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Sodium Valproate Ameliorates Neuronal Apoptosis in a Kainic Acid Model of Epilepsy via Enhancing PKC-Dependent GABAAR γ2 Serine 327 Phosphorylation

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Abstract

GABA is a dominant inhibitory neurotransmitter in the brain and A type GABA receptor (GABAAR) phosphorylation is critical for GABA-mediated inhibitory effect. However, its role in the neuroprotective effect of sodium valproate (VPA), a prevalent drug for treating patients with epilepsy, remains elusive. The present study was conducted to explore the role of GABAAR phosphorylation in the neuroprotection of VPA against a kainic acid-induced epileptic rat model and the potential molecular mechanisms. Neuronal apoptosis was evaluated by TUNEL assay, PI/Annexin V double staining, caspase-3 activity detection and Bax and Bcl-2 proteins expression via Western blot analysis. The primary rat hippocampal neurons were cultivated and cell viability was measured by CCK8 detection following KA- or free Mg2+-induced neuronal impairment. Our results found that VPA treatment significantly reduced neuronal apoptosis in the KA-induced rat model (including reductions of TUNEL-positive cells, caspase-3 activity and Bax protein expression, and increase of Bcl-2 protein level). In the in vitro experiments, VPA at the concentration of 1 mM for 24 h also increased cell survival and suppressed cell apoptosis in KA- or no Mg2+-induced models via CCK8 assay and PI/Annexin V double staining, respectively. What is more important, the phosphorylation of γ2 subunit at serine 327 residue for GABAAR was found to be robustly enhanced both in the KA-induced epileptic rat model and neuronal cultures following KA exposure after VPA treatment, while no evident alteration was found in terms of GABAAR β3 phosphorylation (408 or 409 serine residue). Additionally, pharmacological inhibition of protein kinase C (PKC) clearly abrogated the neuroprotective potential of VPA against KA- or free Mg2+-associated neuronal injury, indicating a critical role of PKC in the effect of GABAAR γ2 serine 327 phosphorylation in VPA’s protection. In summary, our work reveals that VPA mitigates neuronal apoptosis in KA-triggered epileptic seizures, at least, via augmenting PKC-dependent GABAAR γ2 phosphorylation at serine 327 residue.

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Acknowledgements

Our present work was financially supported by National Natural Science Foundation of China (Nos. 81302750, 81671293 and 81503073), Clinical Research Fund of Peking University Unamed-Central South University Xiangya Hospital (No. xywm 2015I16), Natural Science Foundation of Hunan Province (No. 2017JJ3479) and Open Foundation of Innovative Platform in Colleges and University of Hunan Province of China ([2015]54).

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  1. Qin Li and Qiu-Qi Li contributed equally to this work.

Authors and Affiliations

  1. Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, 410008, China

    Qin Li, Qiu-Qi Li, Ji-Ning Jia, Shan Cao, Zhi-Bin Wang, Xu Wang, Chao Luo, Hong-Hao Zhou, Zhao-Qian Liu & Xiao-Yuan Mao

  2. Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, Changsha, 410078, China

    Qin Li, Qiu-Qi Li, Ji-Ning Jia, Shan Cao, Zhi-Bin Wang, Xu Wang, Chao Luo, Hong-Hao Zhou, Zhao-Qian Liu & Xiao-Yuan Mao

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  1. Qin Li
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Correspondence to Xiao-Yuan Mao.

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Fifty Sprague–Dawley rats were used in our present investigation and randomly divided into each group. Efforts were made to minimize animal suffering and the number of animals. Throughout the investigation, all procedures involving animals were in strict accordance with the guidelines established by the Animal Care Committee of Central South University.

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Li, Q., Li, QQ., Jia, JN. et al. Sodium Valproate Ameliorates Neuronal Apoptosis in a Kainic Acid Model of Epilepsy via Enhancing PKC-Dependent GABAAR γ2 Serine 327 Phosphorylation. Neurochem Res 43, 2343–2352 (2018). https://doi.org/10.1007/s11064-018-2659-8

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