Neuroprotection induced by protease-activated receptor 2 activation is independent of Gq signalling

Protease-activated receptor 2 is proposed to be a novel target for several inflammation-related diseases but its role in the central nervous system remains unclear. Protease-activated receptor 2 activation is protective in cell death and excitotoxicity assays whereas examination into the role of protease-activated receptor 2 <i>in vivo</i> has been hindered due to the lack of suitable pharmacological tools. Recently, a small molecule protease-activated receptor 2 activator, AC264613 (AC), was reported to be a potent and selective protease-activated receptor 2 activator that crosses the blood-brain barrier. Furthermore, peptide mimetic molecules, for example GB88, were developed that were reported to act as protease-activated receptor 2 biased antagonists. Here, we examine their signalling pathways and neuroprotective properties in central nervous system preparations. AC induced significant increases in intracellular Ca²⁺ in both neurons and astrocytes of primary hippocampal cultures, whereas in contrast, GB88 induced a small but significant reduction in intracellular Ca²⁺ in both cell types. However, both AC and GB88 induced receptor internalisation when examined using fluorescently tagged protease-activated receptor 2. Both AC and GB88 did not induce neurotoxicity in organotypic hippocampal slice cultures when applied alone but reduced neurotoxicity when co-applied with kainate in excitotoxicity assays. Furthermore, both AC and GB88 reduced neurotoxicity when applied post kainate insult indicating they exhibit neuroprotective properties even after excitotoxicity is induced. These data indicate that protease-activated receptor 2 activation is neuroprotective but this is independent of Gq-induced Ca²⁺ activation. Given that AC crosses the blood-brain barrier, this highlights its use as a novel tool to examine the protective properties of protease-activated receptor 2 in <i>in vivo</i> models of central nervous system disorders.