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International audience; Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a routine method in many laboratories. Normalization of data from experimental conditions is critical for data processing and is usually achieved by the use of a single reference gene. Nevertheless, as pointed by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, several reference genes should be used for reliable normalization. Ageing is a physiological process that results in a decline of many expressed genes. Reliable normalization of RT-qPCR data becomes crucial when studying ageing. Here, we propose a RT-qPCR study from four mouse brain regions (cortex, hippocampus, striatum and cerebellum) at different ages (from 8 weeks to 22 months) in which we studied the expression of nine commonly used reference genes. With the use of two different algorithms, we found that all brain structures need at least two genes for a good normalization step. We propose specific pairs of gene for efficient data normalization in the four brain regions studied. These results underline the importance of reliable reference genes for specific brain regions in ageing.

International audience; 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 Ca²⁺ 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.

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.

International audience; 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.

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.

International audience; 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 μmol/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.

International audience; 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. Overview The World Health Organization (WHO; www.who.int) defines stroke as 'rapidly developing clinical signs of focal (or global) disturbance of cerebral function, with symptoms lasting 24 h or longer or leading to death, with no apparent cause other than that of vascular origin'. Stroke might be classified as either ischemic or hemorrhagic. Ischemic stroke is a leading cause of disability and the third cause of mortality after heart disease and cancer (American Heart Association report, 2008, www.americanheart.org), accounting for 5.5 million deaths worldwide, two-thirds of which are in developing countries [1]. Additionally, each year millions of ischemic stroke survivors have to adapt to a life with restrictions in the activities of daily living, and many of them depend on other people's support to survive. Three approaches have been investigated for the treatment of patients with acute ischemic stroke: neuroprotec-tion, thrombolysis and mechanical removal of the clot. In contrast to neuroprotection, which has been largely unsuccessful , thrombolysis with tissue-type plasminogen activa-tor (tPA) [2] and mechanical removal of the clot [3,4] have been approved by the Food and Drug Administration (FDA; www.fda.gov) for the treatment of patients with acute ischemic stroke. In a study performed by the National Institute of Neurological Disorders and Stroke (NINDS; www.ninds.nih.gov), treatment with tPA within 3 h of the onset of symptoms of acute ischemic stroke resulted in an 11–13% absolute increase in the number of patients with good outcome and in a reduction in the proportion of patients severely disabled or dead at 3 months [2]. Based on these results, intravenous tPA is currently the standard of care for patients with acute ischemic stroke presenting within 3 h of onset of the ischemic event. By contrast, there is a growing body of evidence indicating that tPA also has a deleterious effect in the ischemic brain. Indeed, in the NINDS study the use of tPA was associated with a 10-fold increase in the incidence of intracerebral hemorrhage, which had a 60% mortality [2]. Likewise, animal studies have demonstrated that either genetic deficiency of tPA [5,6] or inhibition of tPA with neuroserpin [7] results in a decrease in the volume of the ischemic lesion after experimental occlusion of the middle cerebral artery. Taken together, these studies indicate a pleiotropic role for tPA in the ischemic brain that is highly dependent of the compartment whereupon tPA acts (intravascular versus extravascular). Here, we review both the beneficial and deleterious effects of tPA in the ischemic brain and discusses potential approaches to improve the treatment of patients with acute ischemic stroke.

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. © 2008 ISCBFM All rights reserved., This work was supported by grants from INSERM, the French Ministry of Research and Technology, the Regional Council of Lower Normandy, the Regional Platform for Cell Imaging of Upper Normandy, the Medical Research Foundation, the University of Caen, Fonds Europeéns de Développement Régional, the Paul Hamel Foundation, the Diagnosis in Molecular Imaging Program (FP6-project DiMI-LSHB-CT-2005-512146-), and PAION Deutschland GmbH.

International audience; Although the mechanism of action of tissue-type plasmino-gen 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. Keywords: apoptosis, serine protease, serum deprivation, tissue-type plasminogen activator.