Potential signalling pathways underlying corticotrophin-releasing hormone-mediated neuroprotection from excitotoxicity in rat hippocampus Caroline R. Elliott-Hunt, Jurate Kazlauskaite, Geraint J. C. Wilde, Dimitris K. Grammatopoulos and Edward W. Hillhouse Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, University of Warwick, Coventry, UK Abstract In several neurological disorders including cerebral ischaemia, glutamate has been implicated as a neurotoxic agent in the mechanisms leading to neuronal cell death. The role of cor- ticotrophin-releasing hormone (CRH), the 41-amino acid peptide, which activates the HPA axis in response to stressful stimuli, remains controversial. In this study, we report that CRH in low physiological concentrations (2 pM), prevented glutamate-induced neurotoxicity via receptor-mediated mechanisms when administered to organotypic hippocampal cultures both during and after the glutamate-induced insult. Detailed investigations on the mechanisms mediating this neuroprotective effect showed that activation of the adenylate cyclase pathway and induction of MAP kinase phosphoryla- tion mediate the CRH action. In addition we showed that CRH can inhibit the phosphorylation of JNK/SAPK by glutamate. Most importantly, we showed that CRH can afford neuropro- tection against neurotoxicity up to 12 h following the insult, suggesting that CRH is acting at a late stage in the neuronal death cycle, and this might be important in the development of novel neuroprotective agents in order to improve neuronal survival following the insult. Keywords: cAMP, corticotrophin-releasing hormone (CRH), glutamate, hippocampal, mitogen-activating protein kinase (MAPK), neuroprotection. J. Neurochem. (2002) 80, 416±425. Neurotransmitters such as glutamate have been implicated as potential neurotoxic agents in several neurological disorders including ischaemia, epilepsy and neurodegenerative diseases. The molecular mechanisms underlying glutamate toxicity in these disorders are not well understood, but it has been shown that accumulation of Ca 2+ induces neuronal death by both necrosis and apoptosis. In addition, NMDA receptor blockade can reduce infarction volume and neurological defects in permanent and reperfusion models of focal ischaemia (Steinberg et al. 1989); the therapeutic potential of NMDA receptor antagonists, however, is limited because of their adverse side-effects (Keyser et al. 1999). Corticotrophin-releasing hormone (CRH) is a 41-amino acid hypothalamic polypeptide, which regulates endocrine and behavioural responses to stressful stimuli through activation of the pituitary-adrenocortical system (Vale et al. 1981). The limbic system, in particular the amygdala and the hippocampus, is rich in neuronal populations that either synthesize CRH or express CRH receptors (De Souza et al. 1985; Avishai-Eliner et al. 1996). CRH is part of a family of peptides, which include the mammalian peptide urocortin. Both these peptides exert their biological actions via activation of a family of seven transmembrane domain spanning heterotrimeric G protein-coupled receptors. Mole- cular characterization of CRH receptors has identi®ed numerous receptor subtypes arising from two distinct genes. Several in vivo studies have implicated CRH as a mediator of brain cell death, an observation supported by experiments showing that CRH antagonists, such as astressin, D-Phe CRH (12±41) or a-helical CRH (9±41) can reduce neuronal damage (Lyons et al. 1991; Fox et al. 1993; Strijbos et al. 1994; Maecker et al. 1997; Roe et al. 1998). However, the role of Received May 30, 2001; revised manuscript received October 8, 2001; accepted October 31, 2001. Address correspondence and reprint requests to C. R. Elliott-Hunt, University Research Centre for Neuroendocrinology, Dorothy Crowfoot Hodgkin Laboratories, University of Bristol, B.R.I., Marlborough Street, Bristol, BS2 8HW, UK. E-mail: C.R.Elliott-Hunt@bristol.ac.uk Abbreviations used: CRH, corticotrophin-releasing hormone; DTT, dithiothreitol; ECL, enhanced chemiluminescence; MAPK, mitogen- activating protein kinase; MEK, MAP kinase kinase; PBS, phosphate- buffered saline; SSC, saline±sodium citrate buffer. Journal of Neurochemistry , 2002, 80, 416±425 416 Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry , 80, 416±425