Hypoxic Preconditioning Maintains GLT-1 Against Transient Global Cerebral Ischemia Through Upregulating Cx43 and Inhibiting c-Src.

Kongping Li,Zhe Shi,Donghai Liang,Liu Liu,Weiwen Sun,Huarong Zhou,Dandan Liu,En Xu,Yafu Tan,Wensheng Xu,Lixuan Zhan

doi:10.3389/fnmol.2018.00344

Abstract

Transient global cerebral ischemia (tGCI) causes excessive release of glutamate from neurons. Astrocytic glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) together play a predominant role in maintaining glutamate at normal extracellular concentrations. Though our previous studies reported the alleviation of tGCI-induced neuronal death by hypoxic preconditioning (HPC) in hippocampal Cornu Ammonis 1 (CA1) of adult rats, the underlying mechanism has not yet been fully elaborated. In this study, we aimed to investigate the roles of GLT-1 and GS in the neuroprotection mediated by HPC against tGCI and to ascertain whether these roles can be regulated by connexin 43 (Cx43) and cellular-Src (c-Src) activity. We found that HPC decreased the level of extracellular glutamate in CA1 after tGCI via maintenance of GLT-1 expression and GS activity. Inhibition of GLT-1 expression with dihydrokainate (DHK) or inhibition of GS activity with methionine sulfoximine (MSO) abolished the neuroprotection induced by HPC. Also, HPC markedly upregulated Cx43 and inhibited p-c-Src expression in CA1 after tGCI, whereas inhibition of Cx43 with Gap26 dramatically reversed this effect. Furthermore, inhibition of p-c-Src with 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3, 4-d) pyrimidine (PP2) decreased c-Src activity, increased protein levels of GLT-1 and Cx43, enhanced GS activity, and thus reduced extracellular glutamate level in CA1 after tGCI. Collectively, our data demonstrated that reduced extracellular glutamate induced by HPC against tGCI through preventing the reduction of GLT-1 expression and maintaining GS activity in hippocampal CA1, which was mediated by upregulating Cx43 expression and inhibiting c-Src activity.

Highlights

Global cerebral ischemia (GCI) may be caused by acute heart failure, cardiac arrest and shock etc., and lead to delayed neuronal damage in the hippocampal Cornu Ammonis 1 (CA1) subregion
476 rats were used for the experiments, six of which in transient GCI (tGCI) group and five in hypoxic preconditioning (HPC) group died during the process of ischemia; four in tGCI group and two in HPC group died during reperfusion; four died after intraperitoneal injection; four died after intracerebroventricular injection and six died after microdialysis
Compared to Sham group, glutamate transporters-1 (GLT-1) immunoreactivity sharply decreased at all time points in tGCI groups, while HPC thoroughly prevented this reduction induced by tGCI (Figure 2B)

Summary

Introduction

Global cerebral ischemia (GCI) may be caused by acute heart failure, cardiac arrest and shock etc., and lead to delayed neuronal damage in the hippocampal Cornu Ammonis 1 (CA1) subregion. The released glutamate from neurons and astrocytes shall be mainly taken up into astrocytes by GLT-1 and converted to glutamine by glutamine synthetase (GS), a key enzyme for the regulation of the extracellular glutamate in the glutamateglutamine cycle localized primarily in astrocytes. This disorder of glutamate-glutamine cycle related to a metabolic trafficking mechanism between glia and neurons plays an important role in neuronal death after cerebral ischemia (Wang et al, 2013; Momosaki et al, 2015). What still remains unclear is whether the expression of GLT-1 and/or activity of GS is related to HPC-induced neuroprotection against tGCI

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