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Title: Understanding the genetic and molecular mechanisms of neurological diseases associated with cortical spreading depression : REST and P2X7 receptor
Author: Ma, D.
ISNI:       0000 0004 7428 6953
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Cortical spreading depression (CSD) is a transient propagating wave of neuronal and glial excitation, followed by depression, which is implicated in migraine, traumatic brain injury (TBI) and stroke. Yet mechanisms of these diseases associated with CSD are not fully understood. The transcription factor, repressor element-1 silencing factor (REST), plays a critical role in the processes of central nervous system (CNS) and REST binding to MIR137 internal promoter can be reduced by multiple CSD events. The first objective of this project was to explore if REST signalling is involved in CSD. CSD was induced by KCl and in vivo electrophysiology was used for recording CSD. Polymerase chain reaction (PCR) was applied for measuring gene expression levels. The results showed that multiple CSD events did not alter gene expression of REST and its truncated isoform REST4 in cortices of rats, although a slight but insignificant up-regulation of REST4 was observed. Interestingly, there was a significant reduction in NR2A coding gene Grin2a in the ipsilateral cortices of rats at 24 hours after multiple CSD. Using qPCR and western blot methods, this project further examined whether multiple CSD events would induce the expression of a2, the major subunit of gamma-aminobutyric acid A (GABAA) receptor and its coding gene. The results showed that both the mRNA and protein level of GABAA a2 was not altered at 3 and 24 hours post multiple CSD, although an elevation trend of GABAA a2 protein level was observed. Collectively, due to small sample size, these data did not support the role of REST in CSD however, it would worth further investigation by increasing sample number to four for statistical significance. Given that N-methyl-D-aspartate (NMDA) receptor plays a key role in CSD genesis and propagation and NMDA-receptor mediated currents and glutamate release can be reduced by blockade of P2X7 receptor (P2X7R); whilst inhibition of P2X7R channel and pore formation reduces CSD susceptibility in rats; the 2nd part of my project aims to study the role of C-terminal domain of P2X7R in CSD using an anti-P2X7R antibody. CSD was induced by KCl and was recorded using intrinsic optical imaging in vitro and electrophysiology method in vivo. The results showed that A740003, which antagonizes the P2X7R channel and pore complex, suppressed CSD with a marked prolongation of CSD latency and a reduced magnitude in the mouse brain slice. Consistently, the anti-P2X7R antibody also suppressed the occurrence of CSD in the mouse brain slice and in rats. Further q-PCR analysis showed that the CSD-induced gene expression of key neuroinflammatory factors, tumor necrosis factor-a (TNF-a) and interleukin-1ß (IL-1ß) was not altered by the antibody. These data demonstrate that this anti-P2X7R antibody is capable of suppressing CSD and does not alter gene expression of TNF-a and IL-1ß immediately post CSD. It is necessary to clarify the role of C-terminal domain of P2X7R in CSD by expressing a C-terminal truncated variant of P2X7R in the P2rx7 gene deficient rats/mice in future. In summary, this study demonstrated that multiple CSD events does not alter REST gene expression but can induce a slight but insignificant up-regulation of REST4 in ipsilateral cerebral cortices of rats. Further work by increasing sample number is necessary to confirm REST4 implications to TBI and stroke. The fact that the anti-P2X7R antibody suppresses CSD suggests a possible role of P2X7R in CSD involving the C-terminal domain, however, further studies using C-terminal domain knockout animals will help to elucidate the contribution of this domain to CSD.
Supervisor: Wang, Minyan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral