Woldeab B. Haile

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.

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.

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 tPAs 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 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.

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.

TUMOR NECROSIS FACTOR-LIKE WEAK INDUCER OF APOPTOSIS AND FIBROBLAST GROWTH FACTOR-INDUCIBLE 14 MEDIATE CEREBRAL ISCHEMIA-INDUCED POLY(ADP-ribose) POLYMERASE-1 ACTIVATION AND NEURONAL DEATH

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fibroblast growth factor-inducible 14 (Fn14) are expressed in neurons. Here we demonstrate that TWEAK induces a dose-dependent increase in neuronal death and that this effect is independent of tumor necrosis factor alpha (TNF-α) and mediated by nuclear factor-kappa B (NF-κB) pathway activation. Incubation with TWEAK induces apoptotic cell death in wild-type (Wt) but not in Fn14 deficient (Fn14(-/-)) neurons. Intracerebral injection of TWEAK induces accumulation of poly(ADP-ribose) polymers (PAR) in Wt but not in Fn14(-/-) mice. Exposure to oxygen-glucose deprivation (OGD) conditions increases TWEAK and Fn14 mRNA expression in Wt neurons, and decreases cell survival in Wt but not in Fn14(-/-) or TWEAK deficient (TWEAK(-/-)) neurons. Experimental middle cerebral artery occlusion (MCAO) increases the expression of TWEAK and Fn14 mRNA and active caspase-3, and the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) with accumulation of PAR in the ischemic area in Wt but not Fn14(-/-) mice. Together, these results suggest a model where in response to hypoxia/ischemia the interaction between TWEAK and Fn14 in neurons induces PARP-1 activation with accumulation of PAR polymers and cell death via NF-κB pathway activation. This is a novel pathway for hypoxia/ischemia-induced TWEAK-mediated cell death and a potential therapeutic target for ischemic stroke.

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-κB 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.

Tissue-type plasminogen activator mediates neuroglial coupling in the central nervous system.

The interaction between neurons, astrocytes and endothelial cells plays a central role coupling energy supply with changes in neuronal activity. For a long time it was believed that glucose was the only source of energy for neurons. However, a growing body of experimental evidence indicates that lactic acid, generated by aerobic glycolysis in perivascular astrocytes, is also a source of energy for neuronal activity, particularly when the supply of glucose from the intravascular space is interrupted. Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved kinase that couples cellular activity with energy consumption via induction of the uptake of glucose and activation of the glycolytic pathway. The uptake of glucose by the blood-brain barrier is mediated by glucose transporter-1 (GLUT1), which is abundantly expressed in endothelial cells and astrocytic end-feet processes. Tissue-type plasminogen activator (tPA) is a serine proteinase that is found in endothelial cells, astrocytes and neurons. Genetic overexpression of neuronal tPA or treatment with recombinant tPA protects neurons from the deleterious effects of metabolic stress or excitotoxicity, via a mechanism independent of tPAs ability to cleave plasminogen into plasmin. The work presented here shows that exposure to metabolic stress induces the rapid release of tPA from murine neurons but not from astrocytes. This tPA induces AMPK activation, membrane recruitment of GLUT1, and GLUT1-mediated glucose uptake in astrocytes and endothelial cells. Our data indicate that this is followed by the synthesis and release of lactic acid from astrocytes, and that the uptake of this lactic acid via the monocarboxylate transporter-2 promotes survival in neurons exposed to metabolic stress.

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.

BackgroundCerebral 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.MethodsHere 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.ResultsWe 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.ConclusionsOur 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 mediates neuronal detection and adaptation to metabolic stress

Adenosine monophosphate-activated protein kinase (AMPK) is an energy sensor that regulates cellular adaptation to metabolic stress. Tissue-type plasminogen activator (tPA) is a serine proteinase found in the intravascular space, where its main role is as thrombolytic enzyme, and in neurons, where its function is less well understood. Here, we report that glucose deprivation induces the mobilization and package of neuronal tPA into presynaptic vesicles. Mass spectrometry and immunohistochemical studies show that the release of this tPA in the synaptic space induces AMPK activation in the postsynaptic terminal, and an AMPK-mediated increase in neuronal uptake of glucose and neuronal adenosine 5′(tetrahydrogen triphosphate; ATP) synthesis. This effect is independent of tPAs proteolytic properties, and instead requires the presence of functional N-methyl-D-aspartate receptors (NMDARs). In agreement with these observations, positron emission tomography (PET) studies and biochemical analysis with synaptoneurosomes indicate that the intravenous administration of recombinant tPA (rtPA) after transient middle cerebral artery occlusion (tMCAO) induces AMPK activation in the synaptic space and NMDAR-mediated glucose uptake in the ischemic brain. These data indicate that the release of neuronal tPA or treatment with rtPA activate a cell signaling pathway in the synaptic space that promotes the detection and adaptation to metabolic stress.