Urokinase-Type Plasminogen Activator Triggers Wingless/Int1-Independent Phosphorylation of the Low-Density Lipoprotein Receptor-Related Protein-6 in Cerebral Cortical Neurons.

BACKGROUND: Urokinase-type plasminogen activator (uPA) is a serine proteinase found in excitatory synapses located in the II/III and V cortical layers. The synaptic release of uPA promotes the formation of synaptic contacts and the repair of synapses damaged by various forms of injury, and its abundance is decreased in the synapse of Alzheimer's disease (AD) patients. Inactivation of the Wingless/Int1 (Wnt)-ß-catenin pathway plays a central role in the pathogenesis of AD. Soluble amyloid-ß (Aß) prevents the phosphorylation of the low-density lipoprotein receptor-related protein-6 (LRP6), and the resultant inactivation of the Wnt-ß-catenin pathway prompts the amyloidogenic processing of the amyloid-ß protein precursor (AßPP) and causes synaptic loss. OBJECTIVE: To study the role of neuronal uPA in the pathogenesis of AD. METHODS: We used in vitro cultures of murine cerebral cortical neurons, a murine neuroblastoma cell line transfected with the APP-695 Swedish mutation (N2asw), and mice deficient on either plasminogen, or uPA, or its receptor (uPAR). RESULTS: We show that uPA activates the Wnt-ß-catenin pathway in cerebral cortical neurons by triggering the phosphorylation of LRP6 via a plasmin-independent mechanism that does not require binding of Wnt ligands (Wnts). Our data indicate that uPA-induced activation of the Wnt-ß-catenin pathway protects the synapse from the harmful effects of soluble Aß and prevents the amyloidogenic processing of AßPP by inhibiting the expression of ß-secretase 1 (BACE1) and the ensuing generation of Aß40 and Aß42 peptides. CONCLUSION: uPA protects the synapse and antagonizes the inhibitory effect of soluble Aß on the Wnt-ß-catenin pathway by providing an alternative pathway for LRP6 phosphorylation and ß-catenin stabilization.

Tissue-type plasminogen activator induces TNF-α-mediated preconditioning of the blood-brain barrier.

Ischemic tolerance is a phenomenon whereby transient exposure to a non-injurious preconditioning stimulus triggers resistance to a subsequent lethal ischemic insult. Despite the fact that not only neurons but also astrocytes and endothelial cells have a unique response to preconditioning stimuli, current research has been focused mostly on the effect of preconditioning on neuronal death. Thus, it is unclear if the blood-brain barrier (BBB) can be preconditioned independently of an effect on neuronal survival. The release of tissue-type plasminogen activator (tPA) from perivascular astrocytes in response to an ischemic insult increases the permeability of the BBB. In line with these observations, treatment with recombinant tPA increases the permeability of the BBB and genetic deficiency of tPA attenuates the development of post-ischemic edema. Here we show that tPA induces ischemic tolerance in the BBB independently of an effect on neuronal survival. We found that tPA renders the BBB resistant to an ischemic injury by inducing TNF-α-mediated astrocytic activation and increasing the abundance of aquaporin-4-immunoreactive astrocytic end-feet processes in the neurovascular unit. This is a new role for tPA, that does not require plasmin generation, and with potential therapeutic implications for patients with cerebrovascular disease.

Microglial-mediated PDGF-CC activation increases cerebrovascular permeability during ischemic stroke.

Treatment of acute ischemic stroke with the thrombolytic tissue plasminogen activator (tPA) can significantly improve neurological outcomes; however, thrombolytic therapy is associated with an increased risk of intra-cerebral hemorrhage (ICH). Previously, we demonstrated that during stroke tPA acting on the parenchymal side of the neurovascular unit (NVU) can increase blood-brain barrier (BBB) permeability and ICH through activation of latent platelet-derived growth factor-CC (PDGF-CC) and signaling by the PDGF receptor-α (PDGFRα). However, in vitro, activation of PDGF-CC by tPA is very inefficient and the mechanism of PDGF-CC activation in the NVU is not known. Here, we show that the integrin Mac-1, expressed on brain microglia/macrophages (denoted microglia throughout), acts together with the endocytic receptor LRP1 in the NVU to promote tPA-mediated activation of PDGF-CC. Mac-1-deficient mice (Mac-1-/-) are protected from tPA-induced BBB permeability but not from permeability induced by intracerebroventricular injection of active PDGF-CC. Immunofluorescence analysis demonstrates that Mac-1, LRP1, and the PDGFRα all localize to the NVU of arterioles, and following middle cerebral artery occlusion (MCAO) Mac-1-/- mice show significantly less PDGFRα phosphorylation, BBB permeability, and infarct volume compared to wild-type mice. Bone-marrow transplantation studies indicate that resident CD11b+ cells, but not bone-marrow-derived leukocytes, mediate the early activation of PDGF-CC by tPA after MCAO. Finally, using a model of thrombotic stroke with late thrombolysis, we show that wild-type mice have an increased incidence of spontaneous ICH following thrombolysis with tPA 5 h after MCAO, whereas Mac-1-/- mice are resistant to the development of ICH even with late tPA treatment. Together, these results indicate that Mac-1 and LRP1 act as co-factors for the activation of PDGF-CC by tPA in the NVU, and suggest a novel mechanism for tightly regulating PDGFRα signaling in the NVU and controlling BBB permeability.

Coagulation factor XII induces pro-inflammatory cytokine responses in macrophages and promotes atherosclerosis in mice.

Atherosclerosis is considered a chronic inflammatory disease of the vessel wall. Coagulation pathways and immune responses contribute to disease development. The role of coagulation factor XII (FXII) in vascular inflammation, however, remains controversial. We here investigated the function of FXII in atherosclerosis using apolipoprotein E and FXII-deficient (F12-/-Apoe-/-) mice. Compared to F12+/+Apoe-/- controls, atherosclerotic lesion formation was reduced in F12-/-Apoe-/- mice. This was associated with a decrease in serum interleukin (IL)-1ß and IL-12 levels and reduced expression of pro-inflammatory cytokines in the aorta in atherosclerotic F12-/-Apoe-/- mice, as well as diminished Th1-cell differentiation in the aorta, blood, and lymphoid organs. No changes in circulating bradykinin, thrombin-antithrombin-complexes or plasminogen were observed. Mechanistically, activated FXII (FXIIa) was revealed to directly induce bone marrow-derived macrophages to secrete pro-inflammatory cytokines, including tumour necrosis factor-α, IL-1ß, IL-12, and IL-6. Exposure of bone marrow-derived antigen presenting cells to FXIIa similarly induced pro-inflammatory cytokines, and an enhanced capacity to trigger antigen-specific interferon γ-production in CD4+ T cells. Notably, bone-marrow derived macrophages were capable of directly activating FXII. Moreover, the induction of cytokine expression by FXIIa in macrophages occurred independently of FXII protease enzymatic activity and was decreased upon phospholipase C treatment, suggesting urokinase-type plasminogen activator receptor (uPAR) to confer FXIIa-induced cell signalling. These data reveal FXII to play an important role in atherosclerotic lesion formation by functioning as a strong inducer of pro-inflammatory cytokines in antigen-presenting cells. Targeting of FXII may thus be a promising approach for treating cardiovascular disease.

ASPECTS decay during inter-facility transfer predicts patient outcomes in endovascular reperfusion for ischemic stroke: a unique assessment of dynamic physiologic change over time.

BACKGROUND: Pretreatment Alberta Stroke Program Early CT Scores (ASPECTS) is associated with clinical outcomes. The rate of decline between subsequent images, however, may be more predictive of outcomes as it integrates time and physiology. METHODS: A cohort of patients transferred from six primary stroke centers and treated with intra-arterial therapy (IAT) was retrospectively studied. Absolute ASPECTS decay was defined as ((ASPECTS First CT-ASPECTS Second CT)/hours elapsed between images). A logistic regression model was performed to determine if the rate of ASPECTS decay predicted good outcomes at 90 days (modified Rankin Scale score of 0-2). RESULTS: 106 patients with a mean age of 66±14 years and a median National Institutes of Health Stroke Scale score of 19 (IQR 15-23) were analyzed. Median time between initial CT at the outside hospital to repeat CT at our facility was 2.7 h (IQR 2.0-3.6). Patients with good outcomes had lower rates of absolute ASPECTS decay compared with those who did not (0.14±0.23 score/h vs 0.49±0.39 score/h; p<0.001). In multivariable modeling, the absolute rate of ASPECTS decay (OR 0.043; 95% CI 0.004 to 0.471; p=0.01) was a stronger predictor of good patient outcome than static pretreatment ASPECTS obtained before IAT (OR 0.64; 95% CI 0.38 to 1.04; p=0.075). In practical terms, every 1 unit increase in ASPECTS decline per hour correlates with a 23-fold lower probability of a good outcome. CONCLUSIONS: Patients with faster rates of ASPECTS decay during inter-facility transfers are associated with worse clinical outcomes. This value may reflect the rate of physiological infarct expansion and thus serve as a tool in patient selection for IAT.

TWEAK and Fn14 in the Neurovascular Unit.

The neurovascular unit (NVU) is a dynamic structure assembled by endothelial cells (EC), a basement membrane (BM), perivascular astrocytes (PA), pericytes, and surrounding neurons. The NVU regulates the passage of substances and cellular elements from the intravascular space into the brain parenchyma. This function, also known as blood-brain barrier (BBB), is regulated by the integrity of tight junctions proteins between EC, and the interaction between PA and the basal lamina. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are abundantly expressed in the NVU. Here we will review data indicating that the interaction between TWEAK and Fn14 in the endothelial cell-BM-astrocyte interface regulates the function of the BBB following an ischemic/hypoxic injury, and that pharmacological inhibition of TWEAK-Fn14 is a promising target for the treatment of patients with neurological diseases that have a direct impact on the structure and function of the NVU.

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 tPA's 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.

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.

LRAD3, a novel low-density lipoprotein receptor family member that modulates amyloid precursor protein trafficking.

We have identified a novel low-density lipoprotein (LDL) receptor family member, termed LDL receptor class A domain containing 3 (LRAD3), which is expressed in neurons. The LRAD3 gene encodes an ∼50 kDa type I transmembrane receptor with an ectodomain containing three LDLa repeats, a transmembrane domain, and a cytoplasmic domain containing a conserved dileucine internalization motif and two polyproline motifs with potential to interact with WW-domain-containing proteins. Immunohistochemical analysis of mouse brain reveals LRAD3 expression in the cortex and hippocampus. In the mouse hippocampal-derived cell line HT22, LRAD3 partially colocalizes with amyloid precursor protein (APP) and interacts with APP as revealed by coimmunoprecipitation experiments. To identify the portion of APP that interacts with LRAD3, we used solid-phase binding assays that demonstrated that LRAD3 failed to bind to a soluble APP fragment (sAPPα) released after α-secretase cleavage. In contrast, C99, the ß-secretase product that remains cell associated, coprecipitated with LRAD3, confirming that regions within this portion of APP are important for associating with LRAD3. The association of LRAD3 with APP increases the amyloidogenic pathway of APP processing, resulting in a decrease in sAPPα production and increased Aß peptide production. Pulse-chase experiments confirm that LRAD3 expression significantly decreases the cellular half-life of mature APP. These results reveal that LRAD3 influences APP processing and raises the possibility that LRAD3 alters APP function in neurons, including its downstream signaling.

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.

A selective TrkB agonist with potent neurotrophic activities by 7,8-dihydroxyflavone.

Brain-derived neurotrophic factor (BDNF), a cognate ligand for the tyrosine kinase receptor B (TrkB) receptor, mediates neuronal survival, differentiation, synaptic plasticity, and neurogenesis. However, BDNF has a poor pharmacokinetic profile that limits its therapeutic potential. Here we report the identification of 7,8-dihydroxyflavone as a bioactive high-affinity TrkB agonist that provokes receptor dimerization and autophosphorylation and activation of downstream signaling. 7,8-Dihydroxyflavone protected wild-type, but not TrkB-deficient, neurons from apoptosis. Administration of 7,8-dihydroxyflavone to mice activated TrkB in the brain, inhibited kainic acid-induced toxicity, decreased infarct volumes in stroke in a TrkB-dependent manner, and was neuroprotective in an animal model of Parkinson disease. Thus, 7,8-dihydroxyflavone imitates BDNF and acts as a robust TrkB agonist, providing a powerful therapeutic tool for the treatment of various neurological diseases.

Anxiety is a better predictor of platelet reactivity in coronary artery disease patients than depression.

AIMS: Depression and anxiety are linked to coronary events but the mechanism(s) remains unclear. We investigated the associations of depression and anxiety with serotonin-mediated platelet hyperactivity in coronary artery disease (CAD) patients in a cross-sectional study. METHODS AND RESULTS: Three months after an acute coronary event, stable CAD patients (n = 83) on aspirin and clopidogrel were evaluated for depression (beck depression inventory) and anxiety (hospital anxiety and depression scale), and their platelet reactivity was measured (optical aggregometry and flow cytometric fibrinogen binding in response to adenosine diphosphate (ADP = 5 microM) and two serotonin + epinephrine doses [5HT:E (L) = 4 microM + 4 microM and 5HT:E (H) = 10 microM + 4 microM]. Platelet reactivity was significantly higher in depressed and anxious than in depressed only or non-depressed-and-non-anxious patients. Aggregation (mean +/- SE) was 41.9 +/- 2.6% vs. 32.2 +/- 2.6% vs. 30.4 +/- 3.7% with 5HT:E (L) and 46.9 +/- 2.7% vs. 35.6 +/- 2.7% vs. 31.7 +/- 3.8% with 5HT:E (H) (P < 0.05 for both). Differences in ADP aggregations were not significant, perhaps because of clopidogrel therapy. Flow cytometry findings were similar. In a multivariate linear regression model adjusted for age, body mass index, and each other, anxiety symptoms independently predicted all 5HT:E-mediated platelet reactivity measures, whereas depression predicted none. CONCLUSION: Anxiety is associated with elevated serotonin-mediated platelet reactivity in stable CAD patients and symptoms of anxiety show strong, independent correlations with platelet function.

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.

Identification of a novel small molecule HIF-1alpha translation inhibitor.

PURPOSE: Hypoxia inducible factor-1 (HIF-1), the central mediator of the cellular response to low oxygen, functions as a transcription factor for a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1 is overexpressed in cancer and has become an important therapeutic target in solid tumors. In this study, a novel HIF-1alpha inhibitor was identified and its molecular mechanism was investigated. EXPERIMENTAL DESIGN: Using a HIF-responsive reporter cell-based assay, a 10,000-member natural product-like chemical compound library was screened to identify novel HIF-1 inhibitors. This led us to discover KC7F2, a lead compound with a central structure of cystamine. The effects of KC7F2 on HIF-1 transcription, translation, and protein degradation processes were analyzed. RESULTS: KC7F2 markedly inhibited HIF-mediated transcription in cells derived from different tumor types, including glioma, breast, and prostate cancers, and exhibited enhanced cytotoxicity under hypoxia. KC7F2 prevented the activation of HIF-target genes such as carbonic anhydrase IX, matrix metalloproteinase 2 (MMP2), endothelin 1, and enolase 1. An investigation into the mechanism of action of KC7F2 showed that it worked through the down-regulation of HIF-1alpha protein synthesis, an effect accompanied by the suppression of the phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 and p70 S6 kinase, key regulators of HIF-1alpha protein synthesis. CONCLUSION: These results show that KC7F2 is a potent HIF-1 pathway inhibitor and its potential as a cancer therapy agent warrants further study.

Synthesis, structural activity-relationships, and biological evaluation of novel amide-based allosteric binding site antagonists in NR1A/NR2B N-methyl-D-aspartate receptors.

The synthesis and structure-activity relationship analysis of a novel class of amide-based biaryl NR2B-selective NMDA receptor antagonists are presented. Some of the studied compounds are potent, selective, non-competitive, and voltage-independent antagonists of NR2B-containing NMDA receptors. Like the founding member of this class of antagonists (ifenprodil), several interesting compounds of the series bind to the amino terminal domain of the NR2B subunit to inhibit function. Analogue potency is modulated by linker length, flexibility, and hydrogen bonding opportunities. However, unlike previously described classes of NR2B-selective NMDA antagonists that exhibit off-target activity at a variety of monoamine receptors, the compounds described herein show much diminished effects against the hERG channel and alpha(1)-adrenergic receptors. Selections of the compounds discussed have acceptable half-lives in vivo and are predicted to permeate the blood-brain barrier. These data together suggest that masking charged atoms on the linker region of NR2B-selective antagonists can decrease undesirable side effects while still maintaining on-target potency.

Interaction of Akt-phosphorylated SRPK2 with 14-3-3 mediates cell cycle and cell death in neurons.

Terminally differentiated neurons are unable to reenter the cell cycle. Aberrant cell cycle activation provokes neuronal cell death, whereas cell cycle inhibition elevates neuronal survival. However, the molecular mechanism regulating the cell cycle and cell death in mature neurons remains elusive. Here we show that SRPK2, a protein kinase specific for the serine/arginine (SR) family of splicing factors, triggers cell cycle progression in neurons and induces apoptosis through regulation of nuclear cyclin D1. Akt phosphorylates SRPK2 on Thr-492 and promotes its nuclear translocation leading to cyclin D1 up-regulation, cell cycle reentry, and neuronal apoptosis. In addition, SRPK2 phosphorylates SC35 and, thus, inactivates p53, resulting in cyclin D1 up-regulation. 14-3-3 binding to SRPK2, regulated by Akt phosphorylation, inhibits these events. We find that SRPK2 is phosphorylated in ischemia-attacked brain, correlating with the observed increase in cyclin D1 levels. Hence, phosphatidylinositol 3-kinase/Akt mediates the cell cycle and cell death machinery in the nervous system through phosphorylation of SRPK2.

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 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 LRP's 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 LRP's 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.

Neuroprotective actions of PIKE-L by inhibition of SET proteolytic degradation by asparagine endopeptidase.

Ischemia and seizure cause excessive neuronal excitation that is associated with brain acidosis and neuronal cell death. However, the molecular mechanism of acidification-triggered neuronal injury is incompletely understood. Here, we show that asparagine endopeptidase (AEP) is activated under acidic condition, cuts SET, an inhibitor of DNase, and triggers DNA damage in brain, which is inhibited by PIKE-L. SET, a substrate of caspases, was cleaved by acidic cytosolic extract independent of caspase activation. Fractionation of the acidic cellular extract yielded AEP that is required for SET cleavage. We found that kainate provoked AEP activation and SET cleavage at N175, triggering DNA nicking in wild-type, but not AEP null, mice. PIKE-L strongly bound SET and prevented its degradation by AEP, leading to resistance of neuronal cell death. Moreover, AEP also mediated stroke-provoked SET cleavage and cell death in brain. Thus, AEP might be one of the proteinases activated by acidosis triggering neuronal injury during neuroexcitotoxicity or ischemia.