No abstract

The deregulation of serine protease activity is a common feature of neurological injury, but little is known regarding their mechanisms of action or whether they can be targeted to facilitate repair. In this study we demonstrate that the thrombin receptor (Protease Activated Receptor 1, (PAR1)) serves as a critical translator of the spinal cord injury (SCI) proteolytic microenvironment into a cascade of pro-inflammatory events that contribute to astrogliosis and functional decline. PAR1 knockout mice displayed improved locomotor recovery after SCI and reduced signatures of inflammation and astrogliosis, including expression of glial fibrillary acidic protein (GFAP), vimentin, and STAT3 signaling. SCI-associated elevations in pro-inflammatory cytokines such as IL-1β and IL-6 were also reduced in PAR1−/− mice and co-ordinate improvements in tissue sparing and preservation of NeuN-positive ventral horn neurons, and PKCγ corticospinal axons, were observed. PAR1 and its agonist’s thrombin and neurosin were expressed by perilesional astrocytes and each agonist increased the production of IL-6 and STAT3 signaling in primary astrocyte cultures in a PAR1-dependent manner. In turn, IL-6-stimulated astrocytes increased expression of PAR1, thrombin, and neurosin, pointing to a model in which PAR1 activation contributes to increased astrogliosis by feedforward- and feedback-signaling dynamics. Collectively, these findings identify the thrombin receptor as a key mediator of inflammation and astrogliosis in the aftermath of SCI that can be targeted to reduce neurodegeneration and improve neurobehavioral recovery.

Kallikrein 6 (Klk6) is a secreted serine protease preferentially expressed by oligodendroglia in CNS white matter. Elevated levels of Klk6 occur in actively demyelinating multiple sclerosis (MS) lesions and in cases of spinal cord injury (SCI), stroke and glioblastoma. Taken with recent evidence establishing Klk6 as a CNS-endogenous activator of protease-activated receptors (PARs), we hypothesized that Klk6 activates a subset of PARs to regulate oligodendrocyte physiology and potentially pathophysiology. Here, primary oligodendrocyte cultures derived from wild type or PAR1-deficient mice and the murine oligodendrocyte cell line, Oli-neu, were used to demonstrate that Klk6 mediates loss of oligodendrocyte processes and impedes morphological differentiation of oligodendrocyte progenitor cells (OPCs) in a PAR1-dependent fashion. Comparable gliopathy was also elicited by the canonical PAR1 agonist, thrombin, as well as PAR1-activating peptides (PAR1-APs). Klk6 also exacerbated ATP-mediated oligodendrogliopathy in vitro, pointing to a potential role in augmenting excitotoxicity. In addition, Klk6 suppressed the expression of proteolipid protein (PLP) RNA in cultured oligodendrocytes by a mechanism involving PAR1-mediated Erk1/2 signaling. Microinjection of PAR1 agonists, including Klk6 or PAR1-APs, into the dorsal column white matter of PAR+/+ but not PAR−/− mice promoted vacuolating myelopathy and a loss of immunoreactivity for myelin basic protein (MBP) and CC-1+ oligodendrocytes. These results demonstrate a functional role for Klk6-PAR1 signaling in oligodendroglial pathophysiology and suggest that PAR1 or PAR1-agonists may represent new targets to moderate demyelination and to promote myelin regeneration in cases of CNS white matter injury or disease.

CNS trauma generates a proteolytic imbalance contributing to secondary injury, including axonopathy and neuron degeneration. Kallikrein 6 (Klk6) is a serine protease implicated in neurodegeneration and here we investigate the role of protease activated receptors 1 (PAR1) and PAR2 in mediating these effects. First we demonstrate Klk6 and the prototypical activator of PAR1, thrombin, as well as PAR1 and PAR2, are each elevated in murine experimental traumatic spinal cord injury (SCI) at acute or subacute time points. Recombinant Klk6 triggered ERK1/2 signaling in cerebellar granule neurons and in the NSC34 spinal cord motoneuron cell line, in a PI3K and MEK-dependent fashion. Importantly, lipopeptide inhibitors of PAR1 or PAR2, and PAR1 genetic deletion, each reduced Klk6-ERK1/2 activation. In addition, Klk6 and thrombin promoted degeneration of cerebellar neurons and exacerbated glutamate neurotoxicity. Moreover, genetic deletion of PAR1 blocked thrombin-mediated cerebellar neurotoxicity and reduced the neurotoxic effects of Klk6. Klk6 also increased glutamate-mediated Bim signaling, PARP cleavage and lactate dehydrogenase (LDH) release in NSC34 motoneurons and these effects were blocked by PAR1 and PAR2 lipopeptide inhibitors. Taken together these data point to a novel Klk6-signaling axis in CNS neurons that is mediated by PAR1 and PAR2 and is positioned to contribute to neurodegeneration.

Astrocytes have emerged as essential regulators of function and response to injury in the brain and spinal cord, yet very little is known about regional differences that exist. Here we compare the expression of key astroglial markers (glial fibrillary acidic protein (GFAP) and Aldehyde Dehydrogenase-1 Family Member L1 (ALDH1L1)) across these disparate poles of the neuraxis, tracking their expression developmentally and in the context of demyelination. In addition, we document changes in the astrocyte regulatory cytokine interleukin 6 (IL-6), and its signaling partner signal transducer and activator of transcription 3 (STAT3), in vivo and in vitro. Results demonstrate that GFAP expression is higher in the developing and adult spinal cord relative to brain. Comparisons between GFAP and ALDH1L1 expression suggest elevations in spinal cord GFAP during the early postnatal period reflect an accelerated appearance of astrocytes, while elevations in adulthood reflect higher expression by individual astrocytes. Notably, increases in spinal cord compared to whole brain GFAP were paralleled by higher levels of IL-6 and STAT3. Equivalent elevations in GFAP, GFAP/ALDH1L1 ratios, and in IL-6, were observed in primary astrocyte cultures derived from spinal cord compared to cortex. Also, higher levels of GFAP were observed in the spinal cord compared to the brain after focal demyelinating injury. Altogether, these studies point to key differences in astrocyte abundance and the expression of GFAP and IL-6 across the brain and spinal cord that are positioned to influence regional specialization developmentally and responses occurring in the context of injury and disease.

Hemorrhagic white matter injuries in the perinatal period are a growing cause of cerebral palsy yet no neuroprotective strategies exist to prevent the devastating motor and cognitive deficits that ensue. We demonstrate the thrombin receptor (protease activated receptor 1, PAR1) exhibits peak expression levels in the spinal cord at term and is a critical regulator of the myelination continuum from initiation to the final levels achieved. Specifically, PAR1 gene deletion resulted in earlier onset of spinal cord myelination, including substantially more Olig2-positive oligodendrocytes, more myelinated axons and higher proteolipid protein (PLP) levels at birth. In vitro, the highest levels of PAR1 were observed in oligodendrocyte progenitor cells (OPCs), being reduced with differentiation. In parallel, the expression of PLP and myelin basic protein (MBP), in addition to Olig2, were all significantly higher in cultures of PAR1−/− oligodendroglia. Moreover, application of a small molecule inhibitor of PAR1 (SCH79797) to OPCs in vitro, inhibited PLP and MBP expression. Enhancements in myelination associated with PAR1 genetic deletion were also observed in adulthood as evidenced by higher amounts of myelin basic protein and thickened myelin sheaths across large, medium and small diameter axons. Enriched spinal cord myelination in PAR1−/− mice was coupled to increases in extracellular-signal-regulated kinase 1/2 and AKT signaling developmentally. Nocturnal ambulation and rearing activity were also elevated in PAR1−/− mice. These studies identify the thrombin receptor as a powerful extracellular regulatory switch that could be readily targeted to improve myelin production in the face of white matter injury and disease.

Kallikrein 6 (Klk6) is a secreted serine protease that is elevated in active multiple sclerosis lesions and patient sera. To further evaluate the involvement of Klk6 in chronic progressive demyelinating disease, we determined its expression in the brain and spinal cord of SJL mice infected with Theiler’s murine encephalomyelitis virus (TMEV) and assessed the effects of Klk6-neutralizing antibodies on disease progression. Klk6 RNA expression was elevated in the brain and spinal cord by 7 days post-infection (dpi). Thereafter, Klk6 expression persisted primarily in the spinal cord reaching a peak of 5-fold over controls at mid-chronic stages (60–120 dpi). Significant elevations in Klk6 RNA were also induced in splenocytes stimulated with viral capsid proteins in vitro and in activated THP-1 monocytes. Klk6-neutralizing antibodies reduced TMEV-driven brain and spinal cord pathology and DTH responses when examined at early chronic time points (40 dpi). Reductions in spinal cord pathology included a decrease in activated monocytes/microglia and reductions in the loss of myelin basic protein (MBP). By 180 dpi, pathology scores no longer differed between groups. These findings point to regulatory activities for Klk6 in the development and progression of CNS inflammation and demyelination that can be effectively targeted through the early chronic stages with neutralizing antibody.

Inflammatory-astrogliosis exacerbates damage in the injured spinal cord and limits repair. Here we identify Protease Activated Receptor 2 (PAR2) as an essential regulator of these events with mice lacking the PAR2 gene showing greater improvements in motor coordination and strength after compression-spinal cord injury (SCI) compared to wild type littermates. Molecular profiling of the injury epicenter, and spinal segments above and below, demonstrated mice lacking PAR2 had significantly attenuated elevations in key hallmarks of astrogliosis (glial fibrillary acidic protein (GFAP), vimentin and neurocan) and in expression of pro-inflammatory cytokines (interleukin-6 (IL-6), tumor necrosis factor (TNF) and interleukin -1 beta (IL- 1β)). SCI in PAR2−/− mice was also accompanied by improved preservation of protein kinase C gamma (PKCγ)-immunopositive corticospinal axons and reductions in GFAP-immunoreactivity, expression of the pro-apoptotic marker BCL2-interacting mediator of cell death (BIM), and in signal transducer and activator of transcription 3 (STAT3). The potential mechanistic link between PAR2, STAT3 and astrogliosis was further investigated in primary astrocytes to reveal that the SCI-related serine protease, neurosin (kallikrein 6) promotes IL-6 secretion in a PAR2 and STAT3-dependent manner. Data point to a signaling circuit in primary astrocytes in which neurosin signaling at PAR2 promotes IL-6 secretion and canonical STAT3 signaling. IL-6 promotes expression of GFAP, vimentin, additional IL-6 and robust increases in both neurosin and PAR2, thereby driving the PAR2-signaling circuit forward. Given the significant reductions in astrogliosis and inflammation as well as superior neuromotor recovery observed in PAR2 knockout mice after SCI, we suggest this receptor and its agonists represent new drug targets to foster neuromotor recovery.