Human Astrocytes Express Functional Protease-Activated Receptors that Increase Their Reactive Phenotype Under Certain Culture Conditions

Background: Protease-activated receptors are G protein-coupled receptors with a novel activation mechanism by which proteases cleave the N-terminus revealing a tethered ligand that binds to the second extracellular loop leading to receptor activation. We have previously shown that protease-activated receptor 2 (PAR2) activation impairs hippocampal synaptic transmission and is neuroprotective in in vitro excitotoxic assays, both of which are astrocyte-dependent. We have also shown that AC264613, a blood-brain barrier-permeable PAR2 activator, results in behavioural changes associated with depression-like behaviour and recently revealed that AC264613 reduces astrocyte reactivity and amyloid load in a mouse model of amyloid pathology. However, whether functional protease-activated receptors are present in human astrocytes and if they alter their reactivity state is unknown. Methods: Commercially available human astrocytes were cultured in serum and serum-free media, and the presence of functional PARs was investigated using standard Ca2+ imaging techniques. Immunocytochemistry was used to examine the consequence of PAR activation on their reactive phenotype. Results: We reveal that functional PAR1 and PAR2 are present in commercially available human astrocytes. Their activation increased glial fibrillary acidic protein (GFAP) expression only after short-term culture but did not affect complement component 3 (C3) or guanylate-binding protein 2 (Gbp2) expression under any conditions tested. In contrast, no change in reactivity was observed following exposure to inflammatory stimuli. Conclusion: Overall, these data highlight that human astrocytes express functional PARs, but their activation may result in different phenotypic changes from those seen in mouse astrocytes.