Fibrinolytic and Non-fibrinolytic Roles of Tissue-type Plasminogen Activator in the Ischemic Brain.

The neurovascular unit (NVU) is assembled by endothelial cells (ECs) and pericytes, and encased by a basement membrane (BM) surveilled by microglia and surrounded by perivascular astrocytes (PVA), which in turn are in contact with synapses. Cerebral ischemia induces the rapid release of the serine proteinase tissue-type plasminogen activator (tPA) from endothelial cells, perivascular astrocytes, microglia and neurons. Owning to its ability to catalyze the conversion of plasminogen into plasmin, in the intravascular space tPA functions as a fibrinolytic enzyme. In contrast, the release of astrocytic, microglial and neuronal tPA have a plethora of effects that not always require the generation of plasmin. In the ischemic brain tPA increases the permeability of the NVU, induces microglial activation, participates in the recycling of glutamate, and has various effects on neuronal survival. These effects are mediated by different receptors, notably subunits of the N-methyl-D-aspartate receptor (NMDAR) and the low-density lipoprotein receptor-related protein-1 (LRP-1). Here we review data on the role of tPA in the NVU under non-ischemic and ischemic conditions, and analyze how this knowledge may lead to the development of potential strategies for the treatment of acute ischemic stroke patients.

Simulation-based education in neurology: lessons from the COVID-19 pandemic.

OBJECTIVES: The COVID-19 pandemic has led to the disruption of all sectors of the economy including education. According to UNESCO over 1.37 million young people including medical students, were affected by the lockdowns in response to COVID-19 and the subsequent closure of the education system. The primary challenge for medical education was to provide clerkships in a biosafety environment. This study aimed to determine the impact of a simulated hospital in a neurology clerkship of 5-year medical students during the coronavirus pandemic and compare their results with a non-pandemic group in Bogotá, Colombia. RESULTS: The students in the pandemic group answered a Likert scale survey regarding their satisfaction with the simulated hospital. Both groups were required to perform an oral, mid-term and final examination. From the results, it is clear that students perceived that exposure to a simulated hospital facilitated their learning process (93.1%) and allowed greater interaction with the teacher compared to a face-to-face environment (77.3%). There were no clinically significant differences in test results. This experience indicates that a simulated hospital is a valuable method to acquire clinical skills in trainees, that could be integrated into the curricular milestones of medical education programs regardless of the pandemic.

Neurological Complications of SARS-CoV-2 Infection and COVID-19 Vaccines: From Molecular Mechanisms to Clinical Manifestations.

Coronavirus Disease 2019 (COVID-19) is an infectious disease, caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), that reached pandemic proportions in 2020. Despite the fact that it was initially characterized by pneumonia and acute respiratory distress syndrome, it is now clear that the nervous system is also compromised in one third of these patients. Indeed, a significant proportion of COVID-19 patients suffer nervous system damage via a plethora of mechanisms including hypoxia, coagulopathy, immune response to the virus, and the direct effect of SARS-CoV-2 on endothelial cells, neurons, astrocytes, pericytes and microglia. Additionally, a low number of previously healthy individuals develop a variety of neurological complications after receiving COVID-19 vaccines and a large proportion of COVID-19 survivors experience longlasting neuropsychiatric symptoms. In conclusion, COVID-19 is also a neurological disease, and the direct and indirect effects of the virus on the nervous system have a significant impact on the morbidity and mortality of these patients. Here we will use the concept of the neurovascular unit, assembled by endothelial cells, basement membrane, perivascular astrocytes, neurons and microglia, to review the effects of SARS-CoV-2 in the nervous system. We will then use this information to review data published to this date on the neurological manifestations of COVID-19, the post- COVID syndrome and COVID-19 vaccines.

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.

The uPA/uPAR system in astrocytic wound healing.

The repair of injured tissue is a highly complex process that involves cell proliferation, differentiation, and migration. Cell migration requires the dismantling of intercellular contacts in the injured zone and their subsequent reconstitution in the wounded area. Urokinase-type plasminogen activator (uPA) is a serine proteinase found in multiple cell types including endothelial cells, smooth muscle cells, monocytes, and macrophages. A substantial body of experimental evidence with different cell types outside the central nervous system indicates that the binding of uPA to its receptor (uPAR) on the cell surface prompts cell migration by inducing plasmin-mediated degradation of the extracellular matrix. In contrast, although uPA and uPAR are abundantly found in astrocytes and uPA binding to uPAR triggers astrocytic activation, it is unknown if uPA also plays a role in astrocytic migration. Neuronal cadherin is a member of cell adhesion proteins pivotal for the formation of cell-cell contacts between astrocytes. More specifically, while the extracellular domain of neuronal cadherin interacts with the extracellular domain of neuronal cadherin in neighboring cells, its intracellular domain binds to ß-catenin, which in turn links the complex to the actin cytoskeleton. Glycogen synthase kinase 3ß is a serine-threonine kinase that prevents the cytoplasmic accumulation of ß-catenin by inducing its phosphorylation at Ser33, Ser37, and Ser41, thus activating a sequence of events that lead to its proteasomal degradation. The data discussed in this perspective indicate that astrocytes release uPA following a mechanical injury, and that binding of this uPA to uPAR on the cell membrane induces the detachment of ß-catenin from the intracellular domain of neuronal cadherin by triggering its extracellular signal-regulated kinase 1/2-mediated phosphorylation at Tyr650. Remarkably, this is followed by the cytoplasmic accumulation of ß-catenin because uPA-induced extracellular signal-regulated kinase 1/2 activation also phosphorylates lipoprotein receptor-related protein 6 at Ser1490, which in turn, by recruiting glycogen synthase kinase 3ß to its intracellular domain abrogates its effect on ß-catenin. The cytoplasmic accumulation of ß-catenin is followed by its nuclear translocation, where it induces the expression of uPAR, which is required for the migration of astrocytes from the injured edge into the wounded area.

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.

Urokinase-type plasminogen activator-mediated crosstalk between N-cadherin and ß-catenin promotes wound healing.

Urokinase-type plasminogen activator (uPA; encoded by Plau) is a serine proteinase that, in the central nervous system, induces astrocytic activation. ß-Catenin is a protein that links the cytoplasmic tail of cadherins to the actin cytoskeleton, thus securing the formation of cadherin-mediated cell adhesion complexes. Disruption of cell-cell contacts leads to the detachment of ß-catenin from cadherins, and ß-catenin is then degraded by the proteasome following its phosphorylation by GSK3ß. Here, we show that astrocytes release uPA following a scratch injury, and that this uPA promotes wound healing via a plasminogen-independent mechanism. We found that uPA induces the detachment of ß-catenin from the cytoplasmic tail of N-cadherin (NCAD; also known as CDH2) by triggering its phosphorylation at Tyr654. Surprisingly, this is not followed by degradation of ß-catenin because uPA also induces the phosphorylation of the low density lipoprotein receptor-related protein 6 (LRP6) at Ser1490, which then blocks the kinase activity of GSK3ß. Our work indicates that the ensuing cytoplasmic accumulation of ß-catenin is followed by its nuclear translocation and ß-catenin-triggered transcription of the receptor for uPA (Plaur), which in turn is required for uPA to induce astrocytic wound healing.

Plasminogen Activators in Neurovascular and Neurodegenerative Disorders.

The neurovascular unit (NVU) is a dynamic structure assembled by endothelial cells surrounded by a basement membrane, pericytes, astrocytes, microglia and neurons. A carefully coordinated interplay between these cellular and non-cellular components is required to maintain normal neuronal function, and in line with these observations, a growing body of evidence has linked NVU dysfunction to neurodegeneration. Plasminogen activators catalyze the conversion of the zymogen plasminogen into the two-chain protease plasmin, which in turn triggers a plethora of physiological events including wound healing, angiogenesis, cell migration and inflammation. The last four decades of research have revealed that the two mammalian plasminogen activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), are pivotal regulators of NVU function during physiological and pathological conditions. Here, we will review the most relevant data on their expression and function in the NVU and their role in neurovascular and neurodegenerative disorders.

Urokinase-type plasminogen activator promotes N-cadherin-mediated synaptic recovery in the ischemic brain.

Urokinase-type plasminogen activator (uPA) is a serine proteinase that catalyzes the generation of plasmin on the cell surface and activates cell signaling pathways that promote remodeling and repair. Neuronal cadherin (NCAD) is a transmembrane protein that in the mature brain mediates the formation of synaptic contacts in the II/III and V cortical layers. Our studies show that uPA is preferentially found in the II/III and V cortical laminae of the gyrencephalic cortex of the non-human primate. Furthermore, we found that in murine cerebral cortical neurons and induced pluripotent stem cell (iPSC)-derived neurons prepared from healthy human donors, most of this uPA is associated with pre-synaptic vesicles. Our in vivo experiments revealed that in both, the gyrencephalic cortex of the non-human primate and the lissecephalic murine brain, cerebral ischemia decreases the number of intact synaptic contacts and the expression of uPA and NCAD in a band of tissue surrounding the necrotic core. Additionally, our in vitro data show that uPA induces the synthesis of NCAD in cerebral cortical neurons, and in line with these observations, intravenous treatment with recombinant uPA three hours after the onset of cerebral ischemia induces NCAD-mediated repair of synaptic contacts in the area surrounding the necrotic core.

Preparation of Synaptoneurosomes to Study the Synapse in the Murine Cerebral Cortex.

The synapse is a complex structure where the transmission of information takes place. Synaptic dysfunction is one of the earliest pathophysiological events in several diseases, such as traumatic brain injury, cerebral ischemia, and neurodegenerative diseases. Thus, a methodology to study synaptic structure and function is crucial for the development of potential strategies for the treatment of many neurological diseases. Synaptoneurosomes (SNs) are structures assembled by the sealed presynaptic bouton and the attached post-synaptic density. Despite the fact that for a long time it has been recognized that SNs are a powerful tool to study synaptic function, composition, and structure, its use has been limited by the requirement of relatively large amounts of material to successfully isolate them. Here we describe a three-step centrifugation procedure performed under hypotonic conditions to isolate SNs from small volumes of the cerebral cortex. Graphic abstract: Schematic flowchart for the preparation of synaptoneurosomes.

The plasminogen activating system in the pathogenesis of Alzheimer's disease.

Dementia is a clinical syndrome that affects approximately 47 million people worldwide and is characterized by progressive and irreversible decline of cognitive, behavioral and sesorimotor functions. Alzheimer's disease (AD) accounts for approximately 60-80% of all cases of dementia, and neuropathologically is characterized by extracellular deposits of insoluble amyloid-ß (Aß) and intracellular aggregates of hyperphosphorylated tau. Significantly, although for a long time it was believed that the extracellular accumulation of Aß was the culprit of the symptoms observed in these patients, more recent studies have shown that cognitive decline in people suffering this disease is associated with soluble Aß-induced synaptic dysfunction instead of the formation of insoluble Aß-containing extracellular plaques. These observations are translationally relevant because soluble Aß-induced synaptic dysfunction is an early event in AD that precedes neuronal death, and thus is amenable to therapeutic interventions to prevent cognitive decline before the progression to irreversible brain damage. The plasminogen activating (PA) system is an enzymatic cascade that triggers the degradation of fibrin by catalyzing the conversion of plasminogen into plasmin via two serine proteinases: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Experimental evidence reported over the last three decades has shown that tPA and uPA play a role in the pathogenesis of AD. However, these studies have focused on the ability of these plasminogen activators to trigger plasmin-induced cleavage of insoluble Aß-containing extracellular plaques. In contrast, recent evidence indicates that activity-dependent release of uPA from the presynaptic terminal of cerebral cortical neurons protects the synapse from the deleterious effects of soluble Aß via a mechanism that does not require plasmin generation or the cleavage of Aß fibrils. Below we discuss the role of the PA system in the pathogenesis of AD and the translational relevance of data published to this date.

Urokinase-Type Plasminogen Activator Protects Cerebral Cortical Neurons from Soluble Aß-Induced Synaptic Damage.

Soluble amyloid ß (Aß)-induced synaptic dysfunction is an early event in the pathogenesis of Alzheimer's disease (AD) that precedes the deposition of insoluble Aß and correlates with the development of cognitive deficits better than the number of plaques. The mammalian plasminogen activation (PA) system catalyzes the generation of plasmin via two activators: tissue-type (tPA) and urokinase-type (uPA). A dysfunctional tPA-plasmin system causes defective proteolytic degradation of Aß plaques in advanced stages of AD. In contrast, it is unknown whether uPA and its receptor (uPAR) contribute to the pathogenesis of this disease. Neuronal cadherin (NCAD) plays a pivotal role in the formation of synapses and dendritic branches, and Aß decreases its expression in cerebral cortical neurons. Here we show that neuronal uPA protects the synapse from the harmful effects of soluble Aß. However, Aß-induced inactivation of the eukaryotic initiation factor 2α halts the transcription of uPA mRNA, leaving unopposed the deleterious effects of Aß on the synapse. In line with these observations, the synaptic abundance of uPA, but not uPAR, is decreased in the frontal cortex of AD patients and 5xFAD mice, and in cerebral cortical neurons incubated with soluble Aß. We found that uPA treatment increases the synaptic expression of NCAD by a uPAR-mediated plasmin-independent mechanism, and that uPA-induced formation of NCAD dimers protects the synapse from the harmful effects of soluble Aß oligomers. These data indicate that Aß-induced decrease in the synaptic abundance of uPA contributes to the development of synaptic damage in the early stages of AD.SIGNIFICANCE STATEMENT Soluble amyloid ß (Aß)-induced synaptic dysfunction is an early event in the pathogenesis of cognitive deficits in Alzheimer's disease (AD). We found that neuronal urokinase-type (uPA) protects the synapse from the deleterious effects of soluble Aß. However, Aß-induced inactivation of the eukaryotic initiation factor 2α decreases the synaptic abundance of uPA, leaving unopposed the harmful effects of Aß on the synapse. In line with these observations, the synaptic expression of uPA is decreased in the frontal cortex of AD brains and 5xFAD mice, and uPA treatment abrogates the deleterious effects of Aß on the synapse. These results unveil a novel mechanism of Aß-induced synaptic dysfunction in AD patients, and indicate that recombinant uPA is a potential therapeutic strategy to protect the synapse before the development of irreversible brain damage.

Dietary and Serum Omega-6/Omega-3 Fatty Acids Are Associated with Physical and Metabolic Function in Stroke Survivors.

The purpose of this study was to quantify habitual dietary and systemic omega-6 and omega-3 fatty acids and their ratios and to determine their relationship with physical and metabolic function in a cohort of chronic adult stroke survivors. Twenty-five older chronic stroke survivors (age: 63 ± 8 years; BMI: 31 ± 7 kg/m2; mean ± SD) were assessed for fitness (VO2peak), gait speed (GS), 3 m timed up and go (TUG), and six-minute walk distance (6MWD). Plasma lipid and glucose profiles were measured, and HOMA-IR calculated. Dietary (5-day food records) and serum (mass spectrometry) omega-6/omega-3 profiles were assessed. Participants were severely deconditioned (VO2peak: 19 ± 4 mL/kg/min; GS: 0.88 ± 0.28 m/s; TUG: 12.6 ± 5.9 s; 6MWD: 295 ± 121 m) and at elevated metabolic risk (HOMA-IR: 6.3 ± 4.5). The dietary intake ratio of omega-6/omega-3 fatty acids averaged 12.6 ± 7.1 and the serum concentration ratio was 1.21 ± 0.37, which were correlated (r = 0.88, p < 0.01). Higher dietary intake and serum concentrations of omega-6/omega-3 fatty acids were associated with lower 6MWD and higher HOMA-IR, while a higher serum omega-6/omega-3 concentration index was associated with lower VO2peak (p's < 0.05). These preliminary data suggest that both dietary omega-6 and omega-3 fatty acids (quantitated as their intake ratio) and the serum concentration ratio of omega-6/omega-3 may be important indices of physical dysfunction and insulin resistance in chronic stroke survivors.

Urokinase-type plasminogen activator (uPA) regulates the expression and function of growth-associated protein 43 (GAP-43) in the synapse.

Growth-associated protein 43 (GAP-43) plays a central role in the formation of presynaptic terminals, synaptic plasticity, and axonal growth and regeneration. During development, GAP-43 is found in axonal extensions of most neurons. In contrast, in the mature brain, its expression is restricted to a few presynaptic terminals and scattered axonal growth cones. Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin and activates signaling pathways that promote cell migration, proliferation, and survival. In the developing brain, uPA induces neuritogenesis and neuronal migration. In contrast, the expression and function of uPA in the mature brain are poorly understood. However, recent evidence reveals that different forms of injury induce release of uPA and expression of uPAR in neurons and that uPA/uPAR binding triggers axonal growth and synapse formation. Here we show that binding of uPA to uPAR induces not only the mobilization of GAP-43 from the axonal shaft to the presynaptic terminal but also its activation in the axonal bouton by PKC-induced calcium-dependent phosphorylation at Ser-41 (pGAP-43). We found that this effect requires open presynaptic N-methyl-d-aspartate receptors but not plasmin generation. Furthermore, our work reveals that, following its activation by uPA/uPAR binding, pGAP-43 colocalizes with presynaptic vesicles and triggers their mobilization to the synaptic release site. Together, these data reveal a novel role of uPA as an activator of the synaptic vesicle cycle in cerebral cortical neurons via its ability to induce presynaptic recruitment and activation of GAP-43.

Urokinase-type plasminogen activator is a modulator of synaptic plasticity in the central nervous system: implications for neurorepair in the ischemic brain.

The last two decades have witnessed a rapid decrease in mortality due to acute cerebral ischemia that paradoxically has led to a rapid increase in the number of patients that survive an acute ischemic stroke with various degrees of disability. Unfortunately, the lack of an effective therapeutic strategy to promote neurological recovery among stroke survivors has led to a rapidly growing population of disabled patients. Thus, understanding the mechanisms of neurorepair in the ischemic brain is a priority with wide scientific, social and economic implications. Cerebral ischemia has a harmful effect on synaptic structure associated with the development of functional impairment. In agreement with these observations, experimental evidence indicates that synaptic repair underlies the recovery of neurological function following an ischemic stroke. Furthermore, it has become evident that synaptic plasticity is crucial not only during development and learning, but also for synaptic repair after an ischemic insult. The plasminogen activating system is assembled by a cascade of enzymes and their inhibitors initially thought to be solely involved in the generation of plasmin. However, recent work has shown that in the brain this system has an important function regulating the development of synaptic plasticity via mechanisms that not always require plasmin generation. Urokinase-type plasminogen activator (uPA) is a serine proteinase and one of the plasminogen activators, that upon binding to its receptor (uPAR) not only catalyzes the conversion of plasminogen into plasmin on the cell surface, but also activates cell signaling pathways that promote cell migration, proliferation and survival. The role of uPA is the brain is not fully understood. However, it has been reported while uPA and uPAR are abundantly found in the developing central nervous system, in the mature brain their expression is restricted to a limited group of cells. Remarkably, following an ischemic injury to the mature brain the expression of uPA and uPAR increases to levels comparable to those observed during development. More specifically, neurons release uPA during the recovery phase from an ischemic injury, and astrocytes, axonal boutons and dendritic spines recruit uPAR to their plasma membrane. Here we will review recent evidence indicating that binding of uPA to uPAR promotes the repair of synapses damaged by an ischemic injury, with the resultant recovery of neurological function. Furthermore, we will discuss data indicating that treatment with recombinant uPA is a potential therapeutic strategy to promote neurological recovery among ischemic stroke survivors.

Temporal profiling of Kv1.3 channel expression in brain mononuclear phagocytes following ischemic stroke.

BACKGROUND: Microglia and CNS-infiltrating monocytes/macrophages (CNS-MPs) perform pro-inflammatory and protective anti-inflammatory functions following ischemic stroke. Selective inhibition of pro-inflammatory responses can be achieved by Kv1.3 channel blockade, resulting in a lower infarct size in the transient middle cerebral artery occlusion (tMCAO) model. Whether beneficial effects of Kv1.3 blockers are mediated by targeting microglia or CNS-infiltrating monocytes/macrophages remains unclear. METHODS: In the 30-min tMCAO mouse model, we profiled functional cell-surface Kv1.3 channels and phagocytic properties of acutely isolated CNS-MPs at various timepoints post-reperfusion. Kv1.3 channels were flow cytometrically detected using fluorescein-conjugated Kv1.3-binding peptide ShK-F6CA as well as by immunohistochemistry. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was performed to measure Kv1.3 (Kcna3) and Kir2.1 (Kcnj2) gene expression. Phagocytosis of 1-µm microspheres by acutely isolated CNS-MPs was measured by flow cytometry. RESULTS: In flow cytometric assays, Kv1.3 channel expression by CD11b+ CNS-MPs was increased between 24 and 72 h post-tMCAO and decreased by 7 days post-tMCAO. Increased Kv1.3 expression was restricted to CD11b+CD45lowLy6clow (microglia) and CD11b+CD45highLy6Clow CNS-MPs but not CD11b+CD45highLy6chigh inflammatory monocytes/macrophages. In immunohistochemical studies, Kv1.3 protein expression was increased in Iba1+ microglia at 24-48 h post-tMCAO. No change in Kv1.3 mRNA in CNS-MPs was observed following tMCAO. CONCLUSIONS: We conclude that resident microglia and a subset of CD45highLy6clow CNS-MPs are the likely cellular targets of Kv1.3 blockers and the delayed phase of neuroinflammation is the optimal therapeutic window for Kv1.3 blockade in ischemic stroke.

Tissue-type plasminogen activator regulates p35-mediated Cdk5 activation in the postsynaptic terminal.

Neuronal depolarization induces the synaptic release of tissue-type plasminogen activator (tPA). Cyclin-dependent kinase-5 (Cdk5) is a member of the family of cyclin-dependent kinases that regulates cell migration and synaptic function in postmitotic neurons. Cdk5 is activated by its binding to p35 (also known as Cdk5r1), a membrane-anchored protein that is rapidly degraded by the proteasome. Here, we show that tPA prevents the degradation of p35 in the synapse by a plasminogen-dependent mechanism that requires open synaptic N-methyl-D-aspartate (NMDA) receptors. We show that tPA treatment increases the abundance of p35 and its binding to Cdk5 in the postsynaptic density (PSD). Furthermore, our data indicate that tPA-induced p35-mediated Cdk5 activation does not induce cell death, but instead prevents NMDA-induced ubiquitylation of postsynaptic density protein-95 (PSD-95; also known as Dlg4) and the removal of GluR1 (also known as Gria1)-containing α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors from the PSD. These results show that the interaction between tPA and synaptic NMDA receptors regulates the expression of AMPA receptor subunits in the PSD via p35-mediated Cdk5 activation. This is a novel role for tPA as a regulator of Cdk5 activation in cerebral cortical neurons.

Urokinase-type plasminogen activator (uPA) protects the tripartite synapse in the ischemic brain via ezrin-mediated formation of peripheral astrocytic processes.

Cerebral ischemia has a harmful effect on the synapse associated with neurological impairment. The "tripartite synapse" is assembled by the pre- and postsynaptic terminals, embraced by astrocytic elongations known as peripheral astrocytic processes (PAPs). Ischemic stroke induces the detachment of PAPs from the synapse, leading to synaptic dysfunction and neuronal death. Ezrin is a membrane-associated protein, required for the formation of PAPs, that links the cell surface to the actin cytoskeleton. Urokinase-type plasminogen activator (uPA) is a serine proteinase that upon binding to its receptor (uPAR) promotes neurite growth during development. In the adult brain, neurons release uPA and astrocytes recruit uPAR to the plasma membrane during the recovery phase from an ischemic stroke, and uPA/uPAR binding promotes functional improvement following an ischemic injury. We found that uPA induces the synthesis of ezrin in astrocytes, with the subsequent formation of PAPs that enter in direct contact with the synapse. Furthermore, either the release of neuronal uPA or intravenous treatment with recombinant uPA (ruPA) induces the formation of PAPs in the ischemic brain, and the interaction of these PAPs with the pre- and postsynaptic terminals protects the integrity of the "tripartite synapse" from the harmful effects of the ischemic injury.

The Plasminogen Activation System Promotes Neurorepair in the Ischemic Brain.

The plasminogen activation (PA) system was originally thought to exclusively promote the degradation of fibrin by catalyzing the conversion of plasminogen into plasmin via two serine proteinases: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). However, experimental evidence accumulated over the last 30 years indicates that tPA and uPA are also found in the central nervous system (CNS), where they have a plethora of functions that not always require plasmin generation or fibrin degradation. For example, plasminogen-dependent and - independent effects of tPA and uPA play a central role in the pathophysiological events that underlie one of the leading causes of mortality and disability in the world: cerebral ischemia. Indeed, recent work indicates that while the rapid release of tPA from the presynaptic compartment following the onset of cerebral ischemia protects the synapse from the deleterious effects of the ischemic injury, the secretion of uPA and its binding to its receptor (uPAR) during the recovery phase promotes the repair of synapses that have been lost to the acute ischemic insult. This restorative role of uPA has high translational significance because to this date there is no effective approach to induce neurorepair in the ischemic brain. Here we will discuss recent evidence that bridges the gap between basic research in the field of the PA system and the bedside of ischemic stroke patients, indicating that uPA and uPAR are potential targets for the development of therapeutic strategies to promote neurological recovery among ischemic stroke survivors.

The role of plasminogen activators in stroke treatment: fibrinolysis and beyond.

Although recent technical advances in thrombectomy have revolutionised acute stroke treatment, prevalence of disability and death related to stroke remain high. Therefore, plasminogen activators-eukaryotic, bacterial, or engineered forms that can promote fibrinolysis by converting plasminogen into active plasmin and facilitate clot breakdown-are still commonly used in the acute treatment of ischaemic stroke. Hence, plasminogen activators have become a crucial area for clinical investigation for their ability to recanalise occluded arteries in ischaemic stroke and to accelerate haematoma clearance in haemorrhagic stroke. However, inconsistent results, insufficient evidence of efficacy, or reports of side-effects in trial settings might reduce the use of plasminogen activators in clinical practice. Additionally, the mechanism of action for plasminogen activators could extend beyond the vessel lumen and involve plasminogen-independent processes, which would suggest that plasminogen activators have also non-fibrinolytic roles. Understanding the complex mechanisms of action of plasminogen activators can guide future directions for therapeutic interventions in patients with stroke.