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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: Glia. 2021 Apr 22;69(9):2111–2132. doi: 10.1002/glia.24012

The Thrombin Receptor Modulates Astroglia-Neuron Trophic Coupling and Neural Repair after Spinal Cord Injury

Ha Neui Kim 1,2, Erin M Triplet 1,2,3, Maja Radulovic 1,2, Samantha Bouchal 1, Laurel S Kleppe 1, Whitney L Simon 1, Hyesook Yoon 1,2, Isobel A Scarisbrick 1,2,3,*
PMCID: PMC8672305  NIHMSID: NIHMS1760708  PMID: 33887067

Abstract

Excessive activation of the thrombin receptor, Protease Activated Receptor 1 (PAR1) is implicated in diverse neuropathologies from neurodegenerative conditions to neurotrauma. PAR1 knockout mice show improved outcomes after experimental spinal cord injury (SCI), however, information regarding the underpinning cellular and molecular mechanisms is lacking. Here we demonstrate that genetic blockade of PAR1 in female mice results in improvements in sensorimotor co-ordination after thoracic spinal cord lateral compression injury. We document improved neuron preservation with increases in Synapsin-1 pre-synaptic proteins and GAP43, a growth cone marker, after a 30 d recovery period. These improvements were coupled to signs of enhanced myelin resiliency and repair, including increases in the number of mature oligodendrocytes, their progenitors and the abundance of myelin basic protein. These significant increases in substrates for neural recovery were accompanied by reduced astrocyte (Serp1) and microglial/monocyte (CD68 and iNOS) pro-inflammatory markers, with coordinate increases in astrocyte (S100A10 and Emp1) and microglial (Arg1) markers reflective of pro-repair activities. Complementary astrocyte-neuron co-culture bioassays suggest astrocytes with PAR1 loss-of-function promote both neuron survival and neurite outgrowth. Additionally, the pro-neurite outgrowth effects of switching off astrocyte PAR1 were blocked by inhibiting TrkB, the high affinity receptor for brain derived neurotrophic factor. Altogether, these studies demonstrate unique modulatory roles for PAR1 in regulating glial-neuron interactions, including the capacity for neurotrophic factor signaling, and underscore its position at neurobiological intersections critical for the response of the CNS to injury and the capacity for regenerative repair and restoration of function.

Keywords: Protease activated receptor 1, Spinal cord trauma, Myelin, Astroglia, Synapse, Growth Cone, Brain derived neurotrophic factor, TrkB

Introduction

Aberrant proteinase activity is an integral player driving neuropathogenesis, including inflammatory responses and astrogliosis, neuron and axonal injury, as well as demyelination (Burda et al. 2013; Gingrich and Traynelis 2000; Radulovic et al. 2016; Yoon et al. 2020; Yoon et al. 2017; Yoon et al. 2013) and thereby represents a potential therapeutic target. The cellular effects of select proteases are mediated by activation of one or more of the four-member protease activated receptor (PAR1-4) family, with increasing recognition of the abundance and regulatory actions of PAR1 in the CNS (Junge et al. 2004; Nicole et al. 2005; Radulovic et al. 2016; Vandell et al. 2008). PAR1 is a G-protein coupled receptor that is canonically activated by thrombin-mediated cleavage in its extracellular N-terminus to promote intracellular signaling (Coughlin 2005). While thrombin is expressed at low levels in the intact CNS it is increased with injury and disease, including in models of Alzheimer dementia (Citron et al. 2016), spinal cord injury (SCI) (Citron et al. 2000; Festoff et al. 2004; Radulovic et al. 2016; Yoon et al. 2013), experimental autoimmune encephalomyelitis (EAE) (Kim et al. 2015a), mild traumatic brain injury (mTBI) (Itsekson-Hayosh et al. 2015), amyotrophic lateral sclerosis (ALS) (Festoff et al. 2000; Shavit-Stein et al. 2020), epilepsy (Maggio et al. 2008), and stroke (Junge et al. 2003; Rajput et al. 2014). PAR1 is also activated, albeit with lower affinity, by select other proteinases deregulated in CNS pathology, including kallikrein 6 (Klk6) (Burda et al. 2013; Choi et al. 2018; Vandell et al. 2008; Yoon et al. 2018; Yoon et al. 2013; Yoon and Scarisbrick 2016), plasmin (Mannaioni et al. 2008), MMP-1 (Allen et al. 2016) and Granzyme B (Lee et al. 2017). Given this, we and others hypothesize that PAR1 represents a final common mediator that can be targeted to alleviate the pro-inflammatory and often neurotoxic impact of excess proteinase activity (Scarisbrick 2008).

We have been particularly interested in therapeutic inhibition of PAR1 activation and signaling after SCI, since deregulated proteinase activity is a well-studied component of the injury cascade occurring secondary to neurotrauma. Moreover, mice with constitutive PAR1 gene knockout show improved neurobehavioral recovery after contusion-compression SCI (Radulovic et al. 2016) and in an impactor model (Whetstone et al. 2017). This pattern of improved neuropathology in mice with constitutive PAR1 gene knockout also extends to models of CNS demyelination (Yoon et al. 2020), ischemic stroke (Junge et al. 2003; Rajput et al. 2014), the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease (Hamill et al. 2007), mild TBI (mTBI) (Itsekson-Hayosh et al. 2015), and the superoxide dismutase 1 (SOD1) model of ALS (Shavit-Stein et al. 2020). Collectively these reports highlight PAR1 as a fundamental mediator of neuropathology likely driven by activities across neural, glial and immune compartments.

In this study, we used a compression model of moderate thoracic SCI and neuron-astrocyte co-culture bioassays to test the hypothesis that genetic knockout of PAR1 reduces signs of neural injury at least in part through its actions in modulating astrocyte-neuron interactions. Specifically, we compared changes in neuronal, synaptic and myelin-associated markers at 30 d after SCI between wild type and PAR1 knockout mice and any associated changes in markers of astroglial or microglia/monocyte pro-injury versus pro-repair phenotype. Finally, we specifically investigated the ability of astrocytes with PAR1 loss-of-function to support neuron survival and neurite outgrowth in vitro. Our findings show for the first time that the improved outcomes in rodent models of SCI seen across several laboratories after genetic knockout of PAR1, include not only reductions in pro-inflammatory glial signatures, but also improvements in glial pro-repair signals capable of promoting improvements in neuron survival, serotonergic axon profiles, and markers of synapse and growth cone abundance. Findings in cell culture link improvements in neurite density to the ability of astrocyte PAR1 to regulate neurotrophic factor activity through a TrkB-dependent signaling mechanism. These findings are exciting because they suggest that genetic knockout of PAR1 function improves glial-neuronal interactions to limit neural injury and promote improvements in neural repair after neurotrauma.

Materials and Methods

Incomplete compression experimental SCI model

To investigate the contributions of PAR1 to the development of neuropathological outcomes after SCI, we induced SCI in 10 wk old adult female PAR1+/+ (C57BL6/J mice, #000664) or PAR1−/− (B6.129S4-F2rtm1Ajc/J, #002862) mice. PAR1 knockout mice have been backcrossed to C57BL6/J mice (#000664) for more than 30 generations (Burda et al. 2013; Choi et al. 2018; Radulovic et al. 2016; Yoon et al. 2020; Yoon et al. 2015). All mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were housed under environmentally-controlled conditions (22-24°C with a 12 h light/dark cycle). All experiments were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC) and completed in strict accordance with NIH Guidelines.

Prior to study initiation, age-matched wild type and PAR1−/− female mice were randomly assigned to SCI groups (receiving lateral compression SCI; n=8) or to non-surgical control groups (n=4) with mouse identity blinded for genotype prior to baseline behavioral outcome collection, subsequent surgery and downstream analyses. To calculate sample size, we used the standard deviation for our primary outcome measure, that is Basso Mouse Scale (BMS) scores observed in prior SCI studies (Basso et al. 1995; Radulovic et al. 2016). Using a Power analysis for ANOVA with a Power of 0.8 and α=0.05 (SigmaStat statistical program version 13.0 (Systat Software; San Jose, CA, USA) we estimated that 6 mice per group would be necessary to detect a 2-point difference in mean BMS scores between groups, which would be a highly meaningful difference in motor function. An additional 2 mice were added per group to account for mortality, which after post-operative losses resulted in 7 PAR1+/+ mice and 6 PAR1−/− mice used in the final analysis.

Lateral compression SCI was induced in mice under deep anesthesia with xylazine (10 mg/kg, Akorn, Inc., Lake Forest, IL) and ketamine (100 mg/kg, Fort Dodge, IA) delivered intraperitoneally (i.p.). Baytril (10 mg/kg, Bayer Health Care, Shawnee Mission, KS) was also administered i.p. prior to surgery to prevent infection. A laminectomy was performed leaving the dura intact and Dumont-type 2 forceps with a 0.25 mm spacer were applied to laterally compress the spinal cord at T8-9 for 14 s (Plemel et al. 2008). Immediately after surgery, 0.2 ml of saline was administered intraperitoneally to replace lost blood volume. Mice were allowed to recover on a heating pad with daily intraperitoneal injections of Buprenex (0.05 mg/kg, Reckitt Benckiser Healthcare, England) to minimize pain every 12 h for 72 h post-surgery following the initial pre-operative dose. Groups of wild type and PAR1−/− non-surgical control groups that did not receive a laminectomy were also prepared. Bladders of injured mice were manually voided twice daily until recovery of spontaneous bladder release. Food and water were provided ad libitum for the duration of the experiment.

Neurobehavioral outcome measure

Open field test.

The open field test was used to assess seven categories of locomotor recovery according to the BMS (Basso et al. 2006). Scores were collected for each mouse prior to SCI (0 days post injury), one day after injury and weekly thereafter until a 30 d after injury endpoint. Briefly, mice were placed in a round, open field enclosed with Plexiglas and their movements were video recorded for 3 mins. Two observers evaluated ankle movement, plantar placement, stepping, coordination, paw position, trunk stability and tail position, generating a BMS score (maximum of 9) (Radulovic et al. 2016).

Neuropathological outcomes

Immunohistochemistry.

Spinal cord histopathological outcomes were quantified at 30 days after injury with changes compared between PAR1+/+ and PAR1−/− (PAR1+/+ Ctrl n=4, PAR1+/+ SCI n=7 and PAR1−/− Ctrl n=3, PAR1−/− SCI n=6). At the 30 day endpoint, mice were deeply anesthetized with pentobarbital (150 mg/kg, i.p. Abbott Laboratories, Chicago, IL) and transcardially perfused with 4% paraformaldehyde (pH 7.2). Spinal cords were dissected and cut transversely into 2 mm segments. The spinal cord segment encompassing the lesion epicenter, in addition to 2 segments above (+4 mm) and 2 below (−4 mm), were embedded in a single paraffin block and cut transversely at 6 μm. Sections were stained with hematoxylin and eosin (H&E) to differentiate white and gray matter regions. Spinal cord sections were immunostained to quantify and characterize myelin and myelinating cells, astrocytes, microglia and neurons in addition to synaptic and growth cone markers (see Table 1). In most cases, sections were counterstained with methyl green to visualize nuclei. All secondary antibodies were from Jackson Laboratories and included goat anti-rat (112-066-072), donkey anti-mouse (715-066-151), and donkey anti-rabbit (711-065-152).

Table 1. Antibodies used for Immunohistochemistry and Immunofluorescence.

5-HT (5-Hydroxytryptamine (Serotonin)); Arg1 (Arginase 1); Beta III Tubulin (Tubulin beta 3); C3D (Complement Component C3d); CD163 (Cluster of Differentiation 163); CD68 (Cluster of Differentiation 68); DAPI (4′,6-diamidino-2-phenylindole); Emp1 (Epithelial Membrane Protein 1); GAP43 (Growth Associated Protein 43); GFAP (Glial fibrillary acidic protein); GST-3 (Glutathione S-Transferase); Iba-1 (Ionized calcium-Binding adapter molecule 1); iNOS (Inducible Nitric Oxide Synthase); MBP (Myelin Basic Protein); NeuN (Neuron-Specific Nuclear Protein); Olig2 (Oligodendrocyte transcription factor); PAR1 (Protease-activated Receptor 1); S100A10 (S100 calcium-binding protein A10); Serp1 (Serping1 (C1HN)); Synapsin-1 (Synapsin).

Primary antibody Source Company RRID number Dilution
5-HT Rabbit Sigma-Aldrich (S5545) AB_572263 1:4000
Arg1 Mouse Santa Cruz (SC-271430) AB_10648473 1:200
Beta III Tubulin Chicken Millipore (AB9354) AB_570918 1:500
C3D Goat R&D Systems (AF2655) AB_2066622 1:500
CD163 Mouse Abcam (ab111250) AB_10888308 1:100
CD68 Rabbit Abcam (ab125212) AB_10975465 1:1000
DAPI N/A ThermoFisher Scientific (D1306) AB_2629482 1:10000
Emp1 Rabbit Abcam (ab202975) - 1:200
GAP43 Rabbit Abcam (ab75810) AB_1310252 1:200
GFAP Chicken Abcam (ab4674) AB_304558 1:2000
GFAP Rabbit DAKO (Z0334) AB_10013382 1:5000
GFAP-Cy3 Mouse Sigma-Aldrich (C9205) AB_476889 1:200
GST-3 Rabbit Abcam (ab153949) AB_2877700 1:5000
Iba-1 Goat Abcam (ab48004) AB_870576 1:5000
iNOS Rat Thermo Fisher (14-5920-82) AB_2572890 1:200
MBP Rat Millipore (MAB386) AB_94975 1:750
NeuN Rabbit Abcam (ab177487) AB_2532109 1:2000
NeuN Mouse Millipore (MAB337) AB_2313673 1:2000
Olig2 Rabbit Millipore (ab9610) AB_10141047 1:500
Olig2 Rabbit Abcam (ab9610) AB_570666 1:500
PAR1 Mouse Santa Cruz (SC-13503) AB_2101175 1:100
S100A10 Rabbit Protein Tech (11250-1-AP) AB_2269906 1:7500
Serp1 Rabbit Santa Cruz (SC-377062) - 1:200
Synapsin-1 Rabbit Abcam (ab64581) AB_1281135 1:100

Stained tissue sections, including one from the SCI epicenter and two for each of the 2 mm blocks above and below were digitally captured (Olympus BX51 microscope and DP72 camera equipped with CellSens software 1.9, Olympus, Center Valley, PA), under constant illumination. Area measurements were made from H&E-stained sections. Gray and white matter regions were assessed individually and in combination (from either the whole spinal cord, the ventral horn or the ventrolateral white matter). Depending on the protein being analyzed, images were captured at 5x (GFAP, MBP), 20x (5-HT, GST-3, Iba-1, NeuN, Olig2, vGlut1), or 40x (GAP43, Synapsin-1). The entire spinal cord section, white matter region, and gray matter region were selected and/or separated using Photoshop (Adobe Photoshop CC, 2018; San Jose, CA). All density measurements were expressed per area of tissue quantified (whole cord, white matter, or gray matter) and then analyzed using KS-400 image analysis software (Carl Zeiss Vision, Hallbergmoos, Germany) (Scarisbrick et al. 1999). The number of oligodendrocytes and neurons were counted manually in both lateral ventral horns from 20x images. Motor neurons were defined by NeuN expression and having a size greater than 20 μm. Only cells with a visible methyl green-stained nucleus were counted and all counts were normalized to tissue area.

To confirm the presence or absence of PAR1 expression in neurons and glial subsets in the intact and injured spinal cord, sections parallel to those for neural antigen quantification were examined for RNA expression by in situ hybridization using RNAscope 2.5 HD Duplex reagents (#322430, Advanced Cell Diagnostics, Newark, CA). Probes specific for PAR1 (Mm-F2r-C1, 471081-C1), Aldh1l1 (Mm-Aldh1l1-C2, 405891-C2), Iba-1 (Mm-Aif1-C2, 319141-C2), NeuN (Mm-Rbfox3-C2, 313311-C2), and Olig2 (Mm-Olig2-C2, 447091-C2) were hybridized as previously detailed (Wang et al. 2012). The C1-tagged PAR1 probe was visualized using horseradish peroxidase-based green chromogenic development and the C2-tagged Aldh1l1, Iba-1, NeuN or Olig2 probes using alkaline phosphatase-based fast red color development. Sections were counterstained with hematoxylin and cover slipped with Vectamount (H5000, Vector Labs, Burlingame, CA). Stained tissue sections were captured digitally (Olympus BX51 microscope and DP72 camera equipped with CellSens software 1.9) at 100x under oil immersion.

Immunofluorescence.

Immunofluorescence outcomes were quantified to evaluate the expression of PAR1 in association with four types of CNS resident cells (astrocytes, microglia, neurons, or myelinating cells) in the intact or injured spinal cord. The spinal cord sections were stained by immunofluorescence with primary antibodies (detailed in Table 1) and secondary antibodies (all from Jackson Laboratories: donkey anti-mouse AF488 (715-546-150), donkey anti-goat Cy3 (750-166-147), donkey anti-goat AF647 (Cy5) (705-606-147), donkey anti-rabbit AF488 (711-166-152), donkey anti-rabbit Cy3 (711-166-152), goat anti-rat AF647 (Cy5) (112-606-143) and donkey anti-chicken AF67 (Cy5) (703-606-155)). All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and were cover slipped with Fluoromount-G medium (00-4958-02, ThermoFisher Scientific). Depending on the antibody being considered, images were taken 20x (C3D, CD163, CD68, GFAP, Iba-1, S100A10) using an Olympus BX51 microscope (Olympus), or 40x and 100x (GFAP, Iba-1, NeuN, Olig2, PAR1) using an inverted confocal microscope (LSM780, Carl Zeiss Microscopy, LLC., Thornwood, NY).

Unbiased stereology for spinal cord volume estimation.

Stereological analyses of myelin basic protein (MBP, Millipore, MABP 386) volume was performed using StereoInvestigator software (Microbrightfield, Inc). A total of six, 6 μm sections spaced 2 mm apart and spanning the lesion epicenter as well as spinal segments above and below were stained for MBP to identify areas of myelin loss. Entire sections from each level of injury were imaged digitally at 5X magnification on an Olympus BX51 microscope (Olympus) under constant illumination and the area of gray and white matter calculated. A Cavalieri probe (grid size, 400 x 400 μm) was arranged in a grid pattern, spaced 100 μm apart over the spinal cord gray and white matter and areas of disrupted white matter that fell within the region of interest were counted. In this analysis, the coefficient of error (CE) was <0.03. The CE is the standard error of the mean of repeated estimates divided by the mean, with lower values ensuring that adequate sample sites and markers of myelin integrity were acquired (Schmitz and Hof 2005).

Cell Culture Studies

Astrocyte-neuron co-cultures.

To determine the potential effect of astrocytic PAR1 deletion on cortical neurons in vitro, primary astrocytes were purified from mixed glial cultures obtained from the cortices of postnatal day 1-3 male or female PAR1+/+ or PAR1−/− pups, with sexes combined for study. Mixed glial preps were grown in media containing DMEM, 1 mM sodium pyruvate, 20 mM HEPES, 5 μg/ml insulin, 100 U/ml penicillin and 100 μg/ml streptomycin (P/S), and 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA). All reagents except FBS were purchased from Gibco (Invitrogen, Carlsbad, CA). After 10-12 d in vitro (DIV), mixed glial cultures were shaken overnight (225 RPM) to remove microglia and oligodendrocytes, as previously detailed (Burda et al. 2013; Radulovic et al. 2016). Astrocytes were trypsinized (0.025%) and plated at a density of 30,000 cells/well on poly-L-lysine-(Sigma-Aldrich) coated cover glass in 24 well plates and grown for 2 to 3 d in mixed glial media. 24 h before cortical neurons were added to the culture, media was changed to neuron plating media, consisting of Neurobasal A media supplemented with 2% B27, 500 μM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% FBS, 1% N2 from Gibco (Invitrogen). Primary cortical neurons were isolated from embryonic day 15 C57BL6/J mice (Radulovic et al. 2013) and plated at a density of 100,000 cells per well in neuron plating media on a bed of PAR1+/+ or PAR1−/− astrocytes. After 24 h, neuron plating media was replaced with serum-free Neurobasal A media containing 2% B27, 500 μM L-glutamine, 100 U/ml penicillin and treated with or without 10 μM of the TrkB antagonist, ANA12 (Fisher Science, Waltham, MA) for 24 or 72 h.

Astrocyte-neuron co-cultures were fixed with cold 2% PFA for a minimum of 15 min and then blocked with 20% NGS and 0.25% Triton-x for 20 min at RT. Cells were incubated with primary antibodies chicken anti-Tubulin (Tuj1) and mouse anti-GFAP-Cy3 followed by secondary antibody: Alexa Fluor 647 donkey anti-chicken (Jackson ImmunoResearch Laboratories, 703-605-155, 1:100). Cells were then incubated with DAPI (D1306; Thermo Fisher Scientific; Waltham, MA, USA) prior to being slide-mounted with Fluoromount-G® mounting media (0100-01; SouthernBiotech; Birmingham, AL) and imaged at 20x using an Olympus BX51 microscope (Olympus). Images were taken randomly from 5 different fields for each cover glass, with at least 3 separate replicates per experiment. The area of Tuj1 or GFAP immunofluorescence was quantified using ImageJ software (NIH) and expressed as mean Tuj1+ or GFAP+ area (μm2) or area per DAPI positive neuron or astrocyte, respectively. Results provided are representative of two independent experiments.

Statistical Analysis

Statistical analysis was carried out using the SigmaStat statistical program version 13.0 (Systat Software; San Jose, CA, USA). All data is represented as mean ± SEM. To evaluate the impact of PAR1 deletion on sensorimotor outcomes after injury over time, BMS scores were analyzed using a Two-Way Repeated Measures ANOVA with fixed effects (Factor A: day and Factor B: genotype) and the Student Newman-Keuls (NK) post-hoc test. Quantitative measurements of neuropathology were made using Two-Way ANOVA (Factor A: genotype and Factor B: injury region). The histological, RNA and cell culture analyses across multiple groups were determined using a One-way ANOVA. All data sets were tested for normality of sample distribution with a Shapiro-Wilk test and for equal variance with Brown-Forsythe test. Data which passed both (P > 0.05) were further assessed with the NK post-hoc test. In instances where the Shapiro-Wilk failed, multiple groups were determined using a non-parametric ANOVA on RANKS with Dunn’s test. Statistical significance was set at P < 0.05. All degrees of freedom, t values and F statistics from these analyses are provided without Bonferroni correction (Armstrong 2014). Associations between variables was tested by Pearson’s correlation coefficient (R), with significance set at p < 0.05. Specific tests used are indicated in figure legends.

Quantification of results in all histological and cell culture experiments was performed without knowledge of the treatment group.

Results

PAR1 knockout mice show improved sensorimotor outcomes after SCI.

PAR1 knockout mice exhibit improved locomotor outcomes in the BMS open field test after moderate spinal cord compression injury in relation to their wild type comparators (Fig. 1). PAR1 knockout mice showed accelerated improvements in sensorimotor outcomes by 3 d after SCI and at the 7, 14, 21 and 30 d time points examined (Two-Way Repeated Measures ANOVA, NK, P < 0.001, day F(6,77) = 272.942, genotype F(1,77) = 43.68, PAR1+/+ n=7, PAR1−/− n=8) (Fig. 1B). At 30 d after injury endpoint, the BMS scores of PAR1−/− mice averaged 1.7 points higher than PAR1+/+, reflecting improvements in coordinated stepping and parallel paw position. Despite these improvements, no genotype related differences in the volume of spared spinal cord gray matter or white matter were observed with the Cavalieri estimator probe in the injury Epicenter, Above or Below (Fig. 1DE). These sensorimotor improvements after SCI in a model elicited by lateral compression of the spinal cord using modified forceps with a 0.25 mm gap fully replicate improved outcomes in mice with constitutive PAR1 knockout in prior studies, including in a model of severe contusion compression SCI model mediated by a modified aneurysm clip with an 8 g closing force (Fejota) (Joshi and Fehlings 2002) or after weight drop contusion injury (Whetstone et al. 2017).

Figure 1. PAR1 knockout mice show improved locomotor outcomes after spinal cord injury.

Figure 1.

(A) Cartoon shows lateral compression SCI model induced by application of forceps with a 0.25 mm gap. (B) Recovery of sensorimotor function assessed by the Basso Mouse Scale (BMS) score was improved in PAR1−/− mice compared to PAR1+/+ mice (PAR1+/+, n=7 and PAR1−/−, n=6 at 30 d post injury, Two-Way Repeated Measures ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, NK). (C) Representative images of myelin basic protein (MBP) immunostained spinal cord sections taken from 2 mm blocks encompassing the injury epicenter (0), and 4 mm above and below. (D and E) The Cavalieri estimator probe showed expected reductions in spared gray matter (GM) and white matter (WM) volume at 30 d post injury, but no differences were detected between genotypes (**P < 0.01 and ***P < 0.001, non-surgical control groups, Student’s t-test; SCI groups, Two-Way ANOVA, NK). (C to E) PAR1+/+ Ctrl, n=4; PAR1+/+ SCI, n=7; PAR1−/− Ctrl, n=3; PAR1−/− SCI, n=6.

Signs of neuronal sparing and regenerative responses are increased after SCI in PAR1 knockout mice.

To determine the neural substrate of improved sensorimotor recovery in PAR1 knockout mice after SCI, we quantified the number of neuronal nuclear antigen (NeuN) positive neurons in the spinal cord ventral horn, in addition to the density of serotonergic axons, the synaptic marker Synapsin-1 and a marker of growth cones, GAP43 (Fig. 2). Comparisons were made between PAR1 knockout and wild type mice across the injury Epicenter, Above and Below at 30 d after injury and in age-matched uninjured controls. The number of neurons positive for NeuN in the spinal cord ventral horn was 3.8-fold higher in the injury Epicenter of PAR1−/− compared to PAR1+/+ at 30 d after injury (Fig. 2AB, Two-Way ANOVA, NK, P = 0.008, genotype F(1,33) = 3.7, injury region F(2,33) = 21.9, PAR1+/+ n=7, PAR1−/− n=6).

Figure 2. PAR1 knockout mice show signs of neural resiliency in gray matter after spinal cord injury.

Figure 2.

(A and B) There was a greater preservation of the number of NeuN+ neurons in the SCI epicenter ventral horn of PAR1−/− (P < 0.01). (C and D) The density of serotonergic 5-HT+ axons was greater in PAR1−/− compared to a PAR1+/+ prior to SCI (P < 0.04). (E and F) Synapsin-1 immunoreactivity was greater prior to SCI in PAR1 knockouts with improved preservation in the injury epicenter after SCI, relative to PAR1+/+ mice (P < 0.05). (G and H) GAP43-immunoreactivity was higher at 30 after SCI in the injury epicenter of PAR1−/− relative to PAR1+/+ mice (P < 0.001). The data are presented as means ± SEM. (PAR1+/+ uninjured control (C), n=4, PAR1+/+ SCI, n=7 per group and PAR1−/− uninjured control (C), n=3, PAR1−/− SCI, n=6 per group, except Synapsin 1 n=5, *P < 0.05, **P < 0.01, ***P < 0.001, non-surgical control groups, Student’s t-test; SCI groups, Two-Way ANOVA, NK). (+4 (above), 0 (epicenter), −4 (below), represent mm from the injury epicenter. Scale bar = 200 μm (A) and 40 μm (C, E and G).

Serotonergic (5HT+) raphespinal axons were 2.2-fold higher in the ventral horn of PAR1−/− mice prior to SCI (Fig. 2CD, Student’s t-test, P < 0.05, PAR1+/+ n=4, PAR1−/− n=3), but did not differ at 30 d post injury between genotypes. The area of Synapsin-1 immunoreactivity, a neuronal phosphoprotein coating synaptic vesicles and used as a pre-synaptic marker, was 1.2-fold higher in the ventral horn of PAR1−/− compared to wild type mice prior to SCI (Student’s t-test, P < 0.05, PAR1+/+ n=4, PAR1−/− n=3)) and 2.5-fold higher in the injury Epicenter 30 d after injury (Two-Way ANOVA, NK, P = 0.003, genotype F(1,30) = 2.708, injury region F(2,30) = 17.87, PAR1+/+ n=7, PAR1−/− n=5) (Fig. 2EF). The density of GAP43, a marker of neuronal growth cones, was 2.9-fold higher in the injury Epicenter of PAR1−/− compared to PAR1+/+ at 30 d after injury (Fig. 2GH, Two-Way ANOVA, NK, P = 0.001, genotype F(1,30) = 7.6, injury region F(2,30) = 3.21, PAR1+/+ n=7, PAR1−/− n=6). These findings suggest that constitutive PAR1 loss-of-function confers neuroprotective benefits to neurons and pre-synaptic profiles after SCI and enhances signs of neural repair.

The abundance of myelin and myelin-producing cells are improved after SCI in PAR1 knockout mice.

PAR1 knockout mice show accelerated myelin development and improved remyelination in models of demyelinating disease (Yoon et al. 2020; Yoon et al. 2015). As improvements in myelinating oligodendroglia after SCI can contribute to improved repair (Pukos et al. 2019), we quantified changes in the number of Olig2+ and GST-3+ oligodendrocytes, in addition to the density of MBP (Fig. 3). The number of Olig2+ cells (a transcription factor highly expressed by oligodendrocyte progenitors and young oligodendrocytes) was elevated at 30 d after SCI in the injury epicenter of PAR1−/− mice, with the most significant increases (1.8-fold) occurring in gray matter (Fig. 3D, Two-Way ANOVA, NK, P = 0.017, genotype F(1,32) = 1.046, injury region F(2,32) = 16.28, PAR1+/+ n=7, PAR1−/− n=6). The number of oligodendrocytes positive for GST-3, a marker of mature oligodendroglia was also significantly elevated in PAR1−/− compared to PAR1+/+ after SCI, but in this case the elevations were restricted to white matter (1.3-fold, Two-Way ANOVA, NK, P = 0.015, genotype F(1,33) = 3.4, injury region F(2,33) = 3.49, PAR1+/+ n=7, PAR1−/− n=6) (Fig. 3H). The density of MBP, a major myelin protein involved in myelin formation and stabilization, was increased in the injury epicenter of PAR1−/− compared to PAR1+/+ mice at 30 d after injury, with 1.6-fold increases in gray matter (Two-Way ANOVA, NK, P < 0.036, genotype F(1,33) = 0.76, injury region F(2,33) = 6.66, PAR1+/+ n=7, PAR1−/− n=6) and 1.5-fold increases in white matter (Two-Way ANOVA, NK, P < 0.003, genotype F(1,33) = 1.9, injury region F(2,33) = 23.91, PAR1+/+ n=7, PAR1−/− n=6) (Fig. 3KL). These improvements in oligodendrocyte progenitors, mature myelinating oligodendroglia and corresponding increases in MBP across the gray and white matter are positioned to contribute to the improvements in sensorimotor recovery observed after SCI in mice with constitutive PAR1 knockout.

Figure 3. PAR1 knockout mice show signs of oligodendrocyte preservation after spinal cord injury.

Figure 3.

(A-B) Representative images show immunoreactivity for immature (Olig2+) and mature (GST-3+) oligodendrocytes at 30 days after SCI within the spinal cord gray matter (outlined) of the PAR1+/+ and PAR1−/− mice. (C-E) The number of Olig2+ oligodendrocyte progenitor cells was greater in the gray matter (GM) of the injury epicenter of PAR1−/− compared to wild type mice at 30 days after injury. (F-H) GST-3+ mature oligodendrocytes were greater in the white matter (WM) of the injury epicenter of PAR1−/− at 30 days after injury. (I-L) The density of MBP immunoreactivity was greater in the epicenter of PAR1−/− compared to WT mice after SCI across both gray and white matter (white matter pictured in I). The data are presented as means ± SEM. (PAR1+/+ non-surgical control (C), n=4, PAR1+/+ SCI, n=7 per group and PAR1−/− non-surgical control (C), n=3, PAR1−/− SCI, n=6 per group, *P < 0.05, **P < 0.01, ***P < 0.001, Uninjured control groups, Student’s t-test; SCI groups, Two-way ANOVA, NK). (+4 (above), 0 (epicenter), −4 (below), represent mm from the injury epicenter. Scale bar = 100 μm.

The potential association between increases in markers of myelin and coordinate improvements in markers of neural integrity after SCI in PAR1 knockouts was examined using the Pearson correlation coefficient. In PAR1−/−, the area of spinal cord white matter or combined white and gray matter positive for MBP was positively correlated with counts of NeuN positive neurons (GM+WM: R = 0.59, P = 0.03; WM: R = 0.69, P = 0.009). Also, the number of Olig2+ oligodendrocytes across the spinal cord white and gray matter or only in the gray matter, and the number of GST-3+ oligodendrocytes in the white matter, was also positively correlated with counts of ventral horn NeuN+ neurons in PAR1 knockouts (Olig2, GM+WM: R = 0.60, P = 0.03; GM: R = 0.69, P = 0.009, GST-3, WM: R = 0.61, P = 0.02, PAR1+/+ n=7, PAR1−/− n=6). There was also a trend towards a correlation between the Synapsin-1+ area in gray and white matter and MBP area. MBP area was not found to be correlated with BMS scores.

PAR1 gene knockout skews astroglial reactivity after SCI towards a pro-repair phenotype.

Since the level of PAR1 activation can regulate astrogliosis, a key cellular contributor to the neural injury and repair microenvironment after SCI, we applied immunofluorescence approaches to quantify astroglial reactivity. While astrocytes display a continuum of reactivity depending on the type of injury and range of markers used, here we examined GFAP as a general marker of astroglial reactivity and C3D and Serp1 or S100A10 and Emp1, as markers of a pro-injury or pro-repair phenotypes, respectively (Fig. 4, 5, S1) (Liddelow et al. 2017). The density of GFAP in white matter was 1.3-fold higher in spinal segments just Above the injury Epicenter in PAR1−/− compared to PAR1+/+ mice at 30 d after SCI (Two-Way ANOVA, P = 0.022, genotype F(1,33) = 14.07, injury region F(2,33) = 2.77). In PAR1 knockouts, the level of Serp1 was reduced in the injury Epicenter gray and white matter, while the level of Emp1 in white matter was elevated across all regions of injury examined at 30 days after SCI (Two-Way ANOVA, NK, P = 0.02, genotype F(1,33) = 14.1, injury region F(2,33) = 2.8). The ratio between the densities of S100A10 to GFAP was 1.8-fold higher in spinal cord white matter at the injury Epicenter in PAR1−/− compared to wild types at 30 d after injury (Two-way ANOVA, NK, P=0.006, genotype F(1, 31) = 0.09, injury region F(2,31) = 1.5, PAR1+/+ n=7, PAR1−/− n=6). These findings suggest that in the moderate compression injury model used here, reducing PAR1 signaling increases the area of GFAP+ astroglia which express lower levels of the pro-inflammatory marker Serp1 and increased levels of Emp1 and S100A10+ markers of a pro-repair phenotype at 30 d after SCI.

Figure 4. Knockout of PAR1 increases pro-repair astrocyte signatures after spinal cord injury.

Figure 4.

(A) Representative immunofluorescence images show triple labeling of astrocytes, including reactive astrocytes (GFAP), pro-inflammatory astrocytes (C3D), and pro-repair astrocytes (S100A10) in the injury epicenter (0 mm) or in spinal cord above (+2 mm) or below (−2 mm), across PAR1+/+ and PAR1−/− mice at 30 days after injury. Dashed line delimits gray and white matter and boxed area on cartoon shows regions quantified. (B-C) GFAP immunoreactivity was higher in white matter (WM) of above the injury epicenter (+2) in PAR1−/− mice compared to PAR1+/+. (D-F) C3D immunoreactivity did not differ significantly between PAR1−/− and PAR1+/+ mice. (G-I) The abundance of S100A10/GFAP immunoreactivity was greater in WM at the injury Epicenter of PAR1−/− compared to PAR+/+ mice. The data are presented as means ± SEM. (PAR1+/+ SCI, n=7 per group and PAR1−/− SCI, n=6 per group, *P < 0.05, **P < 0.01, Two-Way ANOVA, NK). DAPI staining to visualize all cells is also shown. Scale bar = 100 μm.

Figure 5. Knockout of PAR1 increases pro-repair astrocyte signatures after spinal cord injury.

Figure 5.

(A) Representative immunofluorescence images show triple labeling of astrocytes, including reactive astrocytes (GFAP), pro-inflammatory astrocytes (Serp1), and pro-repair astrocytes (Emp1) in the injury epicenter (0 mm) or in spinal cord above (+2 mm) or below (−2 mm), across PAR1+/+ and PAR1−/− mice at 30 days after injury. Dashed line delimits gray and white matter and boxed area on cartoon shows regions quantified. Quantification of GFAP immunofluorescence is shown in Fig. 4 (BC). (B-D) Serp1 immunoreactivity was reduced in gray (GM) and white matter (WM) of the injury epicenter (0) in PAR1−/− mice compared to PAR1+/+. (E-G) Emp1 immunoreactivity was higher in white matter (WM) in the injury epicenter (0), above (+2) and below (−2) in PAR1−/− mice. The data are presented as means ± SEM. (PAR1+/+ SCI, n=7 per group and PAR1−/− SCI, n=6 per group, *P < 0.05, Two-Way ANOVA, NK). DAPI staining to visualize all cells is also shown. Scale bar = 100 μm.

PAR1 gene knockout attenuates microglia/monocyte pro-inflammatory responses after SCI.

Microglia and bone marrow derived monocytes are well studied regulators of the response of the spinal cord to injury, including astroglial reactivity (Liddelow et al. 2017). To quantify the regulatory impact of PAR1 knockout on microglial/monocyte reactivity we quantified immunofluorescence for Iba-1. In parallel, we co-localized Iba-1 with CD68 or iNOS, pro-inflammatory markers, and with CD163 or Arg1 markers of a pro-repair phenotype (Fig. 6). In PAR1 knockouts, the density of Iba-1+ microglia was reduced across both spinal cord white and gray matter at all levels of injury examined at 30 d after SCI (Two-Way ANOVA, NK, P < 0.001, genotype F(1,33) = 23.37, injury region F(2,33) = 16.23). The density of CD68 was 7.9-fold reduced in the gray matter (Two-Way ANOVA, NK, P < 0.001, genotype F(1,33) = 60.1, injury region F(2,33) = 68.45 ) and 1.5-fold reduced in the spinal cord white matter (Two-Way ANOVA, NK, P = 0.07, genotype F(1,33) = 2.4, injury region F(2,33) = 9.83). In parallel, the density of iNOS was 3-fold reduced PAR1+/+ n=7, PAR1−/− n=6) of PAR1−/− compared to PAR1+/+ mice at 30 d after SCI, while levels of CD163 were unchanged, suggesting that the reductions in microglial immunostaining with PAR1 knockout reflect mainly reduction of the pro-inflammatory phenotype. These findings support the hypothesis that constitutive knockout of PAR1 reduces pro-inflammatory microglial/monocyte reactivity after SCI.

Figure 6. Knockout of PAR1 reduces pro-inflammatory microglial signatures after spinal cord injury.

Figure 6.

(A) Representative immunofluorescence images show triple labeling for a pan-microglial/monocyte marker (Iba-1) along with a marker of a pro-inflammatory (CD68) or pro-repair (CD163) phenotype, in the injury epicenter (0 mm) or in spinal cord above (+2 mm) or below (−2 mm), across PAR1+/+ and PAR1−/− mice at 30 days after injury. Dashed line delimits gray and white matter and boxed area on cartoon shows regions quantified. (B-C) Iba-1 immunoreactivity was reduced in the gray matter (GM) and white matter (WM) across all regions of injury in PAR1−/− compared to PAR1+/+ mice. (D-F) CD68 immunoreactivity was lower in GM of the injury epicenter and in white matter (WM) Below in PAR1−/− compared to PAR+/+ mice. (G-I) CD163 immunoreactivity did not differ across genotypes. The data are presented as means ± SEM. (PAR1+/+ SCI, n=7 per group and PAR1−/− SCI, n=6 per group, *P < 0.05, ***P < 0.001, Two-Way ANOVA, NK). DAPI staining to visualize all cells is also shown. Scale bar = 100 μm.

PAR1 is expressed across neuronal and glial compartments and dynamically regulated by traumatic injury.

PAR1 is known to be expressed at relatively high levels in the adult CNS (Vandell et al. 2008), but its neuronal and glial expression patterns in the spinal cord prior to and after injury are not well documented. To gain insights into the potential cellular mechanisms by which genetic knockout of PAR1 may improve neurobehavioral outcomes after SCI, we used immunofluorescence (Fig. S2) and in situ hybridization multiplex (Fig. S3) approaches to visualize PAR1 across the neuron and glial compartments before and at 30 d after SCI. In each case, PAR1 expression was seen in association with NeuN+ spinal cord neurons, Olig2+ oligodendrocytes, GFAP+ astrocytes and Iba1+ microglia. These data complement prior studies showing elevations in PAR1 and its known agonists (thrombin and kallikrein 6) at 30 d after SCI (Radulovic et al. 2016; Yoon et al. 2013) and clarify that the cellular distribution includes both neuron and glial compartments. These findings suggest that knockout of PAR1 expression across both neurons and glia is positioned to contribute to the improvements in recovery after SCI documented here and in other SCI models (Radulovic et al. 2016; Whetstone et al. 2017).

Knockout of astroglial PAR1 improves neurite outgrowth in vitro in a TrkB-dependent manner.

To determine the significance of the increases in astroglial pro-repair phenotype vivo after SCI in PAR1 knockout mice, we tested whether astrocytes purified from PAR1 knockout mice promote greater neuron survival and/or neurite outgrowth in vitro, and whether these effects were reliant on the BDNF receptor TrkB. Our focus on BDNF relates to our published findings that PAR1 knockout astrocytes express higher levels of BDNF (Yoon et al. 2020) and that BDNF is a growth factor of significance to neural repair, including demonstrated beneficial effects in the context of SCI (Charsar et al. 2019; Namiki et al. 2000; Vavrek et al. 2006). Wild type or PAR1 knockout cortical astrocyte cultures were seeded with wild type cortical neurons in the presence or absence of the TrkB inhibitor, ANA12 (Fig. 8). Astrocytes with PAR1 gene knockout promoted significantly enhanced outgrowth of Tuj1+ neurites (2.3-fold, P < 0.04, One-Way ANOVA, NK) and Tuj1+ neurites/neuron by 72 h in vitro (1.6-fold, P < 0.01, One-Way ANOVA, NK). The neurite-outgrowth promoting effects of PAR1 knockout astrocytes were fully blocked by inhibiting TrkB (P < 0.05, One-Way ANOVA, NK). PAR1 knockout astrocytes also promoted neuron survival at 24 and 72 h time points in culture (1.6-fold, P < 0.04, One-Way ANOVA, NK). The pro-survival effects of PAR1−/− astrocytes towards PAR1+/+ cortical neurons were not blocked upon TrkB inhibition. Quantification of the density of GFAP immunofluorescence showed reduced expression in PAR1 knockout astrocytes at 24 h in culture (2.9-fold, P < 0.003, One-Way ANOVA, NK). GFAP levels were not affected by ANA12.

Figure 8. PAR1 knockout astrocytes promote neurite outgrowth in a BDNF dependent manner.

Figure 8.

(A) Cartoon depicts the experimental design in which cultures of PAR1−/− or PAR1+/+ cortical astrocytes were seeded with PAR1+/+ cortical neurons, in the presence or absence of the TrkB inhibitor ANA12. (B-H) Representative immunofluorescence images, and associated histograms, show that PAR1−/− astrocytes promoted an increase in total Tuj1+ neurite area (C), and Tuj1+ area/DAPI+ neuron (E) after 72 h in culture, in a TrkB-dependent manner. PAR1−/− astrocytes also improved neuron number at 24 h, in a TrkB independent manner (D). PAR1−/− astrocytes showed reduced GFAP-immunoreactivity at early (24 h), but not later (72 h) time points in culture. All histograms show mean ± SE (*P < 0.05 and **P < 0.01, One-way Repeated Measures ANOVA, NK). Scale bar = 100 μm.

Discussion

The emerging role of CNS PAR1 as a mediator of neural injury, with PAR1 gene knockout improving outcomes across several neuropathologies, points to the need for a better understanding of the underlying cellular and molecular mechanisms. The importance of this relates in part to the existence of an FDA approved orally bioavailable PAR1 small molecule inhibitor that could be repurposed for neural repair (French et al. 2015). Through an investigation of the impact of PAR1 gene deletion in vivo and in astrocyte-neuron co-culture bioassays, our findings suggest that improved signs of neural resiliency and repair in PAR1 knockout mice after SCI may be linked, at least in part, to associated improvements in astrocyte pro-repair responses and enhanced signaling through TrkB. Taken together, these findings suggest that PAR1 gene knockout uncovers novel neurotrophic coupling mechanisms across the astrocyte-neuron compartments that are positioned to enhance neural resiliency and repair in cases of neurotrauma and potentially in other neuropathology’s as well.

PAR1 knockout mice show improved sensorimotor outcomes after SCI.

The current findings showing that constitutive genetic knockout of PAR1 improves recovery of sensorimotor function in experimental models of spinal cord trauma add to a growing body of evidence supporting the concept that PAR1 over activation by proteinases released in the injury microenvironment contributes to pathogenesis (Eftekhari et al. 2018; Gingrich and Traynelis 2000; Radulovic et al. 2016; Yoon et al. 2013). Moreover, excess proteinases may limit the capacity for neural repair in a PAR-dependent manner (Burda et al. 2013; Yoon et al. 2018; Yoon et al. 2017). Demonstrating that PAR1 knockout mice show improved recovery of function in yet a third model of experimental SCI is significant since trauma to the spinal cord occurs through a variety of mechanisms, necessitating development of reproducible and robust neuroprotective and reparative strategies. Moreover, across these models, beneficial effects of constitutive PAR1 knockout have been reported in both female (Radulovic et al. 2016) and male mice (Whetstone et al. 2017). PAR1 knockout mice also show reduced infarct volume after cerebral ischemia (Junge et al. 2003; Rajput et al. 2014) and improved signs of myelin regeneration and axonal protection in two experimental models of CNS demyelinating disease (Yoon et al. 2020). Pointing to therapeutic value, other experimental approaches to limit canonical PAR1 activation, including pharmacologic inhibition of thrombin or PAR1 show promise in other neuropathologies including experimental models of ALS (Festoff et al. 2000; Shavit-Stein et al. 2020), EAE (Kim et al. 2015a), and mTBI (Itsekson-Hayosh et al. 2015). Altogether these in vivo lines of evidence support blockade of the PAR1 signaling axis as a potential target to improve neural outcomes and that testing pharmacologic PAR1 inhibition, including current FDA-approved therapeutics, in experimental models of SCI is an important future direction.

Signs of neuronal sparing and regenerative responses are increased after SCI in PAR1 knockout mice.

We provide first in the field evidence that knocking out PAR1 not only confers neuroprotective benefits after SCI, but also increases signs of axonal repair. The preservation of spinal cord neurons after SCI in PAR1 knockouts reported after severe contusion compression injury (Radulovic et al. 2016) was similarly documented here in a moderate compression SCI paradigm. Genetic deletion of PAR1 is also neuroprotective in the MPTP model of Parkinson’s disease (Hamill et al. 2007). Here we additionally observed that neuron preservation was coupled to improvements in the density of Synapsin-1, a pre-synaptic marker. The coordinate increases in GAP43 immunoreactivity, a marker of growth cones, suggests PAR1 knockout uncovers active repair mechanisms in the injured adult spinal cord. The overarching concept that PAR1 knockout improves neurite growth receives additional support from co-culture bioassays in which PAR1 knockout astrocytes promoted increases in neurite density. To our knowledge these are the first studies to demonstrate that genetic blockade of PAR1 increases neurite growth in vitro and signs of neural repair in vivo.

While the increases in GAP43+ growth cones in the injury epicenter of PAR1 knockouts is an encouraging sign of repair, we have not differentiated whether increases in Synapsin-1 after SCI in PAR1 knockouts reflects neuroprotection, repair or a combination of these integrally related events. Distinguishing between these possibilities is further complicated by new findings that both the density of Synapsin-1 and that of serotonergic (5-HT+) axon profiles are higher in the intact spinal cord of PAR1 knockouts. These baseline increases in neural elements may reflect yet to be investigated developmental effects of PAR1 knockout in the neuron compartment. It is therefore not possible at this time to distinguish whether improvements in neural reserve in PAR1 knockouts after SCI reflect developmental differences or possible neuroprotective and/or repair mechanisms (Radulovic et al. 2016; Yoon et al. 2013). Nevertheless, it is exciting that increases in GAP43 are seen in the SCI epicenter of PAR1 knockouts and taken with other signs of neuroprotection highlight the need for future efforts that focus on therapeutic blockade of PAR1 function in adulthood after SCI, to differentiate underlying mechanisms. Given that recovery after SCI and the capacity for nerve sprouting is reduced with increasing age (Pestronk et al. 1980; Wilson et al. 2014), determining if blocking PAR1 improves outcomes even in aged mice will also be an important future direction.

The abundance of myelin and myelin-producing cells are improved after SCI in PAR1 knockout mice.

Preserving and regenerating the protective myelin sheath on axons after neural injury is an important strategy to spare and restore function across neurological conditions (Duncan et al. 2020; Lubetzki et al. 2020). Oligodendrocytes not only generate lipid-rich myelin to insulate axons enhancing electrical conduction, but also provide axonal metabolic support (Stassart et al. 2018). Spared white matter is a significant determinant of recovery after SCI (Noble and Wrathall 1985) and residual white matter was improved in PAR1−/− in the contusion model (Whetstone et al. 2017). Although spared tissue volume across the spinal cord gray and white matter was not impacted by PAR1 knockout in the moderate lateral compression injury model examined here, improvements in the area of MBP immunoreactivity in the injury epicenter and co-ordinate increases in numbers of Olig2+ and GST-3+ oligodendrocytes were clear. Moreover, preservation of NeuN+ neurons in PAR1 knockouts after injury was positively correlated with the area of spinal cord MBP immunoreactivity and with improvements in counts of Olig2+ oligodendrocytes. Future studies in which knockout of PAR1 selectively in myelinating cells will be needed to determine if this would be sufficient to confer neuron protection or if PAR1 knockout across the multiplicity of cell types involved in the response of the spinal cord to injury is necessary.

PAR1 gene knockout limits pro-inflammatory while fostering a pro-repair microenvironment.

A model of glial responses to neural injury has been identified wherein microglial and astroglial responses occur across a continuum of pro-inflammatory and pro-repair reactivity (Liddelow and Barres 2017). Here we demonstrate that PAR1 represents a new biological node to shift this continuum after SCI, with PAR1 knockout reducing microglial pro-inflammatory responses while increasing astrocyte pro-repair signatures. For example, in the microglial/monocyte compartments our findings show that the density of Iba-1+ microglia, in addition to CD68+ and iNOS+ signatures of a pro-inflammatory phenotype were reduced in PAR1 knockouts versus wild type comparators, while Arg1, a signature of a pro-repair phenotype was increased. The reductions in pro-inflammatory microglia/monocytes documented lends further corroboration to the likely pro-inflammatory actions that can be engaged by PAR1 over-activation and which are increasingly recognized across organ systems (Le et al. 2018; Saeed et al. 2017; Vergnolle et al. 2004), including the CNS (Kim et al. 2015b; Radulovic et al. 2016; Sachan et al. 2019). For example, prior studies showed that nanomolar concentrations of the PAR1 agonist thrombin activate microglia in culture, including proliferation and potentiation of TNF production (Suo et al. 2002). Improvements in dopamine levels in PAR1 knockouts in the MPTP model of Parkinson’s disease were likewise associated reductions in microglia (Hamill et al. 2007). In the context of severe SCI, reductions in pro-inflammatory cytokines TNF, IL-1β, IL-6 and IL-10 and increases in a pro-repair cytokine TGF-β were observed acutely after SCI in PAR1−/− mice (Radulovic et al. 2016). It is likely that the roles of PAR1 in influencing microglia/monocyte responses depends both on the time and type of injury examined since CD163, a different pro-repair microglia/monocyte marker did not differ between genotypes 30 d after SCI, although increases in this marker were observed in PAR1 knockouts in the cuprizone-induced model of chronic demyelinating disease during the period of myelin regeneration (Yoon et al. 2020).

Astroglial reactivity is regulated by multiple factors, including cytokines secreted by reactive microglia/monocytes. Given the reductions in pro-inflammatory microglia observed in PAR1 knockouts after SCI, therefore, it is of interest that a corresponding increase in S100A10 and Emp1 markers of astrocytes with a pro-repair phenotype (Clarke et al. 2018) was also observed. These findings are consistent with increases in astroglial S100A10 reported in PAR1 knockouts in the context of spinal cord myelin regeneration after acute lysolecithin-demyelinating injury (Yoon et al. 2020). We also observed a coordinate decrease in Serp1 and a trend toward a reduction in Complement 3 (C3d), each an astrocyte pro-inflammatory marker 30 d after SCI. C3d directs proinflammatory signaling, is involved in innate immunity by complement activation and is linked to synaptic pruning and neurodegeneration upon upregulation (Clarke et al. 2018; Liddelow et al. 2017; Yun et al. 2018). C3d increases in the corpus callosum driven by chronic cuprizone consumption were likewise significantly reduced in PAR1 knockouts after demyelination and during repair (Yoon et al. 2020). Taken together these independent lines of research point to PAR1 as a powerful regulator of the continuum of microglial/monocyte and astrocyte reactivity with dynamics influenced by the specific neural injury under consideration and the period of neural recovery examined. Given the role of reactive microglia and astrocyte C3d in synaptic pruning/stripping (Kettenmann et al. 2013), future efforts will be needed to determine the extent to which reductions in pro-inflammatory profiles and increases in pro-repair signatures across both microglial and astrocyte compartments observed in PAR1 knockouts after SCI contribute to the parallel improvements in Synapsin 1 and GAP43 documented here.

GFAP is a well-studied astrocyte marker, that has been in some cases mis-characterized as solely an indicator of pro-inflammatory reactivity. Rather, the functional roles of increased GFAP are poorly understood. What is clear is that GFAP is an intermediate filament protein essential to astrocyte morphology and is dynamically regulated enabling astrocytes to respond to their microenvironment. We previously reported that the spinal cord of adult PAR1 knockouts expresses higher levels of GFAP RNA compared to wild types (Radulovic et al. 2016). After severe SCI however, PAR1 knockouts showed reduced GFAP protein and RNA levels (Radulovic et al. 2016). In a cortical stab wound model, PAR1 knockouts also showed reductions in GFAP responses (Nicole et al. 2005). In the moderate SCI model examined here, the total area of GFAP immunoreactivity was reduced in white matter at the injury epicenter across genotypes, however these reductions were attenuated in PAR1 knockouts. Since astrocytes corral and reduce inflammation (Herrmann et al. 2008; Nobuta et al. 2012; Voskuhl et al. 2009), it is possible that the relative preservation of the GFAP+ astrocytes in PAR1 knockouts contributed to the reductions in Iba1+ microglia and pro-inflammatory CD68 and iNOS immunoreactivity also observed at 30 d after SCI. Altogether these findings suggest that the increases in GFAP in PAR1 knockouts after moderate SCI are coupled to a skewing of astrocyte properties towards an S100A10+, Emp1+ pro-repair phenotype, reductions in pro-inflammatory microglial/monocyte responses, and improved signs of neural repair and sensorimotor outcomes. Astrocyte specific targeting of PAR1 in the context of neural injury and the inclusion of an even wider array of markers of glial phenotype through cell-specific sequencing methods (Liddelow et al. 2017) will be needed to further illuminate PAR1’s roles in astroglial biology.

The fact that PAR1 gene knockout favorably impacts the repair profile across multiple cellular compartments of the SCI microenvironment is exciting, however this also makes a mechanistic understanding more challenging. PAR1 expression is elevated acutely, as early as 24 h after SCI (Citron et al. 2000; Radulovic et al. 2016). Here we document PAR1 RNA and protein expression across neuron and glial compartments of the intact adult spinal cord and persistent expression through 30 d after SCI (Radulovic et al. 2016). Determining whether changes in PAR1 activity in the neuron, microglial/monocyte or astrocyte compartments are necessary or sufficient to improve outcomes after experimental SCI, or in other models of neural injury, will necessitate additional efforts to target PAR1 in cell-type specific manner. The role of PAR1 in early events in the SCI injury cascade will also need to be considered, including acute changes in neutrophils and endothelial permeability (Whetstone et al. 2017). Finally, the abundance of PAR1 in any cellular compartment needs to be considered in the context of coordinate dynamic changes in PAR1 agonists (Radulovic et al. 2013; Radulovic et al. 2016; Scarisbrick et al. 2006).

Knockout of astroglial PAR1 improves neurite outgrowth in vitro in a TrkB-dependent manner.

To test the hypothesis that switching off PAR1 in astrocytes may confer protection and repair neuron responses, we determined the effects of PAR1 knockout astrocytes on the survival of and neurite outgrowth from wild type cortical neurons in co-culture bioassays. Mirroring the improvements in neuron survival and signs of neurite growth in vivo in PAR1 knockouts after SCI, co-culture of PAR1−/−astrocytes with wild type cortical neurons promoted improved neuron survival and an increase in number of Tuj1+ processes per neuron. PAR1 is now a relatively well recognized modulator of astrocyte reactivity (Junge et al. 2004; McCoy et al. 2012; Nicole et al. 2005; Radulovic et al. 2016; Scarisbrick et al. 2012; Sorensen et al. 2003; Vance et al. 2015; Vandell et al. 2008; Yoon et al. 2020; Yoon et al. 2018), with accumulating evidence, including that presented here suggesting activation is pro-inflammatory (Radulovic et al. 2016; Yoon et al. 2020; Yoon et al. 2018). For example, when astrocytes are first plated, they are relatively more reactive compared to later time points in culture and we observed that PAR1 knockout astrocytes showed lower levels of GFAP immunoreactivity at early time points in culture. PAR1 knockout astrocytes also secrete less IL-6 in response to agonist activation in vitro with reductions in signal transducer and activator of transcription 3 (STAT3) activation (Radulovic et al. 2016) as well as β-catenin nuclear translocation and cell stellation (Yoon et al. 2018). In addition, our recent studies show PAR1 knockout astrocytes express higher levels of certain growth factors, including BDNF and IGF1 (Yoon et al. 2020). In these studies, PAR1 knockout astrocytes increased oligodendrocyte differentiation, including expression of MBP in a TrkB-dependent manner. Here we demonstrate that inclusion of ANA12, a TrkB inhibitor in the astrocyte-neuron co-culture reduced increases in neurite outgrowth coupled to PAR1−/− astrocytes, suggesting a pivotal role for astrocyte-derived BDNF. Conversely, inhibition of TrkB did not impact the pro-survival effects of PAR1−/− astrocytes towards neurons, suggesting alternative/additional factors are involved. A role for reduced neurotoxic cytokines such as IL-1β and IL-6, or increases in neuroprotective cytokines (TGFβ) and growth factors (IGF1), already demonstrated to be increased in PAR1−/− astrocytes (Radulovic et al. 2016; Yoon et al. 2020) are possible candidates contributing to the TrkB-independent neuron pro-survival effects of PAR1−/− astrocytes documented here.

Conclusions

Genetic knockout of PAR1 function in the context of SCI has the potential to improve multiple substrates underpinning efficient neural transmission across neural and glial compartments. These findings add to a growing body of evidence pointing to the potential for PAR1 to serve as a pivotal therapeutic target at the interface of neural injury and repair.

Supplementary Material

Kim et al., 2021 Fig. S1
Kim et al., 2021 Fig. S4
Kim et al., 2021 Fig. S3
Kim et al., 2021 Fig. S2

Figure 7. Knockout of PAR1 reduces pro-inflammatory microglial signatures after spinal cord injury.

Figure 7.

(A) Representative immunofluorescence images show triple labeling for a pan-microglial/monocyte marker (Iba-1) along with a marker of pro-inflammatory (iNOS) or pro-repair (Arg1) phenotype, in the injury epicenter (0 mm) or in spinal cord above (+2 mm) or below (−2 mm), across PAR1+/+ and PAR1−/− mice at 30 days after injury. Dashed line delimits gray and white matter and boxed area on cartoon shows regions quantified. Quantification of Iba-1 immunofluorescence is shown in Fig. 6 (BC). (B-D) iNOS immunoreactivity was lower in gray matter (GM) of the injury epicenter and below, and the across all injury zones in white matter (WM) in PAR1−/− compared to PAR+/+ mice. (E-G) Arg1 immunoreactivity was higher in GM and WM across injury zones in PAR1−/− mice. The abundance of Arg1/Iba-1 immunoreactivity was greater in GM and WM of PAR1−/− compared to PAR+/+ mice. The data are presented as means ± SEM. (PAR1+/+ SCI, n=7 per group and PAR1−/− SCI, n=6 per group, *P < 0.05, **P < 0.01, and ***P < 0.001, Two-Way ANOVA, NK). DAPI staining to visualize all cells is also shown. Scale bar = 100 μm.

Figure 9. PAR1 is a molecular switch regulating astroglial-neuron trophic coupling.

Figure 9.

Findings to date support a hypothetical model in which PAR1 activation (Switch ON) by select secreted proteases, including thrombin and kallikrein 6 that are upregulated after neurotrauma, cleave the receptor N-terminus, evoking intracellular signaling across neurons and neuroglia that promotes pro-inflammatory responses and neural injury. Our new findings suggest that that PAR1 loss-of-function (Switch OFF) improves neural outcomes after injury by reducing pro-inflammatory and improving pro-repair glial responses, including the ability of astrocytes to signal through TrkB to support neurite growth. The specific role(s) of other PAR1-regulated astrocyte and microglia-derived growth factors and cytokines in neural injury and repair will require further study.

Main Points.

Neural repair in PAR1 knockout mice is associated with increased astrocyte pro-repair properties.

Genetic knockout of astrocyte PAR1 increases neurite density in a TrkB-dependent manner.

PAR1 regulates neurotrophic coupling across the astrocyte-neuron compartments.

Acknowledgements

The work was supported by a grant from the National Institutes of Health R01NS052741-10 (IAS), the Minnesota State Spinal Cord Injury and Traumatic Brain Injury Research Program (IAS and JRD) and Research Grants (G-1508-05951, RG-1901-33209) from the National Multiple Sclerosis Society (IAS). HNK was supported by a fellowship from the Mayo Clinic Center for MS and Autoimmune Neurology and EMT was supported by the National Institute of General Medical Sciences (T32 GM 65841). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Abbreviations

5-HT

5-hydroxytryptamine receptors

ALDH1L1

aldehyde dehydrogenase 1 family member L1

CNS

Central nervous system

DAPI

4′,6-Diamidino-2-phenylindole

GAP43

Growth Associated Protein 43

GFAP

Glial fibrillary acidic protein

GM

Gray matter

Iba-1

Ionized calcium binding adaptor molecule 1

IL-6

Interleukin 6

MBP

Myelin Basic Protein

NeuN

Neuronal nuclei

Olig2

Oligodendrocyte Transcription Factor 2

PLP

Myelin proteolipid protein

SCI

Spinal cord injury

WM

White matter

Footnotes

Competing Financial Interests

The authors declare no competing interests.

Data Availability:

All data will be made available upon request to the corresponding author.

References

  1. Allen M, Ghosh S, Ahern GP, Villapol S, Maguire-Zeiss KA, Conant K. 2016. Protease induced plasticity: matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1. Sci Rep 6:35497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong RA. 2014. When to use the Bonferroni correction. Ophthalmic Physiol Opt 34:502–8. [DOI] [PubMed] [Google Scholar]
  3. Basso DM, Beattie MS, Breshehan JC. 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1–21. [DOI] [PubMed] [Google Scholar]
  4. Basso DM, Fisher LC, Anderson AJ, Jakeman LB, McTigue DM, Popovich PG. 2006. Basso mouse scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J Neurotrauma 23:635–659. [DOI] [PubMed] [Google Scholar]
  5. Burda JE, Radulovic M, Yoon H, Scarisbrick IA. 2013. Critical role for PAR1 in kallikrein 6-mediated oligodendrogliopathy. Glia 61:1456–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Charsar BA, Brinton MA, Locke K, Chen AY, Ghosh B, Urban MW, Komaravolu S, Krishnamurthy K, Smit R, Pasinelli P and others. 2019. AAV2-BDNF promotes respiratory axon plasticity and recovery of diaphragm function following spinal cord injury. FASEB J 33:13775–13793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Choi CI, Yoon H, Drucker KL, Langley MR, Kleppe L, Scarisbrick IA. 2018. The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation. Sci Rep 8:9360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Citron BA, Ameenuddin S, Uchida K, Suo WZ, SantaCruz K, Festoff BW. 2016. Membrane lipid peroxidation in neurodegeneration: Role of thrombin and proteinase-activated receptor-1. Brain Res 1643:10–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Citron BA, Smirnova IV, Arnold PM, Festoff BW. 2000. Upregulation of neurotoxic serine proteases, prothrombin, and protease- activated receptor 1 early after spinal cord injury. J Neurotrauma 17:1191–203. [DOI] [PubMed] [Google Scholar]
  10. Clarke LE, Liddelow SA, Chakraborty C, Munch AE, Heiman M, Barres BA. 2018. Normal aging induces A1-like astrocyte reactivity. Proc Natl Acad Sci U S A 115:E1896–E1905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Coughlin SR. 2005. Protease-activated receptors in hemostasis, thrombosis and vascular biology. J Thromb Haemost 3:1800–14. [DOI] [PubMed] [Google Scholar]
  12. Duncan GJ, Manesh SB, Hilton BJ, Assinck P, Plemel JR, Tetzlaff W. 2020. The fate and function of oligodendrocyte progenitor cells after traumatic spinal cord injury. Glia 68:227–245. [DOI] [PubMed] [Google Scholar]
  13. Eftekhari R, de Lima SG, Liu Y, Mihara K, Saifeddine M, Noorbakhsh F, Scarisbrick IA, Hollenberg MD. 2018. Microenvironment proteinases, proteinase-activated receptor regulation, cancer and inflammation. Biol Chem 399:1023–1039. [DOI] [PubMed] [Google Scholar]
  14. Festoff BW, Ameenuddin S, Santacruz K, Morser J, Suo Z, Arnold PM, Stricker KE, Citron BA. 2004. Neuroprotective effects of recombinant thrombomodulin in controlled contusion spinal cord injury implicates thrombin signaling. J Neurotrauma 21:907–922. [DOI] [PubMed] [Google Scholar]
  15. Festoff BW, D’Andrea MR, Citron BA, Salcedo RM, Smirnova IV, Andrade-Gordon P. 2000. Motor neuron cell death in wobbler mutant mice follows overexpression of the G-protein-coupled, protease-activated receptor for thrombin. Mol Med 6:410–29. [PMC free article] [PubMed] [Google Scholar]
  16. French SL, Arthur JF, Tran HA, Hamilton JR. 2015. Approval of the first protease-activated receptor antagonist: Rationale, development, significance, and considerations of a novel anti-platelet agent. Blood Rev 29:179–89. [DOI] [PubMed] [Google Scholar]
  17. Gingrich MB, Traynelis SF. 2000. Serine proteases and brain damage - is there a link? Trends in Neuroscience 23:399–407. [DOI] [PubMed] [Google Scholar]
  18. Hamill CE, Caudle WM, Richardson JR, Yuan H, Pennell KD, Greene JG, Miller GW, Traynelis SF. 2007. Exacerbation of dopaminergic terminal damage in a mouse model of Parkinson’s disease by the G-protein-coupled receptor protease-activated receptor 1. Mol Pharmacol 72:653–64. [DOI] [PubMed] [Google Scholar]
  19. Herrmann JE, Imura T, Song B, Qi J, Ao Y, Nguyen TK, Korsak RA, Takeda K, Akira S, Sofroniew MV. 2008. STAT3 is a critical regulator of astrogliosis and scar formation after spinal cord injury. The Journal of neuroscience : the official journal of the Society for Neuroscience 28:7231–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Itsekson-Hayosh Z, Shavit-Stein E, Katzav A, Rubovitch V, Maggio N, Chapman J, Harnof S, Pick CG. 2015. Minimal traumatic brain injury in mice - PAR-1 and thrombin related changes. J Neurotrauma. [DOI] [PubMed] [Google Scholar]
  21. Joshi M, Fehlings M. 2002. Development and characterization of a novel, graded model of clip compressive spinal cord injury in the mouse: Part 2. Quantitative neuroanatomical assessment and analysis of the relationships between axonal tracts, residual tissue, and locomotor recovery. J Neurotrauma 19:191–203. [DOI] [PubMed] [Google Scholar]
  22. Junge CE, Lee CJ, Hubbard KB, Zhang Z, Olson JJ, Hepler JR, Brat DJ, Traynelis SF. 2004. Protease-activated receptor-1 in human brain: localization and functional expression in astrocytes. Exp Neurol 188:94–103. [DOI] [PubMed] [Google Scholar]
  23. Junge CE, Sugawara T, Mannaioni G, Alagarsamy S, Conn PJ, Brat DJ, Chan PH, Traynelis SF. 2003. The contribution of protease-activated receptor 1 to neuronal damage caused by transient focal cerebral ischemia. Proc Natl Acad Sci U S A 100:13019–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kettenmann H, Kirchhoff F, Verkhratsky A. 2013. Microglia: new roles for the synaptic stripper. Neuron 77:10–8. [DOI] [PubMed] [Google Scholar]
  25. Kim HN, Kim YR, Ahn SM, Lee SK, Shin HK, Choi BT. 2015a. Protease activated receptor-1 antagonist ameliorates the clinical symptoms of experimental autoimmune encephalomyelitis via inhibiting breakdown of blood-brain barrier. J Neurochem 135:577–88. [DOI] [PubMed] [Google Scholar]
  26. Kim W, Zekas E, Lodge R, Susan-Resiga D, Marcinkiewicz E, Essalmani R, Mihara K, Ramachandran R, Asahchop E, Gelman B and others. 2015b. Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder. Mol Cell Biol 35:3684–700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Le VB, Riteau B, Alessi MC, Couture C, Jandrot-Perrus M, Rheaume C, Hamelin ME, Boivin G. 2018. Protease-activated receptor 1 inhibition protects mice against thrombin-dependent respiratory syncytial virus and human metapneumovirus infections. Br J Pharmacol 175:388–403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee PR, Johnson TP, Gnanapavan S, Giovannoni G, Wang T, Steiner JP, Medynets M, Vaal MJ, Gartner V, Nath A. 2017. Protease-activated receptor-1 activation by granzyme B causes neurotoxicity that is augmented by interleukin-1beta. J Neuroinflammation 14:131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liddelow SA, Barres BA. 2017. Reactive Astrocytes: Production, Function, and Therapeutic Potential. Immunity 46:957–967. [DOI] [PubMed] [Google Scholar]
  30. Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, Bennett ML, Munch AE, Chung WS, Peterson TC and others. 2017. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541:481–487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lubetzki C, Zalc B, Williams A, Stadelmann C, Stankoff B. 2020. Remyelination in multiple sclerosis: from basic science to clinical translation. Lancet Neurol 19:678–688. [DOI] [PubMed] [Google Scholar]
  32. Maggio N, Shavit E, Chapman J, Segal M. 2008. Thrombin induces long-term potentiation of reactivity to afferent stimulation and facilitates epileptic seizures in rat hippocampal slices: toward understanding the functional consequences of cerebrovascular insults. J Neurosci 28:732–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mannaioni G, Orr AG, Hamill CE, Yuan H, Pedone KH, McCoy KL, Berlinguer Palmini R, Junge CE, Lee CJ, Yepes M and others. 2008. Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1. J Biol Chem 283:20600–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McCoy KL, Gyoneva S, Vellano CP, Smrcka AV, Traynelis SF, Hepler JR. 2012. Protease-activated receptor 1 (PAR1) coupling to G(q/11) but not to G(i/o) or G(12/13) is mediated by discrete amino acids within the receptor second intracellular loop. Cell Signal 24:1351–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Namiki J, Kojima A, Tator CH. 2000. Effect of brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3 on functional recovery and regeneration after spinal cord injury in adult rats. J Neurotrauma 17:1219–31. [DOI] [PubMed] [Google Scholar]
  36. Nicole O, Goldshmidt A, Hamill CE, Sorensen SD, Sastre A, Lyuboslavsky P, Hepler JR, McKeon RJ, Traynelis SF. 2005. Activation of protease-activated receptor-1 triggers astrogliosis after brain injury. The Journal of neuroscience : the official journal of the Society for Neuroscience 25:4319–4329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Noble LJ, Wrathall JR. 1985. Spinal cord contusion in the rat: morphometric analyses of alterations in the spinal cord. Exp Neurol 88:135–49. [DOI] [PubMed] [Google Scholar]
  38. Nobuta H, Ghiani CA, Paez PM, Spreuer V, Dong H, Korsak RA, Manukyan A, Li J, Vinters HV, Huang EJ and others. 2012. STAT3-Mediated astrogliosis protects myelin development in neonatal brain injury. Annals of neurology 72:750–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pestronk A, Drachman DB, Griffin JW. 1980. Effects of aging on nerve sprouting and regeneration. Exp Neurol 70:65–82. [DOI] [PubMed] [Google Scholar]
  40. Plemel JR, Duncan G, Chen KW, Shannon C, Park S, Sparling JS, Tetzlaff W. 2008. A graded forceps crush spinal cord injury model in mice. J Neurotrauma 25:350–70. [DOI] [PubMed] [Google Scholar]
  41. Pukos N, Goodus MT, Sahinkaya FR, McTigue DM. 2019. Myelin status and oligodendrocyte lineage cells over time after spinal cord injury: What do we know and what still needs to be unwrapped? Glia 67:2178–2202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Radulovic M, Yoon H, Larson N, Wu J, Linbo R, Burda JE, Diamandis EP, Blaber SI, Blaber M, Fehlings MG and others. 2013. Kallikrein cascades in traumatic spinal cord injury: in vitro evidence for roles in axonopathy and neuron degeneration. Journal of neuropathology and experimental neurology 72:1072–89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Radulovic M, Yoon H, Wu J, Mustafa K, Scarisbrick IA. 2016. Targeting the thrombin receptor modulates inflammation and astrogliosis to improve recovery after spinal cord injury. Neurobiol Dis 93:226–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rajput PS, Lyden PD, Chen B, Lamb JA, Pereira B, Lamb A, Zhao L, Lei IF, Bai J. 2014. Protease activated receptor-1 mediates cytotoxicity during ischemia using in vivo and in vitro models. Neuroscience 281C:229–240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sachan V, Lodge R, Mihara K, Hamelin J, Power C, Gelman BB, Hollenberg MD, Cohen EA, Seidah NG. 2019. HIV-induced neuroinflammation: impact of PAR1 and PAR2 processing by Furin. Cell Death Differ 26:1942–1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Saeed MA, Ng GZ, Dabritz J, Wagner J, Judd L, Han JX, Dhar P, Kirkwood CD, Sutton P. 2017. Protease-activated Receptor 1 Plays a Proinflammatory Role in Colitis by Promoting Th17-related Immunity. Inflamm Bowel Dis 23:593–602. [DOI] [PubMed] [Google Scholar]
  47. Scarisbrick IA. 2008. The multiple sclerosis degradome: enzymatic cascades in development and progression of central nervous system inflammatory disease. Curr Top Microbiol Immunol 318:133–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Scarisbrick IA, Isackson PJ, Windebank AJ. 1999. Differential expression of brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 in the adult rat spinal cord: regulation by the glutamate receptor agonist kainic acid. J Neurosci 19:7757–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Scarisbrick IA, Radulovic M, Burda JE, Larson N, Blaber SI, Giannini C, Blaber M, Vandell AG. 2012. Kallikrein 6 is a novel molecular trigger of reactive astrogliosis. Biological Chemistry 393:355–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Scarisbrick IA, Sabharwal P, Cruz H, Larsen N, Vandell A, Blaber SI, Ameenuddin S, Papke LM, Fehlings MG, Reeves RK and others. 2006. Dynamic role of kallikrein 6 in traumatic spinal cord injury. Eur J Neuroscience 24:1457–1469. [DOI] [PubMed] [Google Scholar]
  51. Schmitz C, Hof PR. 2005. Design-based stereology in neuroscience. Neuroscience 130:813–31. [DOI] [PubMed] [Google Scholar]
  52. Shavit-Stein E, Abu Rahal I, Bushi D, Gera O, Sharon R, Gofrit SG, Pollak L, Mindel K, Maggio N, Kloog Y and others. 2020. Brain Protease Activated Receptor 1 Pathway: A Therapeutic Target in the Superoxide Dismutase 1 (SOD1) Mouse Model of Amyotrophic Lateral Sclerosis. Int J Mol Sci 21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sorensen SD, Nicole O, Peavy RD, Montoya LM, Lee CJ, Murphy TJ, Traynelis SF, Hepler JR. 2003. Common signaling pathways link activation of murine PAR-1, LPA, and S1P receptors to proliferation of astrocytes. Mol Pharmacol 64:1199–209. [DOI] [PubMed] [Google Scholar]
  54. Stassart RM, Mobius W, Nave KA, Edgar JM. 2018. The Axon-Myelin Unit in Development and Degenerative Disease. Front Neurosci 12:467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Suo Z, Wu M, Ameenuddin S, Anderson HE, Zoloty JE, Citron BA, Andrade-Gordon P, Festoff BW. 2002. Participation of protease-activated receptor-1 in thrombin-induced microglial activation. J Neurochem 80:655–66. [DOI] [PubMed] [Google Scholar]
  56. Vance KM, Rogers RC, Hermann GE. 2015. PAR1-activated astrocytes in the nucleus of the solitary tract stimulate adjacent neurons via NMDA receptors. J Neurosci 35:776–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Vandell AG, Larson N, Laxmikanthan G, Panos M, Blaber SI, Blaber M, Scarisbrick IA. 2008. Protease Activated Receptor Dependent and Independent Signaling by Kallikreins 1 and 6 in CNS Neuron and Astroglial Cell Lines. J Neurochem 107:855–870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Vavrek R, Girgis J, Tetzlaff W, Hiebert GW, Fouad K. 2006. BDNF promotes connections of corticospinal neurons onto spared descending interneurons in spinal cord injured rats. Brain 129:1534–45. [DOI] [PubMed] [Google Scholar]
  59. Vergnolle N, Cellars L, Mencarelli A, Rizzo G, Swaminathan S, Beck P, Steinhoff M, Andrade-Gordon P, Bunnett NW, Hollenberg MD and others. 2004. A role for proteinase-activated receptor-1 in inflammatory bowel diseases. J Clin Invest 114:1444–56. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  60. Voskuhl RR, Peterson RS, Song B, Ao Y, Morales LB, Tiwari-Woodruff S, Sofroniew MV. 2009. Reactive astrocytes form scar-like perivascular barriers to leukocytes during adaptive immune inflammation of the CNS. J Neurosci 29:11511–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wang F, Flanagan J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, Luo Y. 2012. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14:22–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Whetstone WD, Walker B, Trivedi A, Lee S, Noble-Haeusslein LJ, Hsu JC. 2017. Protease-Activated Receptor-1 Supports Locomotor Recovery by Biased Agonist Activated Protein C after Contusive Spinal Cord Injury. PLoS One 12:e0170512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wilson JR, Davis AM, Kulkarni AV, Kiss A, Frankowski RF, Grossman RG, Fehlings MG. 2014. Defining age-related differences in outcome after traumatic spinal cord injury: analysis of a combined, multicenter dataset. Spine J 14:1192–8. [DOI] [PubMed] [Google Scholar]
  64. Yoon H, Choi CI, Triplet EM, Langley MR, Kleppe LS, Kim HN, Simon WL, Scarisbrick IA. 2020. Blocking the Thrombin Receptor Promotes Repair of Demyelinated Lesions in the Adult Brain. J Neurosci 40:1483–1500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Yoon H, Radulovic M, Drucker KL, Wu J, Scarisbrick IA. 2015. The thrombin receptor is a critical extracellular switch controlling myelination. Glia 63:846–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yoon H, Radulovic M, Scarisbrick IA. 2018. Kallikrein-related peptidase 6 orchestrates astrocyte form and function through proteinase activated receptor-dependent mechanisms. Biol Chem 399:1041–1052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Yoon H, Radulovic M, Walters G, Paulsen AR, Drucker K, Starski P, Wu J, Fairlie DP, Scarisbrick IA. 2017. Protease activated receptor 2 controls myelin development, resiliency and repair. Glia 65:2070–2086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Yoon H, Radulovic M, Wu J, Blaber SI, Blaber M, Fehlings MG, Scarisbrick IA. 2013. Kallikrein 6 signals through PAR1 and PAR2 to promote neuron injury and exacerbate glutamate neurotoxicity. J Neurochem 127:283–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Yoon H, Scarisbrick IA. 2016. Kallikrein-related peptidase 6 exacerbates disease in an autoimmune model of multiple sclerosis. Biol Chem 397:1277–1286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Yun SP, Kam TI, Panicker N, Kim S, Oh Y, Park JS, Kwon SH, Park YJ, Karuppagounder SS, Park H and others. 2018. Block of A1 astrocyte conversion by microglia is neuroprotective in models of Parkinson’s disease. Nat Med 24:931–938. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Kim et al., 2021 Fig. S1
Kim et al., 2021 Fig. S4
Kim et al., 2021 Fig. S3
Kim et al., 2021 Fig. S2

Data Availability Statement

All data will be made available upon request to the corresponding author.

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