Galanin Protects from Caspase-8/12-initiated Neuronal Apoptosis in the Ischemic Mouse Brain via GalR1.

Yutao Yang,Zhu Mei,Shuiqiao Liu,Tong Wang,Zhi-Qing David Xu,Hui Li,Yun Li,Junfa Li,Song Han,Xiao-Xiao Li

doi:10.14336/ad.2016.0806

Yutao Yang, Zhu Mei + Show 8 more

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https://doi.org/10.14336/ad.2016.0806

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Abstract

Galanin (GAL) plays key role in many pathophysiological processes, but its role in ischemic stroke remains unclear. Here, the models of 1 h middle cerebral artery occlusion (MCAO)/1-7 d reperfusion (R)-induced ischemic stroke and in vitro cell ischemia of 1 h oxygen-glucose deprivation (OGD)/24 h reoxygenation in primary cultured cortical neurons were used to explore GAL’s effects and its underlying mechanisms. The results showed significant increases of GAL protein levels in the peri-infarct region (P) and infarct core (I) within 48 h R of MCAO mice (p<0.001). The RT-qPCR results also demonstrated significant increases of GAL mRNA during 24-48 h R (p<0.001), and GAL receptors GalR1-2 (but not 3) mRNA levels in the P region at 24 h R of MCAO mice (p<0.001). Furthermore, the significant decrease of infarct volume (p<0.05) and improved neurological outcome (p<0.001-0.05) were observed in MCAO mice following 1 h pre- or 6 h post-treatment of GAL during 1-7 d reperfusion. GalR1 was confirmed as the receptor responsible for GAL-induced neuroprotection by using GalR2/3 agonist AR-M1896 and Lentivirus-based RNAi knockdown of GalR1. GAL treatment inhibited Caspase-3 activation through the upstream initiators Capsases-8/-12 (not Caspase-9) in both P region and OGD-treated cortical neurons. Meanwhile, GAL’s neuroprotective effect was not observed in cortical neurons from conventional protein kinase C (cPKC) γ knockout mice. These results suggested that exogenous GAL protects the brain from ischemic injury by inhibiting Capsase-8/12-initiated apoptosis, possibly mediated by GalR1 via the cPKCγ signaling pathway.

Highlights

Galanin (GAL) plays key role in many pathophysiological processes, but its role in ischemic stroke remains unclear
By using primary neurons extracted from cPKCγ KO mice, we found that GAL’s protection during 1 h oxygen-glucose deprivation (OGD)/24 h reoxygenation is no longer effective without the involvement of cPKCγ (PKCγ KO 0 vs. PKCγ KO 10-6 M GAL, p=0.1094; n=18 per group; Fig. 3C); there was a significant decrease in cell survival rates between wildtype (WT) and cPKCγ KO neurons after GAL treatment (WT+10-6 M GAL vs. PKCγ KO+10-6 M GAL; p
Using an intraluminal middle cerebral artery occlusion (MCAO) model in the mouse, we demonstrate for the first time that galanin is neuroprotective in ischemic stroke

Summary

Introduction

Galanin (GAL) plays key role in many pathophysiological processes, but its role in ischemic stroke remains unclear. The models of 1 h middle cerebral artery occlusion (MCAO)/1-7 d reperfusion (R)-induced ischemic stroke and in vitro cell ischemia of 1 h oxygen-glucose deprivation (OGD)/24 h reoxygenation in primary cultured cortical neurons were used to explore GAL’s effects and its underlying mechanisms. GAL’s neuroprotective effect was not observed in cortical neurons from conventional protein kinase C (cPKC) γ knockout mice. These results suggested that exogenous GAL protects the brain from ischemic injury by inhibiting Capsase-8/12-initiated apoptosis, possibly mediated by GalR1 via the cPKCγ signaling pathway. Numerous studies have unraveled molecules and pathways that mediate the process of cell death following ischemia. The cell model of ischemic injury in vitro by way of subjecting primary cultured cortical neurons to oxygenglucose deprivation (OGD) was used to explore the underlying workings of GAL and its receptors during ischemic injury

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