Urokinase-type plasminogen activator promotes dendritic spine recovery and improves neurological outcome following ischemic stroke.

Spines are dendritic protrusions that receive most of the excitatory input in the brain. Early after the onset of cerebral ischemia dendritic spines in the peri-infarct cortex are replaced by areas of focal swelling, and their re-emergence from these varicosities is associated with neurological recovery after acute ischemic stroke (AIS). Urokinase-type plasminogen activator (uPA) is a serine proteinase that plays a central role in tissue remodeling via binding to the urokinase plasminogen activator receptor (uPAR). We report that cerebral cortical neurons release uPA during the recovery phase from ischemic stroke in vivo or hypoxia in vitro. Although uPA does not have an effect on ischemia- or hypoxia-induced neuronal death, genetic deficiency of uPA (uPA(-/-)) or uPAR (uPAR(-/-)) abrogates functional recovery after AIS. Treatment with recombinant uPA after ischemic stroke induces neurological recovery in wild-type and uPA(-/-) but not in uPAR(-/-) mice. Diffusion tensor imaging studies indicate that uPA(-/-) mice have increased water diffusivity and decreased anisotropy associated with impaired dendritic spine recovery and decreased length of distal neurites in the peri-infarct cortex. We found that the excitotoxic injury induces the clustering of uPAR in dendritic varicosities, and that the binding of uPA to uPAR promotes the reorganization of the actin cytoskeleton and re-emergence of dendritic filopodia from uPAR-enriched varicosities. This effect is independent of uPA's proteolytic properties and instead is mediated by Rac-regulated profilin expression and cofilin phosphorylation. Our data indicate that binding of uPA to uPAR promotes dendritic spine recovery and improves functional outcome following AIS.

Tissue-type plasminogen activator protects neurons from excitotoxin-induced cell death via activation of the ERK1/2-CREB-ATF3 signaling pathway.

The release of the serine proteinase tissue-type plasminogen activator (tPA) from cerebral cortical neurons has a neuroprotective effect in the ischemic brain. Because excitotoxicity is a basic mechanism of ischemia-induced cell death, here we investigated the effect of tPA on excitotoxin-induced neuronal death. We report that genetic overexpression of neuronal tPA or treatment with recombinant tPA renders neurons resistant to the harmful effects of an excitotoxic injury in vitro and in vivo. We found that at concentrations found in the ischemic brain, tPA interacts with synaptic but not extrasynaptic NMDARs. This effect is independent of tPA's proteolytic properties and leads to a rapid and transient phosphorylation of the extracellular signal regulated kinases1/2 (ERK1/2), with ERK1/2-mediated activation of the cAMP response element binding protein (CREB) and induction of the neuroprotective CREB-regulated activating transcription factor 3 (Atf3). In line with these observations, Atf3 down-regulation abrogates the protective effect of tPA against excitotoxin-induced neuronal death. Our data indicate that tPA preferentially activates synaptic NMDARs via a plasminogen-independent mechanism turning on a cell signaling pathway that protects neurons from the deleterious effects of excitotoxicity.

Tissue-type plasminogen activator regulates the neuronal uptake of glucose in the ischemic brain.

The ability to sense and adapt to hypoxic conditions plays a pivotal role in neuronal survival. Hypoxia induces the release of tissue-type plasminogen activator (tPA) from cerebral cortical neurons. We found that the release of neuronal tPA or treatment with recombinant tPA promotes cell survival in cerebral cortical neurons previously exposed to hypoxic conditions in vitro or experimental cerebral ischemia in vivo. Our studies using liquid chromatography and tandem mass spectrometry revealed that tPA activates the mammalian target of rapamycin (mTOR) pathway, which adapts cellular processes to the availability of energy and metabolic resources. We found that mTOR activation leads to accumulation of the hypoxia-inducible factor-1α (HIF-1α) and induction and recruitment to the cell membrane of the HIF-1α-regulated neuronal transporter of glucose GLUT3. Accordingly, in vivo positron emission tomography studies with 18-fluorodeoxyglucose in mice overexpressing tPA in neurons show that neuronal tPA induces the uptake of glucose in the ischemic brain and that this effect is associated with a decrease in the volume of the ischemic lesion and improved neurological outcome following the induction of ischemic stroke. Our data indicate that tPA activates a cell signaling pathway that allows neurons to sense and adapt to oxygen and glucose deprivation.

The cytokine tumor necrosis factor-like weak inducer of apoptosis and its receptor fibroblast growth factor-inducible 14 have a neuroprotective effect in the central nervous system.

BACKGROUND: Cerebral cortical neurons have a high vulnerability to the harmful effects of hypoxia. However, the brain has the ability to detect and accommodate to hypoxic conditions. This phenomenon, known as preconditioning, is a natural adaptive process highly preserved among species whereby exposure to sub-lethal hypoxia promotes the acquisition of tolerance to a subsequent lethal hypoxic injury. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are found in neurons and their expression is induced by exposure to sub-lethal hypoxia. Accordingly, in this work we tested the hypothesis that the interaction between TWEAK and Fn14 induces tolerance to lethal hypoxic and ischemic conditions. METHODS: Here we used in vitro and in vivo models of hypoxic and ischemic preconditioning, an animal model of transient middle cerebral artery occlusion and mice and neurons genetically deficient in TWEAK, Fn14, or tumor necrosis factor alpha (TNF-α) to investigate whether treatment with recombinant TWEAK or an increase in the expression of endogenous TWEAK renders neurons tolerant to lethal hypoxia. We used enzyme-linked immunosorbent assay to study the effect of TWEAK on the expression of neuronal TNF-α, Western blot analysis to investigate whether the effect of TWEAK was mediated by activation of mitogen-activated protein kinases and immunohistochemical techniques and quantitative real-time polymerase chain reaction analysis to study the effect of TWEAK on apoptotic cell death. RESULTS: We found that either treatment with recombinant TWEAK or an increase in the expression of TWEAK and Fn14 induce hypoxic and ischemic tolerance in vivo and in vitro. This protective effect is mediated by neuronal TNF-α and activation of the extracellular signal-regulated kinases 1 and 2 pathway via phosphorylation and inactivation of the B-cell lymphoma 2-associated death promoter protein. CONCLUSIONS: Our work indicate that the interaction between TWEAK and Fn14 triggers the activation of a cell signaling pathway that results in the induction of tolerance to lethal hypoxia and ischemia. These data indicate that TWEAK may be a potential therapeutic strategy to protect the brain from the devastating effects of an ischemic injury.

Tissue-type plasminogen activator has a neuroprotective effect in the ischemic brain mediated by neuronal TNF-α.

Cerebral cortical neurons have a heightened sensitivity to hypoxia and their survival depends on their ability to accommodate to changes in the concentration of oxygen in their environment. Tissue-type plasminogen activator (tPA) is a serine proteinase that activates the zymogen plasminogen into plasmin. Hypoxia induces the release of tPA from cerebral cortical neurons, and it has been proposed that tPA mediates hypoxic and ischemic neuronal death. Here, we show that tPA is devoid of neurotoxic effects and instead is an endogenous neuroprotectant that renders neurons resistant to the effects of lethal hypoxia and ischemia. We present in vitro and in vivo evidence indicating that endogenous tPA and recombinant tPA induce the expression of neuronal tumor necrosis factor-α. This effect, mediated by plasmin and the N-methyl-D-aspartate receptor, leads to increased expression of the cyclin-dependent kinase inhibitor p21 and p21-mediated development of early hypoxic and ischemic tolerance.

Neuroserpin protects neurons from ischemia-induced plasmin-mediated cell death independently of tissue-type plasminogen activator inhibition.

The serine proteinase tissue-type plasminogen activator (tPA) and the serine proteinase inhibitor neuroserpin are both expressed in areas of the brain with the highest vulnerability to hypoxia/ischemia. In vitro studies show that neuroserpin inhibits tPA and, to a lesser extent, urokinase-type plasminogen activator and plasmin. Experimental middle cerebral artery occlusion (MCAO) increases tPA activity and neuroserpin expression in ischemic tissue, and genetic deficiency of tPA or either treatment with or overexpression of neuroserpin decreases the volume of the ischemic lesion following MCAO. These findings have led to the hypothesis that neuroserpin's neuroprotection is mediated by inhibition of tPA's alleged neurotoxic effect. Ischemic preconditioning is a natural adaptive process whereby exposure to a sublethal insult induces tolerance against a subsequent lethal ischemic injury. Here we demonstrate that exposure to sublethal hypoxia/ischemia increases the neuroserpin expression in the hippocampal CA1 layer and cerebral cortex, and that neuroserpin induces ischemic tolerance and decreases the volume of the ischemic lesion following MCAO in wild-type and tPA-deficient (tPA-/-) neurons and mice. Plasmin induces neuronal death, and this effect is abrogated by either neuroserpin or the NMDA receptor antagonist MK-801. Neuroserpin also attenuated kainic acid-induced neuronal death. Our data indicate that the neuroprotective effect of neuroserpin is due to inhibition of plasmin-mediated excitotoxin-induced cell death and is independent of neuroserpin's ability to inhibit tPA activity.

Tissue-type plasminogen activator is a neuroprotectant in the mouse hippocampus.

The best-known function of the serine protease tissue-type plasminogen activator (tPA) is as a thrombolytic enzyme. However, it is also found in structures of the brain that are highly vulnerable to hypoxia-induced cell death, where its association with neuronal survival is poorly understood. Here, we have demonstrated that hippocampal areas of the mouse brain lacking tPA activity are more vulnerable to neuronal death following an ischemic insult. We found that sublethal hypoxia, which elicits tolerance to subsequent lethal hypoxic/ischemic injury in a natural process known as ischemic preconditioning (IPC), induced a rapid release of neuronal tPA. Treatment of hippocampal neurons with tPA induced tolerance against a lethal hypoxic insult applied either immediately following insult (early IPC) or 24 hours later (delayed IPC). tPA-induced early IPC was independent of the proteolytic activity of tPA and required the engagement of a member of the LDL receptor family. In contrast, tPA-induced delayed IPC required the proteolytic activity of tPA and was mediated by plasmin, the NMDA receptor, and PKB phosphorylation. We also found that IPC in vivo increased tPA activity in the cornu ammonis area 1 (CA1) layer and Akt phosphorylation in the hippocampus, as well as ischemic tolerance in wild-type but not tPA- or plasminogen-deficient mice. These data show that tPA can act as an endogenous neuroprotectant in the murine hippocampus.

The interaction between tumor necrosis factor-like weak inducer of apoptosis and its receptor fibroblast growth factor-inducible 14 promotes the recruitment of neutrophils into the ischemic brain.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are expressed in endothelial cells and perivascular astrocytes. Here, we show that TWEAK induces a dose-dependent increase in the expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in astrocytes, and that this effect is mediated by its interaction with Fn14 via nuclear factor-kappaB pathway activation. Exposure to oxygen-glucose deprivation (OGD) conditions increases TWEAK and Fn14 mRNA expression in wild-type (Wt) astrocytic cultures. Likewise, incubation under OGD conditions induces the expression of MCP-1 in Wt astrocytes but not in astrocytes deficient on either TWEAK (TWEAK(-/-)) or Fn14 (Fn14(-/-)). We also found that TWEAK induces the passage of neutrophils to the abluminal side of an in vitro model of the blood-brain barrier. Our earlier studies indicate that cerebral ischemia increases the expression of TWEAK and Fn14 in the endothelial cell-basement membrane-astrocyte interface. Here, we report that middle cerebral artery occlusion increases the expression of MCP-1 and the recruitment of neutrophils into the ischemic tissue in Wt but not in TWEAK(-/-) or Fn14(-/-) mice. These novel results indicate that during cerebral ischemia, the interaction between TWEAK and Fn14 leads to the recruitment of leukocytes into the ischemic tissue.

Microglial low-density lipoprotein receptor-related protein 1 mediates the effect of tissue-type plasminogen activator on matrix metalloproteinase-9 activity in the ischemic brain.

Studies in animal models of cerebral ischemia indicate that besides its thrombolytic effect, treatment with tissue-type plasminogen activator (tPA) also induces an increase in matrix metalloproteinase-9 (MMP-9) activity in the ischemic tissue associated with the development of cerebral edema. Earlier, we had shown that the low-density lipoprotein receptor-related protein 1 (LRP1) is a substrate for tPA in the brain. In this study, we investigated the effect of the interaction between tPA and microglial LRP1 on MMP-9 activity after middle cerebral artery occlusion (MCAO). We found that exposure to oxygen-glucose deprivation (OGD) conditions increases MMP-9 activity in wild-type (Wt) and plasminogen-deficient (Plg(-/-)) microglia, but not in tPA (tPA(-/-)) or LRP1-deficient (macLRP-) cells. Treatment with tPA increases MMP-9 expression in tPA(-/-) but not in macLRP- microglia. Middle cerebral artery occlusion increases MMP-9 expression and activity in Wt but not in tPA(-/-) or macLRP- mice, and treatment with tPA increases MMP-9 activity in tPA(-/-) mice but not in macLRP- animals. Finally, MCAO-induced ischemic edema and degradation of the interendothelial right junction protein claudin-5 were significantly attenuated in tPA(-/-) and macLRP- mice. The results of our study indicate that the interaction between tPA and microglial LRP1 increases MMP-9 expression and activity resulting in the degradation of claudin-5 and development of cerebral edema.