Focal ischemia induces expression of protease-activated receptor1 (PAR1) and PAR3 on microglia and enhances PAR4 labeling in the penumbra

doi:10.1016/j.brainres.2005.10.100

Brain Research

Volume 1070, Issue 1, 27 January 2006, Pages 232-241

https://doi.org/10.1016/j.brainres.2005.10.100 Get rights and content

Abstract

Thrombin significantly influences neurodegenerative processes after ischemia. The current literature suggests that the effects are mediated via protease-activated receptors 1, 3 and 4 (PAR1, 3, 4). Therefore, we investigated with immunohistochemical methods whether focal cerebral ischemia altered the expression of PARs in the rodent brain. For this purpose, we used the model of endothelin-induced occlusion of the middle cerebral artery and the model of transcranial permanent occlusion of the middle cerebral artery in mice. In contrast to the exclusively neuronal staining in the brain parenchyma of naïve animals, PAR1 and PAR3 occurred in addition on microglial cells in the penumbra after transient and after permanent focal ischemia. Although microglia activation could be detected for several weeks after the insult, PAR1 and PAR3 were traceable on microglia only 12 and 48 h after the insult, but not on day 7 post-ischemia. PAR4 was expressed, both in naïve and in ischemic animals, exclusively in neuronal cells. However, at the border zone and within the infarct area, enhanced immunohistochemical PAR4 signals were recognized. From our data, we conclude that PAR1 and PAR3 could be involved in thrombin-modulated initiation of post-ischemic inflammation and PAR4 may be associated with neuronal degeneration.

Introduction

The serine protease thrombin is not only involved in the development of ischemic damage by mediating clot formation, but it also affects brain tissue directly. For example, thrombin can induce neuronal cell damage after ischemic insults (Hua et al., 2003, Kitaoka et al., 2002, Kitaoka et al., 2003, Lee et al., 1996, Striggow et al., 2000, Xi et al., 1999). On the other hand, this serine protease can also mediate neuroprotection under certain circumstances: low doses or application as a preconditioning stimulus led to increased neuronal survival (Masada et al., 2000, Striggow et al., 2000).

Protective as well as toxic effects of thrombin have been linked to PARs (for an overview, see Rohatgi et al., 2004, Xi et al., 2003). Our laboratory and others have shown that all PARs (PAR1–4) are widely expressed in the naïve rodent brain (Striggow et al., 2001, Weinstein et al., 1995). PAR1, PAR3 and PAR4 can be activated by thrombin, whereas PAR2 is activated by trypsin and tryptase (Ishihara et al., 1997, Nystedt et al., 1994, Vu et al., 1991, Xu et al., 1998). PARs possess a 7-trans-membrane domain structure and are activated by proteolytic cleavage of the N-terminus resulting in the exposure of a tethered ligand, which ultimately binds to the second extracellular loop and causes receptor activation (for an overview, see Wang and Reiser, 2003). Immunohistochemical analysis of ex vivo slices suggested an expression of PARs exclusively on neuronal structures in the unlesioned rodent brain (Striggow et al., 2001). However, in vitro studies demonstrated that the other major cell types of the central nervous system also express PARs, and functional responses after PAR activation could be elicited in astrocytes and microglial cells (Möller et al., 2000, Suo et al., 2002, Suo et al., 2003, Ubl et al., 1998, Wang et al., 2002a, Wang et al., 2002b). As astrocyte as well as microglia activation is a significant part of the post-ischemic pathophysiology (Danton and Dietrich, 2003, Mergenthaler et al., 2004), it is of interest to investigate whether PARs may be associated with these post-lesional astrocyte and microglia reactions.

For this purpose, we performed endothelin-induced middle cerebral artery occlusion (eMCAO) in rats, which is a technique to induce transient focal ischemia. However, to also detect changes in receptor expression under non-reperfusion conditions, we additionally used the method of transcranial electrocoagulation of the middle cerebral artery (MCA) in mice. In both models, unilateral damage of the cerebral cortex is induced. Additionally, in the rat eMCAO model, striatal areas are affected.

In the current study, we investigated whether the expression pattern of the thrombin receptors PAR1, PAR3 and PAR4 changed after transient and permanent focal ischemia and whether such possible changes could provide a clue about the role of PARs in the pathophysiology of such insults.

Section snippets

Ischemia

To check whether the ischemic conditions in the current work indeed induced damage, in 2–3 rats per time-point, we quantified the infarct area after Nissl staining on systematic randomly sampled slices and calculated the infarct volume by linear trapezoidal extrapolation. The average infarct volume increased with survival time, being approximately 30 mm³ at 12 h post-ischemia, 43 mm³ at 48 h post-ischemia and 77 mm³ at 7 days of survival. Fig. 1A shows a representative example of the infarct

Naïve animals

Former results indicated that in the naïve rodent brain PAR1 is expressed in the neuronal membrane (Striggow et al., 2001). In the current study, immunohistochemistry demonstrated the PAR1 signal also as a thin granular ring in rat and mice brain tissue. These cells were identified as neurons by NeuN double labeling (Fig. 1C, rat brain slice). Such membrane-bound localization of PAR1 was also shown in other cell types (Chevessier et al., 2001). Additionally, we found a pronounced PAR1 signal in

eMCAO

Male Sprague–Dawley rats (250–300 g) were used. The animals were fed with laboratory chow (Altromin, Germany) and water ad libitum and maintained in a thermoregulated environment (17–21°C) during a 12/12 h light/dark cycle. The rats were anesthetized with sodium pentobarbitone (40–50 mg/kg) and placed in a stereotaxic frame. A scalp incision of approximately 1 cm in length was made from a point between the eyes, along the midline towards the back of the skull. The periosteum was then removed

Acknowledgments

This work was supported by Deutsche Forschungsgemeinschaft (DFG grant RE 847/3). The technical support from K. Krautwald is greatly acknowledged.

References (41)

U.K. Hanisch et al.
The microglia-activating potential of thrombin
J. Biol. Chem.
(2004)
V. Junker et al.
Stimulation of [beta]-adrenoceptor activates astrocytes and provides neuroprotection
Eur. J. Pharmacol.
(2002)
K.R. Lee et al.
Seizures induced by intracerebral injection of thrombin: a model of intracerebral hemorrhage
J. Neurosurg.
(1997)
T. Masada et al.
The effects of thrombin preconditioning on focal cerebral ischemia in rats
Brain Res.
(2000)
G. Stoll et al.
Inflammation and glial responses in ischemic brain lesions
Prog. Neurobiol.
(1998)
Z. Suo et al.
Persistent protease-activated receptor 4 signaling mediates thrombin-induced microglial activation
J. Biol. Chem.
(2003)
J.J. Ubl et al.
Co-existence of two types of [Ca²⁺]_I-inducing protease-activated receptors (PAR-1 and PAR-2) in rat astrocytes and C6 glioma cells
Neuroscience
(1998)
T.K. Vu et al.
Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation
Cell
(1991)
Y. Wang et al.
Expression of protease-activated receptors (PARs) in OLN-93 oligodendrolial cells and mechanism of PAR-1-induced calcium signalling
Neuroscience
(2004)
S. Balcaitis et al.
Expression of proteinase-activated receptors in mouse microglial cells
NeuroReport
(2003)

F. Chevessier et al.

Expression of the thrombin receptor (PAR-1) during rat skeletal muscle differentiation

J. Cell. Physiol.

(2001)

G.H. Danton et al.

Inflammatory mechanisms after ischemia and stroke

J. Neuropathol. Exp. Neurol.

(2003)

B. Gabryel et al.

Role of astrocytes in the pathogenesis of ischemic brain injury

Neurotox. Res.

(2001)

P. Grabham et al.

Thrombin receptor activation stimulates astrocyte proliferation and reversal of stellation by distinct pathways: involvement of tyrosine phosphorylation

J. Neurochem.

(1995)

J.R. Hamilton et al.

Increased expression of protease-activated receptor-2 (PAR2) and PAR4 in human coronary artery by inflammatory stimuli unveils endothelium-dependent relaxation to PAR2 and PAR4 agonists

Circ. Res.

(2001)

Y. Hua et al.

Thrombin exacerbates brain edema in focal cerebral ischemia

Acta Neurochir., Suppl.

(2003)

H. Ishihara et al.

Protease-activated receptor 3 is a second thrombin receptor in humans

Nature

(1997)

T. Kitaoka et al.

Delayed argatroban treatment reduces edema in a rat model of intracerebral hemorrhage

Stroke

(2002)

T. Kitaoka et al.

Effect of delayed argatroban treatment on intracerebral hemorrhage-induced edema in the rat

Acta Neurochir., Suppl.

(2003)

K.R. Lee et al.

Edema from intracerebral hemorrhage: the role of thrombin

J. Neurosurg.

(1996)

Cited by (39)

Protease-activated receptor 1 (PAR1) inhibits synaptic NMDARs in mouse nigral dopaminergic neurons
2020, Pharmacological Research
Citation Excerpt :
PAR1 function in normal brain has been less examined, but cumulative evidence supports its role in the regulation of synaptic transmission as well as in learning and memory processes and complex behaviors [24–27]. PAR1 is ubiquitously expressed in the brain, either in neurons or in astrocytes and microglia, although with strong differences between areas and cellular populations [4,28–30]. Substantia nigra pars compacta (SNpc) is among brain areas with highest PAR1 expression [31], with PAR1 localization demonstrated in dopamine (DA) neurons from rodent (rat and mouse) and human midbrain [22,31,32].
Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor (GPCR), whose activation requires a proteolytic cleavage in the extracellular domain exposing a tethered ligand, which binds to the same receptor thus stimulating Gα_q/11-, Gα_i/o- and Gα₁₂₋₁₃ proteins. PAR1, activated by serine proteases and matrix metalloproteases, plays multifaceted roles in neuroinflammation and neurodegeneration, in stroke, brain trauma, Alzheimer’s diseases, and Parkinson’s disease (PD).
Substantia nigra pars compacta (SNpc) is among areas with highest PAR1 expression, but current evidence on its roles herein is restricted to mechanisms controlling dopaminergic (DAergic) neurons survival, with controversial data showing PAR1 either fostering or counteracting degeneration in PD models. Since PAR1 functions on SNpc DAergic neurons activity are unknown, we investigated if PAR1 affects glutamatergic transmission in this neuronal population.
We analyzed PAR1’s effects on NMDARs and AMPARs by patch-clamp recordings from DAergic neurons from mouse midbrain slices. Then, we explored subunit composition of PAR1-sensitive NMDARs, with selective antagonists, and mechanisms underlying PAR1-induced NMDARs modulation, by quantifying NMDARs surface expression.
PAR1 activation inhibits synaptic NMDARs in SNpc DAergic neurons, without affecting AMPARs. PAR1-sensitive NMDARs contain GluN2B/GluN2D subunits. Moreover, PAR1-mediated NMDARs hypofunction is reliant on NMDARs internalization, as PAR1 stimulation increases NMDARs intracellular levels and pharmacological limitation of NMDARs endocytosis prevents PAR1-induced NMDARs inhibition.
We reveal that PAR1 regulates glutamatergic transmission in midbrain DAergic cells. This might have implications in brain’s DA-dependent functions and in neurological/psychiatric diseases linked to DAergic dysfunctions.
Effects of low-dose unfractionated heparin on early brain injury after subarachnoid hemorrhage in mice
2020, Neuroscience Letters
Sphingosine kinase (SphK) 1 has been reported as an important signaling node in anti-apoptotic signaling. Heparin is a pleiotropic drug that antagonizes many pathophysiological mechanisms. In this study, we evaluated if heparin prevents early brain injury (EBI) after subarachnoid hemorrhage (SAH) by anti-apoptotic mechanisms including SphK1.
SAH was induced by endovascular perforation in mice, which were randomly assigned to sham-operated (n = 23), SAH + vehicle (n = 36), SAH + 10U heparin pretreatment (n = 13), SAH + 30U heparin pretreatment (n = 15), SAH + 10U heparin posttreatment (n = 31), and SAH + 30U heparin posttreatment (n = 23). At 24 hours post-SAH, neurological scores, brain water content and Evans blue extravasation were evaluated. Also, the expression of SphK, phosphorylated Akt, and cleaved caspase-3 was determined by Western blotting, and cell death was examined by terminal deoxynucleotidyl transferase–mediated uridine 5’-triphosphate-biotin nick end-labeling staining.
Low-dose heparin posttreatment improved neurobehavioral function, brain edema, blood-brain barrier disruption and cell death in the cortex, associated with an increase in SphK1 and phosphorylated Akt, and a decrease in cleaved caspase-3. High-dose heparin had a tendency for increased SAH severity, which obscured the neuroprotective effects by heparin.
Low-dose heparin posttreatment may decrease the development of post-SAH EBI through anti-apoptotic mechanisms including sphingosine-related pathway activation.
PAR1 activation affects the neurotrophic properties of Schwann cells
2017, Molecular and Cellular Neuroscience
Citation Excerpt :
It has been proposed that PAR1 is a novel regulator of synaptic responses and a modulator of astrocyte-neuron interactions underlying memory formation (Almonte et al., 2013). Furthermore PAR1 expression, especially in glial cells, is modulated under various pathological conditions (for review, see Luo et al., 2007; Sokolova and Reiser, 2008) suggesting that PAR1 may be involved in glial response to brain damage (Striggow et al., 2001; Boven et al., 2003; Pompili et al., 2004, 2006, 2011; Ishida et al., 2006; Henrich-Noack et al., 2006). Under pathological conditions, PAR1 has been shown to mediate either neuronal death or survival (Ramachandran et al., 2012; Zhen et al., 2016).
Protease-activated receptor-1 (PAR1) is the prototypic member of a family of four G-protein-coupled receptors that signal in response to extracellular proteases. In the peripheral nervous system, the expression and/or the role of PARs are still poorly investigated. High PAR1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR1 mRNA increased in response to sciatic nerve transection. In the sciatic nerve, functional PAR1 receptor was reported at the level of non-compacted Schwann cell myelin microvilli of the nodes of Ranvier. Schwann cells are the principal population of glial cells of the peripheral nervous system which myelinate axons playing an important role during axonal regeneration and remyelination. The present study was undertaken in order to determine if the activation of PAR1 affects the neurotrophic properties of Schwann cells. Our results suggest that the stimulation of PAR1 could potentiate the Schwann cell ability to favour nerve regeneration. In fact, the conditioned medium obtained from Schwann cell cultures challenged with a specific PAR1 activating peptide (PAR1 AP) displays increased neuroprotective and neurotrophic properties with respect to the culture medium from untreated Schwann cells. The proteomic analysis of secreted proteins in untreated and PAR1 AP-treated Schwann cells allowed the identification of factors differentially expressed in the two samples. Some of them (such as macrophage migration inhibitory factor, matrix metalloproteinase-2, decorin, syndecan 4, complement C1r subcomponent, angiogenic factor with G patch and FHA domains 1) appear to be transcriptionally regulated after PAR1 AP treatment as shown by RT-PCR.
Astrocyte tissue factor controls CNS hemostasis and autoimmune inflammation
2016, Thrombosis Research
Tissue factor is the primary initiator of the coagulation cascade. Formation of the TF:FVIIa complex activates both FX and FIX, with subsequent thrombin generation, fibrin deposition and activation of platelets. In addition to playing important role in hemostasis and thrombosis, TF and downstream coagulation proteases can mediate intracellular signaling via activation of protease-activated receptors (PARs). Maintaining hemostasis in the brain is of utmost importance: bleeding or thrombosis within this organ can lead to significant morbidity and mortality. Both TF and PARs are widely expressed within the CNS, with TF expressed predominantly by astrocytes and PARs expressed in multiple cell types including astrocytes, neurons, microglia and oligodendrocytes [1, 2, 3, 4]. PARs activation can result in either neuronal survival or death and link the coagulation system with the inflammatory response. In this brief review we summarize the contribution of the coagulation system to brain hemostasis as well as to the pathophysiology of stroke and multiple sclerosis.
Substituted indoles as selective protease activated receptor 4 (PAR-4) antagonists: Discovery and SAR of ML354
2014, Bioorganic and Medicinal Chemistry Letters
Herein we report the discovery and SAR of an indole-based protease activated receptor-4 (PAR-4) antagonist scaffold derived from a similarity search of the Vanderbilt HTS collection, leading to MLPCN probe ML354 (VU0099704). Using a novel PAC-1 fluorescent α_IIbβ3 activation assay this probe molecule antagonist was found to have an IC₅₀ of 140 nM for PAR-4 with 71-fold selectivity versus PAR-1 (PAR-1IC₅₀ = 10 μM).
Serine proteases, serine protease inhibitors, and protease-activated receptors: Roles in synaptic function and behavior
2011, Brain Research
Citation Excerpt :
However, when coexpressed with PAR4, PAR3 forms heterodimers with PAR4 and seems to act as a cofactor for PAR4 activation and signaling through Gαq/11 coupling at low thrombin concentrations (as reviewed in Coughlin, 2000; Macfarlane et al., 2001; and Ramachandran and Hollenberg, 2008). Reminiscent of the studies of PAR1, PAR4 expression was highly upregulated in both neurons and glia in ischemia models (Henrich-Noack et al., 2006, 2010; Rohatgi et al., 2004a). Moreover, reduced infarct volumes were recently observed in PAR4 knockout mice in a transient ischemia/reperfusion model (Mao et al., 2010).
Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes—coagulation, inflammation, and digestion, for example—have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation.

View all citing articles on Scopus

View full text

Research ReportFocal ischemia induces expression of protease-activated receptor1 (PAR1) and PAR3 on microglia and enhances PAR4 labeling in the penumbra

Abstract

Introduction

Section snippets

Ischemia

Naïve animals

eMCAO

Acknowledgments

J. Biol. Chem.

Eur. J. Pharmacol.

J. Neurosurg.

Brain Res.

Prog. Neurobiol.

J. Biol. Chem.

Neuroscience

Cell

Neuroscience

Expression of proteinase-activated receptors in mouse microglial cells

NeuroReport

Expression of the thrombin receptor (PAR-1) during rat skeletal muscle differentiation

J. Cell. Physiol.

Inflammatory mechanisms after ischemia and stroke

J. Neuropathol. Exp. Neurol.

Role of astrocytes in the pathogenesis of ischemic brain injury

Neurotox. Res.

Thrombin receptor activation stimulates astrocyte proliferation and reversal of stellation by distinct pathways: involvement of tyrosine phosphorylation

J. Neurochem.

Increased expression of protease-activated receptor-2 (PAR2) and PAR4 in human coronary artery by inflammatory stimuli unveils endothelium-dependent relaxation to PAR2 and PAR4 agonists

Circ. Res.

Thrombin exacerbates brain edema in focal cerebral ischemia

Acta Neurochir., Suppl.

Protease-activated receptor 3 is a second thrombin receptor in humans

Nature

Delayed argatroban treatment reduces edema in a rat model of intracerebral hemorrhage

Stroke

Effect of delayed argatroban treatment on intracerebral hemorrhage-induced edema in the rat

Acta Neurochir., Suppl.

Edema from intracerebral hemorrhage: the role of thrombin

J. Neurosurg.

Research Report
Focal ischemia induces expression of protease-activated receptor1 (PAR1) and PAR3 on microglia and enhances PAR4 labeling in the penumbra