Benoit D. Roussel

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.

Anti-NR1 N-terminal-domain vaccination unmasks the crucial action of tPA on NMDA-receptor-mediated toxicity and spatial memory.

Fine-tuning of NMDA glutamatergic receptor signalling strategically controls crucial brain functions. This process depends on several ligands and modulators, one of which unexpectedly includes the serine protease tissue-type plasminogen activator (tPA). In vitro, tPA increases NMDA-receptor-mediated calcium influx by interacting with, and then cleaving, the NR1 subunit within its N-terminal domain. Owing to lack of in vivo evidence of the relevance and contribution of this mechanism in physiological and pathological brain processes, active immunisation was developed here in mice, to allow transient and specific prevention of the interaction of tPA with the NR1 subunit. Immunisation significantly reduced the severity of ischemic and excitotoxic insults in the mouse brain. Cognitive function was altered in some, but not all behavioural tasks affected in tPA-deficient mice. Our data demonstrate that in vivo, tPA controls neurotoxicity and the encoding of novel spatial experiences by binding to and cleaving the NMDA receptor NR1 subunit. Interesting therapeutic possibilities for several brain pathologies that involve excitotoxicity may now be envisaged.

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 (KD=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.

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 DSPAα1 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.

Antibodies Preventing the Interaction of Tissue-Type Plasminogen Activator With N-Methyl-d-Aspartate Receptors Reduce Stroke Damages and Extend the Therapeutic Window of Thrombolysis

Background and Purpose— Tissue-type plasminogen activator (tPA) is the only drug approved for the acute treatment of ischemic stroke but with two faces in the disease: beneficial fibrinolysis in the vasculature and damaging effects on the neurovascular unit and brain parenchyma. To improve this profile, we developed a novel strategy, relying on antibodies targeting the proneurotoxic effects of tPA. Methods— After production and characterization of antibodies (&agr;ATD-NR1) that specifically prevent the interaction of tPA with the ATD-NR1 of N-methyl-D-aspartate receptors, we have evaluated their efficacy in a model of murine thromboembolic stroke with or without recombinant tPA-induced reperfusion, coupled to MRI, near-infrared fluorescence imaging, and behavior assessments. Results— In vitro, &agr;ATD-NR1 prevented the proexcitotoxic effect of tPA without altering N-methyl-D-aspartate-induced neurotransmission. In vivo, after a single administration alone or with late recombinant tPA-induced thrombolysis, antibodies dramatically reduced brain injuries and blood–brain barrier leakage, thus improving long-term neurological outcome. Conclusions— Our strategy limits ischemic damages and extends the therapeutic window of tPA-driven thrombolysis. Thus, the prospect of this immunotherapy is an extension of the range of treatable patients.

Tissue-type plasminogen activator rescues neurones from serum deprivation-induced apoptosis through a mechanism independent of its proteolytic activity

Although the mechanism of action of tissue‐type plasminogen activator (tPA) in excitotoxic necrosis is well documented, whether this serine protease can influence the apoptotic cascade remains a subject of debate. Here, we report that tPA protects cultured cortical neurones against apoptotic cell death induced by serum deprivation, an effect associated with a reduction of caspase‐3 activation. Interestingly, blocking tPA proteolytic activity by either tPA stop or neuroserpin did not prevent this neuroprotection. Similarly, prevention of the interaction between tPA and its receptor low‐density lipoprotein receptor‐related protein (LRP) could not alter tPA anti‐apoptotic activity. Interestingly, the survival‐promoting effect of tPA was blocked by the phosphatidylinositol‐3 (PI‐3) kinase inhibitor, LY294002, but not by the mitogen‐activated protein (MAP) kinase inhibitor, U0126. In conclusion, the present demonstration of an anti‐apoptotic effect of tPA, independent of its enzymatic activity, reveals an additional level of complexity in our understanding of this critical mediator of brain physiology and pathology.

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.

Adaptive preconditioning in neurological diseases – therapeutic insights from proteostatic perturbations

In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a ‘proteostasis network’ and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge – the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.

Cerebrovascular protection as a possible mechanism for the protective effects of NXY-059 in preclinical models: An in vitro study

NXY-059, a polar compound with limited transport across the blood-brain barrier, has demonstrated neuroprotection in several animal models of acute ischemic stroke but failed to confirm clinical benefit in the second phase III trial (SAINT-II). To improve the understanding of the mechanisms responsible for its neuroprotective action in preclinical models a series of experiments was carried out in an in vitro blood-brain barrier (BBB) model. A clinically attainable concentration of 250 mumol/L of NXY-059 administered at the onset or up to 4 h after oxygen glucose deprivation (OGD) produced a significant reduction in the increased BBB permeability caused by OGD. Furthermore, OGD produced a huge influx of tissue plasminogen activator across the BBB, which was substantially reduced by NXY-059. This study suggests that the neuroprotective effects of NXY-059 preclinically, may at least in part be attributed to its ability to restore functionality of the brain endothelium.

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.