cells significantly reduced the H2O2-induced accumulation of carbonylated beta-actin/beta-tubulin, P-NFH, and beta-actin/beta-tubulin depolymerization. Conclusions: We concluded that carbonylation of cytoskeleton proteins could lead to depolymerization of their filaments and axonal injury, and proteasome suppression contributes to the accumulation of carbonylated proteins under oxidative conditions. (C) 2018 The Author(s) Published by S. Karger AG, Basel