Xiejun Zhang1,2, Zongyang Li2,3, Qiusheng Zhang1,2, Lei Chen1,2, Xianjian Huang1,2, Yuan Zhang2,3, Xiaojia Liu1, Wenlan Liu2,3, Weiping Li1,2. 1. Department of Neurosurgery, Shenzhen University First Affiliated Hospital, Shenzhen Second People's Hospital, Clinical College of Anhui Medical University, Shenzhen, China. 2. Shenzhen Key Laboratory of Neurosurgery, Shenzhen, China. 3. The Central Laboratory, Shenzhen University First Affiliated Hospital, Shenzhen Second People's Hospital, Clinical College of Anhui Medical University, Shenzhen, China.
Abstract
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 μM, 200 μM, and 300 μM) of H2O2 for 24 h or 48 h. Carbonyl modification of cytoskeletal proteins (β-actin and β-tubulin) and its impact on β-actin/β-tubulin filament dynamics were determined by enzyme-linked immunosorbent assay, immunostaining, and western blotting. Depolymerization of β-actin/β-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 β-actin/β-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 β-actin/β-tubulin filaments was indicated by increased monomer/polymer ratios of each protein. Lastly, overexpression of the proteasome β5 subunit in PC-12 cells significantly reduced the H2O2-induced accumulation of carbonylated β-actin/ β-tubulin, P-NFH, and β-actin/β-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.
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 μM, 200 μM, and 300 μM) of H2O2 for 24 h or 48 h. Carbonyl modification of cytoskeletal proteins (β-actin and β-tubulin) and its impact on β-actin/β-tubulin filament dynamics were determined by enzyme-linked immunosorbent assay, immunostaining, and western blotting. Depolymerization of β-actin/β-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 β-actin/β-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 β-actin/β-tubulin filaments was indicated by increased monomer/polymer ratios of each protein. Lastly, overexpression of the proteasome β5 subunit in PC-12 cells significantly reduced the H2O2-induced accumulation of carbonylated β-actin/ β-tubulin, P-NFH, and β-actin/β-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.