Rink-Jan Lohman, a,b Terence J. O'Brien, b and Thomas M. Cocks a, ⁎ a Department of Pharmacology, The University of Melbourne, Grattan Street, Victoria, 3010, Australia b Department of Medicine, The University of Melbourne, Royal Melbourne Hospital, Parkville, Victoria, 3050, Australia Received 30 October 2007; revised 14 December 2007; accepted 16 December 2007 Available online 5 January 2008 Keywords: Protease-activated receptor-2 (PAR 2 ); SLIGRL; Protease; Trypsin; Brain; Tissue plasminogen activator (tPA); Seizure; Epileptogen- esis; Large dense core vesicles (LDCVs) Introduction There is increasing evidence that inflammatory processes play a role in epileptogenesis that occurs following acquired brain insult (Pawlak and Strickland, 2002, Tsirka, 2002, Vezzani, 2005a). The trypsin receptor, PAR 2 , is considered to be involved in pro- inflammatory events (Smith-Swintosky et al., 1997, Steinhoff et al., 2000, Macfarlane et al., 2001, Vergnolle et al., 2001), although it has been proposed to have cytoprotective roles in many organs, such as the pancreas, gut and airways (Cocks et al., 1999, Kawabata et al., 2005, Bunnett, 2006, Singh et al., 2007). In the brain, PAR 2 is highly expressed in limbic regions involved with memory and emotion such as the amygdala, hippocampus and cortex (Smith-Swintosky et al., 1997, Striggow et al., 2001, Bushell et al., 2006). The expression of PAR 2 has been shown to be up-regulated in these same regions after cerebral ischaemic injury (Striggow et al., 2001), experimental autoimmune encephalitis (Noorbakhsh et al., 2006), HIV infection and in response to pro- inflammatory cytokines such as TNFα (Noorbakhsh et al., 2005). PAR 2 knock-out mice exhibit a greater infarct volume compared to wild-types following ischaemic brain injury (Jin et al., 2005). Furthermore, wild-type mice treated with the synthetic PAR 2 peptide agonists (SLIGRL) are resistant to neuronal death related to HIV dementia, whereas PAR 2 knock-out mice have a more severe neurotoxicity in the same disease model (Noorbakhsh et al., 2005). Taken together, these findings support the proposition that, like in other tissue types such as the pancreas and airways (Cocks et al., 1999, Namkung et al., 2004, Sharma et al., 2005, Hirota et al., 2006, Singh et al., 2007), PAR 2 may have cytoprotective roles within the brain (Jin et al., 2005, Noorbakhsh et al., 2005, Gorbacheva et al., 2006, Bushell, 2007). Low levels of proteases including tissue plasminogen activator (tPA) (Pawlak and Strickland, 2002, Tsirka, 2002, Gorter et al., 2007), neuropsin (Tomimatsu et al., 2002), motopsin (Mitsui et al., 2007), trypsin (Koshikawa et al., 1998) and thrombin (Rohatgi et al., 2004) are normally present in limbic regions of the brain including the hippocampus and are associated with neuronal plasticity and matrix remodelling related to synaptogenesis, long-term potentia- tion (LTP), long-term depression (LTD) and memory (Hoffman et al., 1998, Calabresi et al., 2000, Tomimatsu et al., 2002, Tamura et al., 2006). The most studied of these enzymes, tPA, is involved in a wide range of proteolytic-dependent processes generally regarded as neuromodulatory (Wu et al., 2000, Pawlak et al., 2005). tPA converts plasminogen to the active protease plasmin that degrades extra- cellular matrix proteins such as laminin (Chen and Strickland, 1997) and potentiates NMDA receptor signalling, possibly via NR1- subunit cleavage, and mediates kainic acid-induced seizures, both seemingly independent of plasminogen activation (Nicole et al., 2001, Yepes et al., 2002). In hippocampal pyramidal neurons, increased levels of tPA, which is stored in large dense core vesicles www.elsevier.com/locate/ynbdi Neurobiology of Disease 30 (2008) 84 – 93 ⁎ Corresponding author. Fax: +61 3 8344 0241. E-mail address: thomasmc@unimelb.edu.au (T.M. Cocks). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.nbd.2007.12.010 Protease-activated receptor-2 regulates trypsin expression in the brain and protects against seizures and epileptogenesis Protease-activated receptor-2 (PAR 2 ), primarily involved in inflamma- tion, is highly expressed in limbic regions of the brain such as the hippocampus. Although extracellular proteolysis is involved in normal and stress-related neuronal plasticity associated with learning, memory and inflammatory disease states, little is known about the role of PAR 2 and its physiological agonist, trypsin, in the brain. We show immunohisto- chemically that trypsin co-localises with tissue plasminogen activator within granular-like structures in PAR 2 -positive pyramidal neurons of the rat hippocampus. Central administration of the PAR 2 peptide agonist, SLIGRL, inhibited electrical amygdala kindling-induced epileptogenesis and abolished kindling-induced over-expression of trypsin in the hippocampus. SLIGRL similarly attenuated kindling when administered subcutaneously. Non-enzymatic activation of neuronal PAR 2 using SLIGRL may thus activate feedback mechanisms to inhibit the over- production of trypsin and possibly other proteases during brain insults and thereby attenuate pathogenesis. Prophylactic systemic administration of non-proteolytic PAR 2 agonists may therefore represent a novel approach to protect against epileptogenic brain insults. © 2008 Published by Elsevier Inc.