Migraine pathophysiology: NR2A/SFKs signaling in cortical spreading depression

Bu, F (2017) Migraine pathophysiology: NR2A/SFKs signaling in cortical spreading depression. Doctor of Philosophy thesis, University of Liverpool.

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Abstract

Cortical spreading depression (CSD) is a propagating neuronal/glial excitation, followed by depression in cerebral cortex and subcortical regions. It is known to be the underlying cause of migraine with aura in humans. CSD can also lead to migraine-like behavior by triggering pannexin1 (Panx1) channels opening and induce the release of calcitonin gene-related peptide (CGRP) that plays a key player in migraine patients. Increasing evidence points to an essential role of NR2A-containing NMDA receptors in CSD propagation in vitro; however whether NR2A also mediates CSD genesis and its downstream signaling associated with CSD is unknown. The purpose of this thesis is to clarify the contribution of NR2A-containing receptors to CSD propagation and determine their role in CSD genesis in vivo, and if so, to further explore the mechanism underlying the action of NR2A relevant to Panx1 channels opening and CGRP gene expression in rats. In the present study, CSD was induced both in vitro and in vivo. Multi-disciplinary methods were used including electrophysiology and intrinsic optical imaging for CSD recording, western blot, immunoprecipitation and immunohistochemistry for protein detection, and qPCR for gene expression analysis. The results demonstrated that NR2A-containing receptor inhibition using NR2A-preferring antagonist, TCN-201, suppressed CSD propagation in a concentration-dependent manner in the chick retina. In addition, both the NR2A antagonists, NVP-AAM077 and TCN-201, concentration-dependently suppressed CSD genesis but not propagation in the microdialysis-based CSD model in rats. Differently, perfusion of 0.3 nmol NVP-AAM077 into contralateral cerebroventricle considerably suppressed CSD propagation in rats. These data suggests a key role of NR2A in mediating both CSD genesis and propagation. Further mechanism study showed that CSD not only promoted sarcoma family kinases (SFKs) activation, but also SFKs-Panx1 interaction and neuronal Panx1 channels opening in the ipsilateral cortex of rats. Corresponds to this finding, inhibition of SFKs by intracerebroventricle (i.c.v.) perfusion of 2.5 nmol PP2 not only attenuated both SFKs activation and Panx1 channels opening induced by CSD, but also suppressed CSD propagation. Furthermore, the CSD-induced SFKs activation, SFKs-Panx1 interaction and neuronal Panx1 channels opening were significantly suppressed under NR2A inhibition by i.c.v. perfusion of 0.3 nmol NVP-AAM077. Finally, pre-treatment with 0.3 nmol NVP-AAM077 prevented the elevation of CGRP mRNA 24 hour after multiple CSD in the ipsilateral visual cortex of rats. In summary, this study provides strong evidence that NR2A-containing NMDA receptors contribute to CSD genesis and propagation, and reveals a previously unknown migraine mechanism of NR2A involving SFKs, Panx1 and CGRP during CSD, i.e. NR2A regulates single CSD-induced opening of neuronal Panx1 channels via coupling activated SFKs to Panx1 in cortex, and that NR2A regulates multiple CSD-induced CGRP gene expression in visual cortex. These findings provide new insights into CSD involving NR2A-containing receptor and downstream signals. Selectively antagonizing these elements might constitute a highly specific strategy treating migraine and other diseases associated with CSD.

Item Type:	Thesis (Doctor of Philosophy)
Divisions:	Faculty of Health and Life Sciences > Faculty of Health and Life Sciences
Depositing User:	Symplectic Admin
Date Deposited:	05 Sep 2017 07:51
Last Modified:	19 Jan 2023 07:03
DOI:	10.17638/03007920
Supervisors:	Wang, M
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3007920