Protease-activated receptor 1 (PAR-1) affects glutamatergic transmission in mouse midbrain dopaminergic neurons

Protease-activated receptor 1 (PAR-1) belongs to a family of G protein-coupled receptors (GPCRs) composed of four members, PAR-1, PAR-2, PAR-3 and PAR-4, that play critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression (Ramachandran et al., 2012). PARs are characterized by a unique activation mechanism consisting in a proteolytic cleavage by endogenous proteases at specific sites within the extracellular amino-terminus and in the exposure of an amino-terminal domain, the so called “tethered ligand”, that binds to and activates the cleaved receptor (Nanevicz et al., 1995). Endogenous activators of PAR-1 are highly specific serine proteases, including thrombin, plasmin, activated protein C (APC), factor Xa (FXa), factor VIIa (FVIIa), and various matrix metalloproteases (MMPs), including MMP-1, MMP-2, MMP-3, MMP-8 MMP-9, and MMP-13. PAR-1 is coupled to different G proteins, Gαq11- , Gαi/o- and Gα12-13, thus possible modulating a complex network of intracellular signalling pathways. Furthermore, besides G protein-mediated pathways, PAR1 can activate β-arrestindependent signalling pathways or constitute homo- or heterodimers, by association with other PARs subtypes or different receptors. Such receptor crosstalk mechanisms significantly contribute to a high diversity of PAR signal transduction and receptortrafficking processes that turn out in different physiological effects (Soh et al., 2010). Whereas it was previously believed that PAR-1 could be activated only in pathological conditions, like traumatic brain injuries (TBI) or ischemia, allowing peripheral/circulating proteases to enter in the brain by a compromised blood-brain barrier (BBB), now it is well recognized that different serine proteases and MMPs are synthesized in the brain, thus allowing PAR-1 activation also in physiological conditions. Evidence has revealed the presence of different PARs in the central nervous systems. PAR-1 expression in the brain is widespread, although with strong differences between areas and cellular populations. Its highest expression has been reported in the midbrain dopaminergic (DAergic) nucleus, substantia nigra pars compacta (SNpc), with localization either in DAergic neurons and in astrocytes (Niclou, 1998). PAR-1 activation has been documented to play a role in cell proliferation, differentiation and migration during neural development. Interestingly, 2 recent evidence also suggests PAR1-dependent roles in the modulation of synaptic transmission and plasticity in the hippocampus (Lee et al., 200; Maggio et al., 2013). The expression of PARs in the brain is differentially upregulated or downregulated under pathological conditions, including in neurodegenerative disorders like Parkinson’s disease (PD), Alzheimer’s disease (AD), multiple sclerosis, stroke, and human immunodeficiency virus-associated dementia. Interestingly, PARs activation has been reported to mediate either cell death or cell survival in the brain, also depending on the amplitude and duration of agonist stimulation, brain areas/neuronal populations involved, and the neurotoxic insult. Despite being reported highly expressed in SNpc, and PAR-1activation in SNpc being linked to both neurodegeneration or protection of DAergic neurons in PD animal models (MPTP and 6-OHDA-induced) (Hamill et al., 2007; Cannon et al., 2005, 2006), and in spite of a well-documented expression, PAR-1’s functional roles in the regulation of midbrain DA neurons activity and neurotransmission are completely uncharacterized. Thus, the aim of this work is to investigate on a possible PAR-1 role in the modulation of the glutamatergic transmission in midbrain DA neurons. Performing electrophysiological patch clamp recordings on SNpc DAergic neurons from midbrain slices of young adult mice, we have analysed whether the activation of PAR-1, obtained by using the agonist TFLLR-NH2, affects synaptic and extrasynaptic NMDA receptor (NMDAR) and AMPA receptor (AMPAR) mediated currents. Excitatory postsynaptic currents (EPSCs) were evoked placing a parallel stimulating electrode rostral to the DA neurons, and delivering brief electrical pulses (100–200 μs duration, every 30 s) through a constant-current isolated stimulating unit. AMPARmediated EPSCs (AMPAR-EPSCs) were isolated by using a pharmacological cocktail composed of antagonists of the GABAA, GABAB, D2 and NMDAR; whilst NMDARmediated EPSCs (NMDAR-EPSCs) were isolated by using the same drugs except for the NMDAR blocker which was substituted by the AMPAR blocker. Activation of extrasynaptic NMDAR or AMPAR was achieved by bath applications of NMDA or AMPA, respectively. Results obtained support a modulatory role of PAR-1 in the regulation of the glutamatergic transmission in SNpc DAergic cells, with a selective involvement in the 3 long term regulation of NMDAR-mediated transmission, in terms of hypofunction. Moreover, in order to investigate the mechanism underlying the modulatory effect of PAR-1 activation on NMDAR-mediated transmission, we evaluated, by western blotting analysis of a Triton-insoluble post-synaptic fraction (TIF) and a surface expression assay, the possibility that alterations in the subunit composition of the NMDAR or modifications in surface expression and trafficking to the intracellular compartment may be the cause of the PAR1-dependent synaptic alterations observed in DAergic neurons. Thus, we demonstrate that the long lasting reduction of synaptic NMDAR-mediated currents, induced by PAR-1 activation, is caused by NMDAR internalization into the intracellular compartment. Overall our results disclose a novel functional role for PAR-1 in the modulation of glutamatergic transmission in SNpc DAergic neurons with potential implications in several physiopathological conditions involving the activation of midbrain DAergic system.