Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, C13
PACAP induces neuroprotection through the induction of activity-dependent signalling through the CREB coactivator CRTC1
P. Baxter1, M. Martel1, A. McMahon1, P. C. Kind1, G. E. Hardingham1
1. University of Edinburgh, Edinburgh, United Kingdom.
Pituitary adenylate cyclase-activating peptide (PACAP) is a known neuroprotective peptide widely assumed to exert its effect directly through the cAMP-protein kinase A (PKA_ pathway. Contrary to this, we find that PACAP-induced PKA signalling in cortical neurons is a poor direct activator of neuroprotective pathways, and actually exerts its effects indirectly through inducing synchronized action potential firing. To investigate the actions of PACAP, dissociated cortical cultures were prepared from E17 Sprague-Dawley rat embryos. Using Fluo-3 calcium imaging, PACAP was shown to induce a PKA-dependent increase in action potential (AP) firing and associated calcium transients (n = 6). These were shown to be essential for the anti-apoptotic actions of PACAP, as tetrodotoxin (TTX) blocked PACAP mediated protection against trophic deprivation (n = 3) and staurosporine (n = 4). Furthermore PACAP stimulation induced neuroprotective ERK 1/2 phosphorylation in a TTX sensitive manner (n = 4), and ERK pathway inhibition by PD98059 blocked PACAP induced protection. Through direct activation by PKA, PACAP caused CREB phosphorylation at serine-133, in the presence of TTX. Moreover, using a CRE-Firefly Luciferase-reporter showed that PACAP could induce some CRE-dependent gene expression in the absence of APs (n = 7), though this was significantly reduced. In the presence of TTX PACAP is unable to induce the activation and nuclear import of CREB co-activator CRTC1, which is dependent on firing activity-dependent calcineurin signalling (n = 6). Over-expression of CRTC1 is sufficient to rescue PACAP-induced CRE-mediated gene expression in the face of activity-blockade (n = 4). A Dominant-Negative form of CRTC1, and the inhibitory CREB isoform ICER both blocked long-term PACAP neuroprotection (n = 4). Thus, the enhancement of AP firing may play a significant role in the neuroprotective actions of PACAP and other adenylate cyclase-coupled ligands.
Where applicable, experiments conform with Society ethical requirements