Axonal injury is a common cause of neurological dysfunction. Unfortunately, in contrast to axons from the peripheral nervous system, the limited capacity of regeneration of central nervous system (CNS) axons is a major obstacle for functional recovery in patients suffering neurological diseases that involve the subcortical white matter. Urokinase-type plasminogen activator (uPA) is a serine proteinase that upon binding to the urokinase-type plasminogen activator receptor (uPAR) catalyzes the conversion of plasminogen into plasmin on the cell surface. uPAR expression increases after an injury, and signaling through uPAR promotes tissue remodeling. However, it is yet unknown whether uPA binding to uPAR has an effect on axonal recovery in the CNS. Here, we used in vitro and in vivo models of CNS axonal injury to test the hypothesis that uPA binding to uPAR promotes axonal regeneration in the CNS. We found that newly formed growth cones from axons re-emerging from an axonal injury express uPAR and that binding of uPA to this uPAR promotes axonal recovery by a mechanism that does not require the generation of plasmin. Our data indicate that the binding of recombinant uPA or endogenous uPA to uPAR induces membrane recruitment and activation of β1 integrin via the low density lipoprotein receptor-related protein-1 (LRP1), which leads to activation of the Rho family small GTPase Rac1 and Rac1-induced axonal regeneration. Our results show that the uPA/uPAR/LRP1 system is a potential target for the development of therapeutic strategies to promote axonal recovery following a CNS injury.
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.
Advances in neurocritical care and interventional neuroradiology have led to a significant decrease in acute ischemic stroke (AIS) mortality. In contrast, due to the lack of an effective therapeutic strategy to promote neuronal recovery among AIS survivors, cerebral ischemia is still a leading cause of disability in the world. Ischemic stroke has a harmful impact on synaptic structure and function, and plasticity-mediated synaptic recovery is associated with neurological improvement following an AIS. Dendritic spines (DSs) are specialized dendritic protrusions that receive most of the excitatory input in the brain. The deleterious effect of cerebral ischemia on DSs morphology and function has been associated with impaired synaptic transmission and neurological deterioration. However, these changes are reversible if cerebral blood flow is restored on time, and this recovery has been associated with neurological improvement following an AIS. Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are two serine proteases that besides catalyzing the conversion of plasminogen into plasmin in the intravascular and pericellular environment, respectively, are also are efficient inductors of synaptic plasticity. Accordingly, recent evidence indicates that both, tPA and uPA, protect DSs from the metabolic stress associated with the ischemic injury, and promote their morphological and functional recovery during the recovery phase from an AIS. Here we will review data indicating that plasticity-induced changes in DSs and the associated post-synaptic density play a pivotal role in the recovery process from AIS, making special emphasis on the role of tPA and uPA in this process.
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-β (Aβ) and intracellular aggregates of hyperphosphorylated tau. Significantly, although for a long time it was believed that the extracellular accumulation of Aβ 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 Aβ-induced synaptic dysfunction instead of the formation of insoluble Aβ-containing extracellular plaques. These observations are translationally relevant because soluble Aβ-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 Aβ-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 Aβ via a mechanism that does not require plasmin generation or the cleavage of Aβ 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.
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 modu-lated 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 α1-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.
Urokinase-type plasminogen activator (uPA; encoded by Plau) is a serine proteinase that, in the central nervous system, induces astrocytic activation. β-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 β-catenin from cadherins, and β-catenin is then degraded by the proteasome following its phosphorylation by GSK3β. 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 β-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 β-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 GSK3β. Our work indicates that the ensuing cytoplasmic accumulation of β-catenin is followed by its nuclear translocation and β-catenin-triggered transcription of the receptor for uPA (Plaur), which in turn is required for uPA to induce astrocytic wound healing.
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.
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 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 (rtPA) 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 decrease in the volume of the ischemic lesion and improved neurological outcome following the induction of ischemic stroke. Our data indicate that tPA activates a cell signaling pathway that allows neurons to sense and adapt to oxygen and glucose deprivation.
The 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 β-catenin, which in turn links the complex to the actin cytoskeleton. Glycogen synthase kinase 3β is a serine-Threonine kinase that prevents the cytoplasmic accumulation of β-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 β-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 β-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 3β to its intracellular domain abrogates its effect on β-catenin. The cytoplasmic accumulation of β-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.