Abstract
This study aimed to investigate the protective effect of the M9 region (residues 290–562) of amino-Nogo-A fused to the human immunodeficiency virus trans-activator TAT in an in vitro model of ischemia–reperfusion induced by oxygen–glucose deprivation (OGD) in HT22 hippocampal neurons, and to investigate the role of NADPH oxidase in this protection. Transduction of TAT-M9 was analyzed by immunofluorescence staining and western blot. The biologic activity of TAT-M9 was assessed by its effects against OGD-induced HT22 cell damage, compared with a mutant M9 fusion protein or vehicle. Cellular viability and lactate dehydrogenase (LDH) release were assessed. Neuronal apoptosis was evaluated by flow cytometry. The Bax/Bcl-2 ratio was determined by western blotting. Reactive oxygen species (ROS) levels and NADPH oxidase activity were also measured in the presence or absence of an inhibitor or activator of NADPH oxidase. Our results confirmed the delivery of the protein into HT22 cells by immunofluorescence and western blot. Addition of 0.4 μmol/L TAT-M9 to the culture medium effectively improved neuronal cell viability and reduced LDH release induced by OGD. The fusion protein also protected HT22 cells from apoptosis, suppressed overexpression of Bax, and inhibited the reduction in Bcl-2 expression. Furthermore, TAT-M9, as well as apocynin, decreased NADPH oxidase activity and ROS content. The protective effects of the TAT-M9 were reversed by TBCA, an agonist of NADPH oxidase. In conclusion, TAT-M9 could be successfully transduced into HT22 cells, and protected HT22 cells against OGD damage by inhibiting NADPH oxidase-mediated oxidative stress. These findings suggest that the TAT-M9 protein may be an efficient therapeutic agent for neuroprotection.
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Acknowledgments
This work was supported by the Major Program of National Natural Science Foundation of China (Grant 30930091), the Program for Changjiang Scholars and Innovative Research Team in University (Beijing, China, Grant 2010CXTD01), and the National Natural Science Foundation of China (Grants 81072888, 81071060, 81035375).
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Fan Guo and Huiwen Wang contributed equally to this work.
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Guo, F., Wang, H., Li, L. et al. A Novel Domain of Amino-Nogo-A Protects HT22 Cells Exposed to Oxygen Glucose Deprivation by Inhibiting NADPH Oxidase Activity. Cell Mol Neurobiol 33, 443–452 (2013). https://doi.org/10.1007/s10571-013-9911-1
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DOI: https://doi.org/10.1007/s10571-013-9911-1