Critical role for PAR1 in kallikrein 6-mediated oligodendrogliopathy.
ABSTRACT: 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 (rodent form) 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 PAR1(+/+) but not PAR1(-/-) 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 antagonists of PAR1 or its protease agonists may represent new modalities to moderate demyelination and to promote myelin regeneration in cases of CNS white matter injury or disease.
Project description:While protease-activated receptors (PARs) are known to mediate signaling events in CNS, contributing both to normal function and pathogenesis, the endogenous activators of CNS PARs are poorly characterized. In this study, we test the hypothesis that kallikreins (KLKs) represent an important pool of endogenous activators of CNS PARs. Specifically, KLK1 and KLK6 were examined for their ability to evoke intracellular Ca(2+) flux in a PAR-dependent fashion in NSC34 neurons and Neu7 astrocytes. Both KLKs were also examined for their ability to activate mitogen-activated protein kinases (extracellular signal-regulated kinases, C-Jun N-terminal kinases, and p38) and protein kinase B (AKT) intracellular signaling cascades. Cumulatively, these studies show that KLK6, but not KLK1, signals through PARs. KLK6 evoked intracellular Ca(2+) flux was mediated by PAR1 in neurons and both PAR1 and PAR2 in astrocytes. Importantly, both KLK1 and KLK6 altered the activation state of mitogen-activated protein kinases and AKT, suggestive of important roles for each in CNS neuron and glial differentiation, and survival. The cellular specificity of CNS-KLK activity was underscored by observations that both proteases promoted AKT activation in astrocytes, but inhibited such signaling in neurons. PAR1 and bradykinin receptor inhibitors were used to demonstrate that KLK1-mediated activation of extracellular signal-regulated kinases in neurons occurred in a non-PAR, bradykinin 2 (B2) receptor-dependent fashion, while similar signaling by KLK6 was mediated by the combined activation of PAR1 and B2. Cumulatively results indicate KLK6, but not KLK1 is an activator of CNS PARs, and that both KLKs are poised to signal in a B2 receptor-dependent fashion to regulate multiple signal transduction pathways relevant to CNS physiologic function and dysfunction.
Project description:Clostridium perfringens epsilon toxin (?-toxin) is responsible for a devastating multifocal central nervous system (CNS) white matter disease in ruminant animals. The mechanism by which ?-toxin causes white matter damage is poorly understood. In this study, we sought to determine the molecular and cellular mechanisms by which ?-toxin causes pathological changes to white matter. In primary CNS cultures, ?-toxin binds to and kills oligodendrocytes but not astrocytes, microglia, or neurons. In cerebellar organotypic culture, ?-toxin induces demyelination, which occurs in a time- and dose-dependent manner, while preserving neurons, astrocytes, and microglia. ?-Toxin specificity for oligodendrocytes was confirmed using enriched glial culture. Sensitivity to ?-toxin is developmentally regulated, as only mature oligodendrocytes are susceptible to ?-toxin; oligodendrocyte progenitor cells are not. ?-Toxin sensitivity is also dependent on oligodendrocyte expression of the proteolipid myelin and lymphocyte protein (MAL), as MAL-deficient oligodendrocytes are insensitive to ?-toxin. In addition, ?-toxin binding to white matter follows the spatial and temporal pattern of MAL expression. A neutralizing antibody against ?-toxin inhibits oligodendrocyte death and demyelination. This study provides several novel insights into the action of ?-toxin in the CNS. (i) ?-Toxin causes selective oligodendrocyte death while preserving all other neural elements. (ii) ?-Toxin-mediated oligodendrocyte death is a cell autonomous effect. (iii) The effects of ?-toxin on the oligodendrocyte lineage are restricted to mature oligodendrocytes. (iv) Expression of the developmentally regulated proteolipid MAL is required for the cytotoxic effects. (v) The cytotoxic effects of ?-toxin can be abrogated by an ?-toxin neutralizing antibody.Our intestinal tract is host to trillions of microorganisms that play an essential role in health and homeostasis. Disruption of this symbiotic relationship has been implicated in influencing or causing disease in distant organ systems such as the brain. Epsilon toxin (?-toxin)-carrying Clostridium perfringens strains are responsible for a devastating white matter disease in ruminant animals that shares similar features with human multiple sclerosis. In this report, we define the mechanism by which ?-toxin causes white matter disease. We find that ?-toxin specifically targets the myelin-forming cells of the central nervous system (CNS), oligodendrocytes, leading to cell death. The selectivity of ?-toxin for oligodendrocytes is remarkable, as other cells of the CNS are unaffected. Importantly, ?-toxin-induced oligodendrocyte death results in demyelination and is dependent on expression of myelin and lymphocyte protein (MAL). These results help complete the mechanistic pathway from bacteria to brain by explaining the specific cellular target of ?-toxin within the CNS.
Project description: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.
Project description:The inwardly rectifying K+ channel Kir4.1 is broadly expressed by CNS glia and deficits in Kir4.1 lead to seizures and myelin vacuolization. However, the role of oligodendrocyte Kir4.1 channels in controlling myelination and K+ clearance in white matter has not been defined. Here, we show that selective deletion of Kir4.1 from oligodendrocyte progenitors (OPCs) or mature oligodendrocytes did not impair their development or disrupt the structure of myelin. However, mice lacking oligodendrocyte Kir4.1 channels exhibited profound functional impairments, including slower clearance of extracellular K+ and delayed recovery of axons from repetitive stimulation in white matter, as well as spontaneous seizures, a lower seizure threshold, and activity-dependent motor deficits. These results indicate that Kir4.1 channels in oligodendrocytes play an important role in extracellular K+ homeostasis in white matter, and that selective loss of this channel from oligodendrocytes is sufficient to impair K+ clearance and promote seizures.
Project description:Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease hypothesized to promote inflammation via cleavage of protease-activated receptor 1 (PAR1) and PAR2. KLK6 levels are elevated in multiple inflammatory and autoimmune conditions, but no definitive role in pathogenesis has been established. Here, we show that skin-targeted overexpression of KLK6 causes generalized, severe psoriasiform dermatitis with spontaneous development of debilitating psoriatic arthritis-like joint disease. The psoriatic skin and joint phenotypes are reversed by normalization of skin KLK6 levels and attenuated following genetic elimination of PAR1 but not PAR2. Conservation of this regulatory pathway was confirmed in human psoriasis using vorapaxar, an FDA-approved PAR1 antagonist, on explanted lesional skin from patients with psoriasis. Beyond defining a critical role for KLK6/PAR1 signaling in promoting psoriasis, our results demonstrate that KLK6/PAR1-mediated inflammation in the skin alone is sufficient to drive inflammatory joint disease. Further, we identify PAR1 as a promising cytokine-independent target in therapy of psoriasis and psoriatic arthritis.
Project description:Oligodendrocytes, which are the main cell type in cerebral white matter, are generated from their precursor cells (oligodendrocyte precursor cells: OPCs). However, the differentiation from OPCs to oligodendrocytes is disturbed under stressed conditions. Therefore, drugs that can improve oligodendrocyte regeneration may be effective for white matter-related diseases. Here we show that a vasoactive peptide adrenomedullin (AM) promotes the in vitro differentiation of OPCs under pathological conditions. Primary OPCs were prepared from neonatal rat brains, and differentiated into myelin-basic-protein expressing oligodendrocytes over time. This in vitro OPC differentiation was inhibited by prolonged chemical hypoxic stress induced by non-lethal CoCl(2) treatment. However, AM promoted the OPC differentiation under the hypoxic stress conditions, and the AM receptor antagonist AM(22-52) canceled the AM-induced OPC differentiation. In addition, AM treatment increased the phosphorylation level of Akt in OPC cultures, and correspondingly, the PI3K/Akt inhibitor LY294002 blocked the AM-induced OPC differentiation. Taken together, AM treatment rescued OPC maturation under pathological conditions via an AM-receptor-PI3K/Akt pathway. Oligodendrocytes play critical roles in white matter by forming myelin sheath. Therefore, AM signaling may be a promising therapeutic target to boost oligodendrocyte regeneration in CNS disorders.
Project description:The serine/threonine kinase Akt regulates multiple cellular functions. The current studies identify a new role for Akt in CNS myelination. In earlier studies on cultured oligodendrocytes, we showed that neuregulin signals through phosphatidylinositol-3'-OH kinase and Akt to enhance survival of oligodendrocytes. However, when transgenic animals were generated that overexpressed constitutively active Akt in oligodendrocytes and their progenitor cells, no enhanced survival of oligodendrocytes or progenitors was found. No alteration in the proliferation or death of progenitors was noted. In contrast, the major impact of Akt overexpression in oligodendrocytes was enhanced myelination. Most interestingly, oligodendrocytes in these mice continued actively myelinating throughout life. Thus, expression of constitutively active Akt in oligodendrocytes and their progenitor cells generated no more oligodendrocytes, but dramatically more myelin. The increased myelination continued as these mice aged, resulting in enlarged optic nerves and white matter areas. In older animals with enlarged white matter areas, the density of oligodendrocytes was reduced, but because of the increased area, the total number of oligodendrocytes remained comparable with wild-type controls. Interestingly, in these animals, overexpression of Akt in Schwann cells did not impact myelination. Thus, in vivo, constitutively active Akt enhances CNS myelination but not PNS myelination and has no impact developmentally on oligodendrocyte number. Understanding the unique aspects of Akt signal transduction in oligodendrocytes that lead to myelination rather than uncontrolled proliferation of oligodendrocyte progenitor cells may have important implications for understanding remyelination in the adult nervous system.
Project description:Oligodendrocyte lineage cells are susceptible to a variety of insults including hypoxia, excitotoxicity, and reactive oxygen species. Demyelination is a well-recognized feature of several CNS disorders including multiple sclerosis, white matter strokes, progressive multifocal leukoencephalopathy, and disorders due to mitochondrial DNA mutations. Although mitochondria have been implicated in the demise of oligodendrocyte lineage cells, the consequences of mitochondrial respiratory chain defects have not been examined. We determine the in vitro impact of established inhibitors of mitochondrial respiratory chain complex IV or cytochrome c oxidase on oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes as well as on differentiation capacity of OPCs from P0 rat. Injury to mature oligodendrocytes following complex IV inhibition was significantly greater than to OPCs, judged by cell detachment and mitochondrial membrane potential (MMP) changes, although viability of cells that remained attached was not compromised. Active mitochondria were abundant in processes of differentiated oligodendrocytes and MMP was significantly greater in differentiated oligodendrocytes than OPCs. MMP dissipated following complex IV inhibition in oligodendrocytes. Furthermore, complex IV inhibition impaired process formation within oligodendrocyte lineage cells. Injury to and impaired process formation of oligodendrocytes following complex IV inhibition has potentially important implications for the pathogenesis and repair of CNS myelin disorders.
Project description:Oligodendrocytes, the myelin-forming glial cells of the central nervous system (CNS), are fundamental players in rapid impulse conduction and normal axonal functions. JunB and c-Jun are DNA-binding components of the AP-1 transcription factor, which is known to regulate different processes such as proliferation, differentiation, stress responses and death in several cell types, including cultured oligodendrocyte/lineage cells. By selectively inactivating Jun B and c-Jun in myelinating oligodendrocytes in vivo, we generated mutant mice that developed normally, and within more than 12 months showed normal ageing and survival rates. In the adult CNS, absence of JunB and c-Jun from mature oligodendrocytes caused low-grade glial activation without overt signs of demyelination or secondary leukocyte infiltration into the brain. Even after exposure to toxic or autoimmune oligodendrocyte insults, signs of altered oligodendrocyte viability were mild and detectable only upon cuprizone treatment. We conclude that JunB and c-Jun expression in post-mitotic oligodendrocytes is mostly dispensable for the maintainance of white matter tracts throughout adult life, even under demyelinating conditions.
Project description:Investigating human oligodendrogenesis and the interaction of oligodendrocytes with neurons and astrocytes would accelerate our understanding of the mechanisms underlying white matter disorders. However, this is challenging because of the limited accessibility of functional human brain tissue. Here, we developed a new differentiation method of human induced pluripotent stem cells to generate three-dimensional brain organoids that contain oligodendrocytes as well as neurons and astrocytes, called human oligodendrocyte spheroids. We found that oligodendrocyte lineage cells derived in human oligodendrocyte spheroids transitioned through developmental stages similar to primary human oligodendrocytes and that the migration of oligodendrocyte lineage cells and their susceptibility to lysolecithin exposure could be captured by live imaging. Moreover, their morphology changed as they matured over time in vitro and started myelinating neurons. We anticipate that this method can be used to study oligodendrocyte development, myelination, and interactions with other major cell types in the CNS.