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 and 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.

Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin on the cell surface. Our previous studies indicate that uPA and uPAR expression increase in the ischemic brain during the recovery phase from an acute ischemic injury and that uPA binding to uPAR promotes neurological recovery after an acute ischemic stroke. Here, we used male mice genetically deficient on either uPA (uPA) or uPAR (uPAR) or with a four-amino acid substitution into the growth factor domain of uPA that abrogates its binding to uPAR () to investigate the mechanism whereby uPA promotes neurorepair in the ischemic brain. We found that neurons release uPA and astrocytes recruit uPAR to their plasma membrane during the recovery phase from a hypoxic injury and that binding of neuronal uPA to astrocytic uPAR induces astrocytic activation by a mechanism that does not require plasmin generation, but instead is mediated by extracellular signal-regulated kinase 1/2 (ERK1/2)-regulated phosphorylation of the signal transducer and activator of transcription 3 (STAT3). We report that uPA/uPAR binding is necessary and sufficient to induce astrocytic activation in the ischemic brain and that astrocytes activated by neuronal uPA promote synaptic recovery in neurons that have suffered an acute hypoxic injury via a mechanism mediated by astrocytic thrombospondin-1 (TSP1) and synaptic low-density lipoprotein receptor-related protein-1 (LRP1). In summary, we show that uPA/uPAR-induced astrocytic activation mediates a cross talk between astrocytes and injured neurons that promotes synaptic recovery in the ischemic brain. To date, there is no therapeutic strategy to promote synaptic recovery in the injured brain. Here, we show that neurons release urokinase-type plasminogen activator (uPA) and astrocytes recruit the uPA receptor (uPAR) to their plasma membrane during the recovery phase from a hypoxic injury. We found that binding of neuronal uPA to astrocytic uPAR promotes astrocytic activation and that astrocytes activated by uPA-uPAR binding promote synaptic recovery in neurons that have suffered a hypoxic injury by a mechanism that does not require plasmin generation, but instead is mediated by ERK1/2-regulated STAT3 phosphorylation, astrocytic thrombospondin-1 (TSP1) and synaptic low-density lipoprotein receptor-related protein-1 (LRP1). Our work unveils a new biological function for uPA-uPAR as mediator of a neuron-astrocyte cross talk that promotes synaptic recovery in the ischemic brain.

Tissue-type plasminogen activator (tPA) is a serine proteinase released by the presynaptic terminal of cerebral cortical neurons following membrane depolarization (Echeverry et al., 2010). Recent studies indicate that the release of tPA triggers the synaptic vesicle cycle and promotes the exocytosis (Wu et al., 2015) and endocytic retrieval (Yepes et al., 2016) of glutamate-containing synaptic vesicles. Here we used electron microscopy, proteomics, quantitative phosphoproteomics, biochemical analyses with extracts of the postsynaptic density (PSD), and an animal model of cerebral ischemia with mice overexpressing neuronal tPA to study whether the presynaptic release of tPA also has an effect on the postsynaptic terminal. We found that tPA has a bidirectional effect on the composition of the PSD of cerebral cortical neurons that is independent of the generation of plasmin and the presynaptic release of glutamate, but depends on the baseline level of neuronal activity and the extracellular concentrations of calcium (Ca2+). Accordingly, in neurons that are either inactive or incubated with low Ca2+ concentrations tPA induces phosphorylation and accumulation in the PSD of the Ca2+/calmodulin-dependent protein kinase IIα (pCaMKIIα), followed by pCaMKIIα-mediated phosphorylation and synaptic recruitment of GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast, in neurons with previously increased baseline levels of pCaMKIIα in the PSD due to neuronal depolarization in vivo or incubation with high concentrations of either Ca2+ or glutamate in vitro, tPA induces pCaMKIIα and pGluR1 dephosphorylation and their subsequent removal from the PSD. We found that these effects of tPA are mediated by synaptic N-methyl-D-aspartate (NMDA) receptors and cyclin-dependent kinase 5 (Cdk5)-induced phosphorylation of the protein phosphatase 1 (PP1) at T320. Our data indicate that by regulating the pCaMKIIα/PP1 balance in the PSD tPA acts as a homeostatic regulator of the postsynaptic response of cerebral cortical neurons to the presynaptic release of glutamate.

Local induction of angiotensin-converting enzyme as a mechanism contributing to progressive renal disease. Angiotensin Converting Enzyme (ACE) or Kininase II has a pivotal role determining the local activity of the renin angiotensin and kallikrein kinin systems. Angiotensin II (Ang II), a main hormone of the renin system, has a well established participation as a renal injury agent in models of renal disease with tubulointerstitial fibrosis. Although, since its discovery, ACE has been known to convert Ang I to Ang II, and to inactivate bradykinin (BK), only recently has been emerged evidence for a role of BK with renal protective and antifibrotic effects opposing Ang II. Pertinent to the tubulointerstitial injury, where infiltration and proliferation of macrophages and fibroblast occur, ACE also regulates the levels of the natural hemoregulatory peptide, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). Owing the importance of tissue ACE, its distribution was studied in several models of renal injury. The results showed increased ACE in proximal tubules and ACE induction in the cell infiltrated tubulointerstitium (macrophages and myofibroblasts) of injured kidneys from hypokalemic, Goldblatt hypertensive, Ang II and phenylefrine infused rats, and in both human diabetic and membranous nephropathies. ACE, in addition to Ang II generation, may play a pathogenic role through the hydrolysis of BK and Ac-SDKP. Thus, local increase in ACE can be a novel mechanism involved in tubulointerstitial renal injury, providing, from an anatomical ground, a pathological basis for the putative deleterious effect of ACE in the diseased kidneys, and the beneficial effect of ACE inhibition.

Cerebral ischemia causes the presynaptic release of tissue-type plasminogen activator (tPA). The postsynaptic density (PSD) is a postsynaptic structure that provides a matrix where signaling transduction of excitatory synapses takes place. The postsynaptic density protein-95 (PSD-95) is the most abundant scaffolding protein in the postsynaptic density (PSD), where it modulates the postsynaptic response to the presynaptic release of glutamate by regulating the anchoring of glutamate receptors to the PSD. We found that tPA induces the local translation of PSD-95 mRNA and the subsequent recruitment of PSD-95 protein to the PSD, via plasminogen-independent activation of TrkB receptors. Our data show that PSD-95 is removed from the PSD during the early stages of cerebral ischemia, and that this effect is abrogated by either the release of neuronal tPA, or intravenous administration of recombinant tPA (rtPA). We report that the effect of tPA on PSD-95 is associated with inhibition of the phosphorylation and recruitment of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to the PSD, known to amplify the effect of the excitotoxic injury, and that this is followed by TrkB-mediated protection of dendritic spines from the harmful effects of the hypoxic insult. These data reveal that tPA is a synaptic protector in the ischemic brain.