Wu, Jianmin

Trauma to the CNS generates a proteolytic imbalance that contributes to secondary injury, including axonopathy and neuron degeneration; however the mechanism by which this occurs is not well understood. Kallikrein 6 (KLK6) is a serine protease implicated in neurotoxicity and in these studies we investigate the role of protease activated receptors (PARs) in mediating these effects. We demonstrate KLK6 and the prototypical activator of PAR1, thrombin, as well as PAR1 and ‐2, are each elevated in a murine experimental spinal cord injury at acute or subacute time points. KLK6 and thrombin each promoted cerebellar granule neuron toxicity and potentiated glutamate excitotoxicity in vitro. Importantly, neural injury in each case was reduced or blocked in neurons derived from PAR1 deficient mice. KLK6 was shown to activate ERK in cerebellar neurons in a PI3K and MEK‐dependent fashion. Novel lipopeptide inhibitors specific for PAR1 or PAR2, in addition to neurons derived from PAR1 deficient mice were utilized to demonstrate both receptors play an important role in KLK6‐elicited ERK signaling and in the activation of the proapoptotic protein Bim. Taken together these data point to novel KLK6‐PAR1 and ‐PAR2 mediated signaling cascades in CNS neurons that are positioned to participate in neuron degeneration and glutamate‐mediated excitotoxicity in cases of CNS injury. Supported by NIH R01 NS052741

AbstractOligodendrocytes are essential regulators of axonal energy homeostasis and electrical conduction and emerging target cells for restoration of neurological function. Here we investigate the role of protease activated receptor 2 (PAR2), a unique protease activated G protein‐coupled receptor, in myelin development and repair using the spinal cord as a model. Results demonstrate that genetic deletion of PAR2 accelerates myelin production, including higher proteolipid protein (PLP) levels in the spinal cord at birth and higher levels of myelin basic protein and thickened myelin sheaths in adulthood. Enhancements in spinal cord myelin with PAR2 loss‐of‐function were accompanied by increased numbers of Olig2‐ and CC1‐positive oligodendrocytes, as well as in levels of cyclic adenosine monophosphate (cAMP), and extracellular signal related kinase 1/2 (ERK1/2) signaling. Parallel promyelinating effects were observed after blocking PAR2 expression in purified oligodendrocyte cultures, whereas inhibiting adenylate cyclase reversed these effects. Conversely, PAR2 activation reduced PLP expression and this effect was prevented by brain derived neurotrophic factor (BDNF), a promyelinating growth factor that signals through cAMP. PAR2 knockout mice also showed improved myelin resiliency after traumatic spinal cord injury and an accelerated pattern of myelin regeneration after focal demyelination. These findings suggest that PAR2 is an important controller of myelin production and regeneration, both in the developing and adult spinal cord.

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 that 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 increased PLP and MBP expression. Enhancements in myelination associated with PAR1 genetic deletion were also observed in adulthood as evidenced by higher amounts of MBP 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. GLIA 2015;63:846–859