Two-Chains Tissue Plasminogen Activator Unifies Met and NMDA Receptor Signalling to Control Neuronal Survival.

Tissue-type plasminogen activator (tPA) plays roles in the development and the plasticity of the nervous system. Here, we demonstrate in neurons, that by opposition to the single chain form (sc-tPA), the two-chains form of tPA (tc-tPA) activates the MET receptor, leading to the recruitment of N-Methyl-d-Aspartate receptors (NMDARs) and to the endocytosis and proteasome-dependent degradation of NMDARs containing the GluN2B subunit. Accordingly, tc-tPA down-regulated GluN2B-NMDAR-driven signalling, a process prevented by blockers of HGFR/MET and mimicked by its agonists, leading to a modulation of neuronal death. Thus, our present study unmasks a new mechanism of action of tPA, with its two-chains form mediating a crosstalk between MET and the GluN2B subunit of NMDARs to control neuronal survival.

Distant Space Processing is Controlled by tPA-dependent NMDA Receptor Signaling in the Entorhinal Cortex.

In humans, spatial cognition and navigation impairments are a frequent situation during physiological and pathological aging, leading to a dramatic deterioration in the quality of life. Despite the discovery of neurons with location-specific activity in rodents, that is, place cells in the hippocampus and later on grid cells in the entorhinal cortex (EC), the molecular mechanisms underlying spatial cognition are still poorly known. Our present data bring together in an unusual combination 2 molecules of primary biological importance: a major neuronal excitatory receptor, N-methyl-D-aspartate receptor (NMDAR), and an extracellular protease, tissue plasminogen activator (tPA), in the control of spatial navigation. By using tPA-deficient mice and a structure-selective pharmacological approach, we demonstrate that the tPA-dependent NMDAR signaling potentiation in the EC plays a key and selective role in the encoding and the subsequent use of distant landmarks during spatial learning. We also demonstrate that this novel function of tPA in the EC is reduced during aging. Overall, these results argue for the concept that encoding of proximal versus distal landmarks is mediated not only by different anatomical pathways but also by different molecular mechanisms, with the tPA-dependent potentiation of NMDAR signaling in the EC that plays an important role.

Sterol metabolism regulates neuroserpin polymer degradation in the absence of the unfolded protein response in the dementia FENIB.

Mutants of neuroserpin are retained as polymers within the endoplasmic reticulum (ER) of neurones to cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. The cellular consequences are unusual in that the ordered polymers activate the ER overload response (EOR) in the absence of the canonical unfolded protein response. We use both cell lines and Drosophila models to show that the G392E mutant of neuroserpin that forms polymers is degraded by UBE2j1 E2 ligase and Hrd1 E3 ligase while truncated neuroserpin, a protein that lacks 132 amino acids, is degraded by UBE2g2 (E2) and gp78 (E3) ligases. The degradation of G392E neuroserpin results from SREBP-dependent activation of the cholesterol biosynthetic pathway in cells that express polymers of neuroserpin (G392E). Inhibition of HMGCoA reductase, the limiting enzyme of the cholesterol biosynthetic pathway, reduced the ubiquitination of G392E neuroserpin in our cell lines and increased the retention of neuroserpin polymers in both HeLa cells and primary neurones. Our data reveal a reciprocal relationship between cholesterol biosynthesis and the clearance of mutant neuroserpin. This represents the first description of a link between sterol metabolism and modulation of the proteotoxicity mediated by the EOR.

HMGB-1 promotes fibrinolysis and reduces neurotoxicity mediated by tissue plasminogen activator.

Owing to its ability to generate the clot-dissolving protease plasmin, tissue plasminogen activator (tPA) is the only approved drug for the acute treatment of ischemic stroke. However, tPA also promotes hemorrhagic transformation and excitotoxic events. High mobility group box-1 protein (HMGB-1) is a non-histone transcription factor and a pro-inflammatory cytokine, which has also been shown to bind to both tPA and plasminogen. We thus investigated the cellular and molecular effects through which HMGB-1 could influence the vascular and parenchymal effects of tPA during ischemia. We demonstrate that HMGB-1 not only increases clot lysis by tPA, but also reduces the passage of vascular tPA across the blood-brain barrier, as well as tPA-driven leakage of the blood-brain barrier. In addition, HMGB-1 prevents the pro-neurotoxic effect of tPA, by blocking its interaction with N-methyl-D-aspartate (NMDA) receptors and the attendant potentiation of NMDA-induced neuronal Ca²âº influx. In conclusion, we show in vitro that HMGB-1 can promote the beneficial effects of tPA while counteracting its deleterious properties. We suggest that derivatives of HMGB-1, devoid of pro-inflammatory properties, could be used as adjunctive therapies to improve the overall benefit of tPA-mediated thrombolysis following stroke.

Characterisation of serpin polymers in vitro and in vivo.

Neuroserpin is a member of the serine protease inhibitor or serpin superfamily of proteins. It is secreted by neurones and plays an important role in the regulation of tissue plasminogen activator at the synapse. Point mutations in the neuroserpin gene cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. This is one of a group of disorders caused by mutations in the serpins that are collectively known as the serpinopathies. Others include α(1)-antitrypsin deficiency and deficiency of C1 inhibitor, antithrombin and α(1)-antichymotrypsin. The serpinopathies are characterised by delays in protein folding and the retention of ordered polymers of the mutant serpin within the cell of synthesis. The clinical phenotype results from either a toxic gain of function from the inclusions or a loss of function, as there is insufficient protease inhibitor to regulate important proteolytic cascades. We describe here the methods required to characterise the polymerisation of neuroserpin and draw parallels with the polymerisation of α(1)-antitrypsin. It is important to recognise that the conditions in which experiments are performed will have a major effect on the findings. For example, incubation of monomeric serpins with guanidine or urea will produce polymers that are not found in vivo. The characterisation of the pathological polymers requires heating of the folded protein or alternatively the assessment of ordered polymers from cell and animal models of disease or from the tissues of humans who carry the mutation.

Thrombolysis by PLAT/tPA increases serum free IGF1 leading to a decrease of deleterious autophagy following brain ischemia.

Cerebral ischemia is a pathology involving a cascade of cellular mechanisms, leading to the deregulation of proteostasis, including macroautophagy/autophagy, and finally to neuronal death. If it is now accepted that cerebral ischemia induces autophagy, the effect of thrombolysis/energy recovery on proteostasis remains unknown. Here, we investigated the effect of thrombolysis by PLAT/tPA (plasminogen activator, tissue) on autophagy and neuronal death. In two in vitro models of hypoxia reperfusion and an in vivo model of thromboembolic stroke with thrombolysis by PLAT/tPA, we found that ischemia enhances neuronal deleterious autophagy. Interestingly, PLAT/tPA decreases autophagy to mediate neuroprotection by modulating the PI3K-AKT-MTOR pathways both in vitro and in vivo. We identified IGF1R (insulin-like growth factor I receptor; a tyrosine kinase receptor) as the effective receptor and showed in vitro, in vivo and in human stroke patients and that PLAT/tPA is able to degrade IGFBP3 (insulin-like growth factor binding protein 3) to increase IGF1 (insulin-like growth factor 1) bioavailability and thus IGF1R activation.Abbreviations: AKT/protein kinase B: thymoma viral proto-oncogene 1; EGFR: epidermal growth factor receptor; Hx: hypoxia; IGF1: insulin-like growth factor 1; IGF1R: insulin-like growth factor I receptor; IGFBP3: insulin-like growth factor binding protein 3; Ka: Kainate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OGD: oxygen and glucose deprivation; OGDreox: oxygen and glucose deprivation + reoxygentation; PepA: pepstatin A1; PI3K: phosphoinositide 3-kinase; PLAT/tPA: plasminogen activator, tissue; PPP: picropodophyllin; SCH77: SCH772984; ULK1: unc-51 like kinase 1; Wort: wortmannin.

Tissue-type plasminogen activator in the ischemic brain: more than a thrombolytic.

Thrombolysis with tissue-type plasminogen activator (tPA) is used for the treatment of patients with acute ischemic stroke. However, a growing body of evidence indicates that, besides the unquestionable benefit from its thrombolytic activity, tPA also has a deleterious effect on the ischemic brain including cytotoxicity and increased permeability of the neurovascular unit with the development of cerebral edema. Because an increasing number of acute stroke patients are treated with tPA, it is important to know the mechanisms of harmful effects of tPA on the ischemic brain. Here, the best studied pathways of tPA neurotoxicity are discussed along with future directions for a safer use of tPA as a thrombolytic agent in the setting of acute ischemic stroke.

Neuroserpin polymers activate NF-kappaB by a calcium signaling pathway that is independent of the unfolded protein response.

The autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies is characterized by the accumulation of ordered polymers of mutant neuroserpin within the endoplasmic reticulum of neurones. We show here that intracellular neuroserpin polymers activate NF-kappaB by a pathway that is independent of the IRE1, ATF6, and PERK limbs of the canonical unfolded protein response but is dependent on intracellular calcium. This pathway provides a mechanism for cells to sense and react to the accumulation of folded structures of mutant serpins within the endoplasmic reticulum. Our results provide strong support for the endoplasmic reticulum overload response being independent of the unfolded protein response.

Age and albumin D site-binding protein control tissue plasminogen activator levels: neurotoxic impact.

Recombinant tissue-type plasminogen activator (tPA) is the fibrinolytic drug of choice to treat stroke patients. However, a growing body of evidence indicates that besides its beneficial thrombolytic role, tPA can also have a deleterious effect on the ischaemic brain. Although ageing influences stroke incidence, complications and outcome, age-dependent relationships between endogenous tPA and stroke injuries have not been investigated yet. Here, we report that ageing is associated with a selective lowering of brain tPA expression in the murine brain. Moreover, our results show that albumin D site-binding protein (DBP) as a key age-associated regulator of the neuronal transcription of tPA. Additionally, inhibition of DBP-mediated tPA expression confers in vitro neuroprotection. Accordingly, reduced levels of tPA in old mice are associated with smaller excitotoxic/ischaemic injuries and protection of the permeability of the neurovascular unit during cerebral ischaemia. Likewise, we provide neuroradiological evidence indicating the existence of an inverse relationship between age and the volume of the ischaemic lesion in patients with acute ischaemic stroke. Together, these results indicate that the relationship among DBP, tPA and ageing play an important role in the outcome of cerebral ischaemia.

Proteostasis During Cerebral Ischemia.

Cerebral ischemia is a complex pathology involving a cascade of cellular mechanisms, which deregulate proteostasis and lead to neuronal death. Proteostasis refers to the equilibrium between protein synthesis, folding, transport, and protein degradation. Within the brain proteostasis plays key roles in learning and memory by controlling protein synthesis and degradation. Two important pathways are implicated in the regulation of proteostasis: the unfolded protein response (UPR) and macroautophagy (called hereafter autophagy). Both are necessary for cell survival, however, their over-activation in duration or intensity can lead to cell death. Moreover, UPR and autophagy can activate and potentiate each other to worsen the issue of cerebral ischemia. A better understanding of autophagy and ER stress will allow the development of therapeutic strategies for stroke, both at the acute phase and during recovery. This review summarizes the latest therapeutic advances implicating ER stress or autophagy in cerebral ischemia. We argue that the processes governing proteostasis should be considered together in stroke, rather than focusing either on ER stress or autophagy in isolation.

Toward safer thrombolytic agents in stroke: molecular requirements for NMDA receptor-mediated neurotoxicity.

Current thrombolytic therapy for acute ischemic stroke with tissue-type plasminogen activator (tPA) has clear global benefits. Nevertheless, evidences argue that in addition to its prohemorrhagic effect, tPA might enhance excitotoxic necrosis. In the brain parenchyma, tPA, by binding to and then cleaving the amino-terminal domain (ATD) of the NR1 subunit of N-methyl-D-aspartate (NMDA) glutamate receptors, increases calcium influx to toxic levels. We show here that tPA binds the ATD of the NR1 subunit by a two-sites system (K(D)=24 nmol/L). Although tenecteplase (TNK) and reteplase also display two-sites binding profiles, the catalytically inactive mutant TNKS478A displays a one-site binding profile and desmoteplase (DSPA), a kringle 2 (K2) domain-free plasminogen activator derived from vampire bat, does not interact with NR1. Moreover, we show that in contrast to tPA, DSPA does not promote excitotoxicity. These findings, together with three-dimensional (3D) modeling, show that a critical step for interaction of tPA with NR1 is the binding of its K2 domain, followed by the binding of its catalytic domain, which in turn cleaves the NR1 subunit at its ATD, leading to a subsequent potentiation of NMDA-induced calcium influx and neurotoxicity. This could help design safer new generation thrombolytic agents for stroke treatment.

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.

Recombinant Desmodus rotundus salivary plasminogen activator crosses the blood-brain barrier through a low-density lipoprotein receptor-related protein-dependent mechanism without exerting neurotoxic effects.

BACKGROUND AND PURPOSE: Desmoteplase, a recombinant form of the plasminogen activator DSPAalpha1 from Desmodus rotundus, may offer improved clinical benefits for acute ischemic stroke treatment over the current therapy, recombinant tissue plasminogen activator (rtPA). Accumulating evidence suggests that clinical use of rtPA could be limited by unfavorable properties, including its ability to cross the blood-brain barrier (BBB), thus potentially adding to the pro-excitotoxic effect of endogenous tPA in cerebral parenchyma. Here, to investigate whether desmoteplase may display a safer profile than the structurally-related tPA, both agents were compared for their ability to cross the BBB and promote neurotoxicity. METHODS: First, the passage of vascular DSPA and rtPA was investigated in vitro in a model of BBB, subjected or not to oxygen and glucose deprivation. Second, we studied DSPA- and rtPA-mediated effects in an in vivo paradigm of excitotoxic necrosis. RESULTS: The rtPA and desmoteplase cross the intact BBB by LRP-mediated transcytosis. Under conditions of oxygen and glucose deprivation, translocation rates of both compounds increased; however, unlike rtPA, desmoteplase transport remained LRP-dependent. Additionally, neither intracerebral nor intravenous desmoteplase administration enhanced NMDA-induced excitotoxic striatal damage in vivo. Interestingly, intravenous but not intrastriatal coadministration of desmoteplase and rtPA reduced the pro-excitotoxic effect of rtPA. CONCLUSIONS: We show that desmoteplase crosses the BBB but does not promote neuronal death. Moreover, intravenous administration of desmoteplase antagonizes the neurotoxicity induced by vascular rtPA. This action may be caused by competition of desmoteplase with rtPA for LRP binding at the BBB, thus effectively blocking rtPA access to the brain parenchyma.

Activation of cell surface GRP78 decreases endoplasmic reticulum stress and neuronal death.

The unfolded protein response (UPR) is an endoplasmic reticulum (ER) -related stress conserved pathway that aims to protect cells from being overwhelmed. However, when prolonged, UPR activation converts to a death signal, which relies on its PERK-eIF2α branch. Overactivation of the UPR has been implicated in many neurological diseases, including cerebral ischaemia. Here, by using an in vivo thromboembolic model of stroke on transgenic ER stress-reporter mice and neuronal in vitro models of ischaemia, we demonstrate that ischaemic stress leads to the deleterious activation of the PERK branch of the UPR. Moreover, we show that the serine protease tissue-type plasminogen activator (tPA) can bind to cell surface Grp78 (78 kD glucose-regulated protein), leading to a decrease of the PERK pathway activation, thus a decrease of the deleterious factor CHOP, and finally promotes neuroprotection. Altogether, this work highlights a new role and a therapeutic potential of the chaperone protein Grp78 as a membrane receptor of tPA capable to prevent from ER stress overactivation.

Progressive myoclonus epilepsy associated with neuroserpin inclusion bodies (neuroserpinosis).

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a conformational proteinopathy characterised by neuronal inclusion bodies composed of the serine protease inhibitor (SERPIN), neuroserpin. Presenting clinically as a familial dementia-epilepsy syndrome, the molecular mechanism of the pathogenic abnormalities in neuroserpin has been characterised at atomic resolution. There is a remarkable genotype-phenotype correlation between the degree of molecular destabilisation of the several variants of the neuroserpin protein, their propensity to self-associate and the age of onset of the dementia-epilepsy complex. As with other serpinopathies there appears to be a mix of cell-autonomous toxicity, due to neuronal accumulation of neuroserpin, and non-cell autonomous toxicity, caused by loss of protease inhibition, in this case the dysregulated protease is likely to be tissue plasminogen activator (tPA). FENIB should be considered in cases of progressive myoclonic epilepsy and dementia particularly where there is family history of neuropsychiatric disease.

Endoplasmic reticulum dysfunction in neurological disease.

Endoplasmic reticulum (ER) dysfunction might have an important part to play in a range of neurological disorders, including cerebral ischaemia, sleep apnoea, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, the prion diseases, and familial encephalopathy with neuroserpin inclusion bodies. Protein misfolding in the ER initiates the well studied unfolded protein response in energy-starved neurons during stroke, which is relevant to the toxic effects of reperfusion. The toxic peptide amyloid ß induces ER stress in Alzheimer's disease, which leads to activation of similar pathways, whereas the accumulation of polymeric neuroserpin in the neuronal ER triggers a poorly understood ER-overload response. In other neurological disorders, such as Parkinson's and Huntington's diseases, ER dysfunction is well recognised but the mechanisms by which it contributes to pathogenesis remain unclear. By targeting components of these signalling responses, amelioration of their toxic effects and so the treatment of a range of neurodegenerative disorders might become possible.

Structural dynamics associated with intermediate formation in an archetypal conformational disease.

In conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of α(1)-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn α(1)-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions.

Pharmacological activation/inhibition of the cannabinoid system affects alcohol withdrawal-induced neuronal hypersensitivity to excitotoxic insults.

Cessation of chronic ethanol consumption can increase the sensitivity of the brain to excitotoxic damages. Cannabinoids have been proposed as neuroprotectants in different models of neuronal injury, but their effect have never been investigated in a context of excitotoxicity after alcohol cessation. Here we examined the effects of the pharmacological activation/inhibition of the endocannabinoid system in an in vitro model of chronic ethanol exposure and withdrawal followed by an excitotoxic challenge. Ethanol withdrawal increased N-methyl-D-aspartate (NMDA)-evoked neuronal death, probably by altering the ratio between GluN2A and GluN2B NMDA receptor subunits. The stimulation of the endocannabinoid system with the cannabinoid agonist HU-210 decreased NMDA-induced neuronal death exclusively in ethanol-withdrawn neurons. This neuroprotection could be explained by a decrease in NMDA-stimulated calcium influx after the administration of HU-210, found exclusively in ethanol-withdrawn neurons. By contrast, the inhibition of the cannabinoid system with the CB1 receptor antagonist rimonabant (SR141716) during ethanol withdrawal increased death of ethanol-withdrawn neurons without any modification of NMDA-stimulated calcium influx. Moreover, chronic administration of rimonabant increased NMDA-stimulated toxicity not only in withdrawn neurons, but also in control neurons. In summary, we show for the first time that the stimulation of the endocannabinoid system is protective against the hyperexcitability developed during alcohol withdrawal. By contrast, the blockade of the endocannabinoid system is highly counterproductive during alcohol withdrawal.

Unravelling the twists and turns of the serpinopathies.

Members of the serine protease inhibitor (serpin) superfamily are found in all branches of life and play an important role in the regulation of enzymes involved in proteolytic cascades. Mutants of the serpins result in a delay in folding, with unstable intermediates being cleared by endoplasmic reticulum-associated degradation. The remaining protein is either fully folded and secreted or retained as ordered polymers within the endoplasmic reticulum of the cell of synthesis. This results in a group of diseases termed the serpinopathies, which are typified by mutations of α(1)-antitrypsin and neuroserpin in association with cirrhosis and the dementia familial encephalopathy with neuroserpin inclusion bodies, respectively. Current evidence strongly suggests that polymers of mutants of α(1)-antitrypsin and neuroserpin are linked by the sequential insertion of the reactive loop of one molecule into ß-sheet A of another. The ordered structure of the polymers within the endoplasmic reticulum stimulates nuclear factor-kappa B by a pathway that is independent of the unfolded protein response. This chronic activation of nuclear factor-kappa B may contribute to the cell toxicity associated with mutations of the serpins. We review the pathobiology of the serpinopathies and the development of novel therapeutic strategies for treating the inclusions that cause disease. These include the use of small molecules to block polymerization, stimulation of autophagy to clear inclusions and stem cell technology to correct the underlying molecular defect.