Jie An

Tissue-type plasminogen activator and the low-density lipoprotein receptor–related protein induce Akt phosphorylation in the ischemic brain

Tissue-type plasminogen activator (tPA) is found in the intravascular space and in the central nervous system. The low-density lipoprotein receptor-related protein (LRP) is expressed in neurons and in perivascular astrocytes. During cerebral ischemia, tPA induces the shedding of LRPs extracellular domain from perivascular astrocytes, and this is followed by the development of cerebral edema. Protein kinase B (Akt) is a serine/threonine kinase that plays a critical role not only in cell survival but also in the regulation of the permeability of the blood-brain barrier. We found that, in the early phases of the ischemic insult, the interaction between tPA and LRP induces Akt phosphorylation (pAkt) in perivascular astrocytes and inhibits pAkt in neurons. Coimmunoprecipitation studies indicate that pAkt and LRPs intracellular domain interact in perivascular astrocytes and that this interaction is dependent on the presence of tPA and results in the development of edema. Together, these results indicate that, in the early stages of cerebral ischemia, the interaction between tPA and LRP in perivascular astrocytes induces the activation of a cell signaling event mediated by pAkt that leads to increase in the permeability of the blood-brain barrier.

The low-density lipoprotein receptor-related protein 1 mediates tissue-type plasminogen activator-induced microglial activation in the ischemic brain.

Microglia are the immune cells of the central nervous system (CNS) that become activated in response to pathological situations such as cerebral ischemia. Tissue-type plasminogen activator (tPA) is a serine proteinase that is found in the intravascular space and the CNS. The low-density lipoprotein receptor-related protein 1 (LRP1) is a member of the low-density lipoprotein receptor gene family found in neurons, astrocytes, and microglia. The present study investigated whether the interaction between tPA and microglial LRP1 plays a role in cerebral ischemia-induced microglial activation. We found that middle cerebral artery occlusion (MCAO) induces microglial activation in both wild-type and plasminogen-deficient (Plg(-/-)) mice. In contrast, MCAO-induced microglial activation is significantly decreased in tPA-deficient (tPA(-/-)) mice and in mice that lack LRP1 in microglial cells (macLRP(-)). We observed a significant increase in microglial activation when tPA(-/-) mice received treatment with murine tPA after MCAO. In contrast, treatment of macLRP(-) mice with tPA did not have an effect on the extent of microglial activation. Finally, both the volume of the ischemic lesion as well as inducible nitric oxide synthase production were significantly decreased in macLRP(-) mice and macLRP(-) microglia. In summary, our results indicate that the interaction between tPA and LRP1 induces microglial activation with the generation of an inflammatory response in the ischemic brain, suggesting a cytokine-like role for tPA in the CNS.

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.

Phosphoinositide 3-Kinase Enhancer Regulates Neuronal Dendritogenesis and Survival in Neocortex

Phosphoinositide 3-kinase enhancer (PIKE) binds and enhances phosphatidylinositol 3-kinase (PI3K)/Akt activities. However, its physiological functions in brain have never been explored. Here we show that PIKE is important in regulating the neuronal survival and development of neocortex. During development, enhanced apoptosis is observed in the ventricular zone of PIKE knock-out (PIKE−/−) cortex. Moreover, PIKE−/− neurons show reduced dendritic complexity, dendritic branch length, and soma size. These defects are due to the reduced PI3K/Akt activities in PIKE−/− neurons, as the impaired dendritic arborization can be rescued when PI3K/Akt cascade is augmented in vitro or in PIKE−/−PTEN−/− double-knock-out mice. Interestingly, PIKE−/− mice display behavioral abnormality in locomotion and spatial navigation. Because of the diminished PI3K/Akt activities, PIKE−/− neurons are more vulnerable to glutamate- or stroke-induced neuronal cell death. Together, our data established the critical role of PIKE in regulating neuronal survival and development by substantiating the PI3K/Akt pathway.

Regulated Intramembrane Proteolysis of the Low-Density Lipoprotein Receptor-Related Protein Mediates Ischemic Cell Death

The low-density lipoprotein receptor-related protein (LRP), a member of the low-density lipoprotein receptor gene family, mediates cellular signal transduction pathways. In this study we investigated the role of LRP in cell death. We found that incubation of mouse embryonic fibroblasts in serum-free media induces caspase-3 activation, an effect that is attenuated in LRP-deficient (LRP(-/-)) mouse embryonic fibroblasts. Since we previously demonstrated that middle cerebral artery occlusion (MCAO) in mice induces shedding of the LRP ectodomain, we investigated here whether cerebral ischemia induces regulated intramembrane proteolysis of LRP and whether this process is related to cell death. We found that MCAO induces an increase in gamma-secretase activity in the ischemic hemisphere and that treatment with the gamma-secretase inhibitor L-685,458 improves the neurological outcome and results in a 50% decrease in the volume of the ischemic lesion. Furthermore, MCAO caused nuclear translocation of the intracellular domain of LRP in neurons within the area of ischemic penumbra, and this effect was attenuated in mice treated with L-685,458. Finally, inhibition of either LRP or gamma-secretase attenuated cerebral ischemia-induced caspase-3 cleavage and apoptotic cell death. In summary, our results indicate that gamma-secretase-mediated regulated intramembrane proteolysis of LRP results in cell death under ischemic conditions.

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.

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.