Fabian Docagne

The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling.

Tissue-plasminogen activator (t-PA) is now available for the treatment of thrombo-embolic stroke but adverse effects have been reported in some patients, particularly hemorrhaging. In contrast, the results of animal studies have indicated that t-PA could increase neuronal damage after focal cerebral ischemia. Here we report for the first time that t-PA potentiates signaling mediated by glutamatergic receptors by modifying the properties of the N-methyl-D-aspartate (NMDA) receptor. When depolarized, cortical neurons release bio-active t-PA that interacts with and cleaves the NR1 subunit of the NMDA receptor. Moreover, the treatment with recombinant t-PA leads to a 37% increase in NMDA-stimulated fura-2 fluorescence, which may reflect an increased NMDA-receptor function. These results were confirmed in vivo by the intrastriatal injection of recombinant-PA, which potentiated the excitotoxic lesions induced by NMDA. These data provide insight into the regulation of NMDA-receptor–mediated signaling and could initiate therapeutic strategies to improve the efficacy of t-PA treatment in man.

Ischemia-Induced Interleukin-6 as a Potential Endogenous Neuroprotective Cytokine against NMDA Receptor-Mediated Excitoxicity in the Brain

In the brain, the expression of the pleiotropic cytokine interleukin-6 (IL-6) is enhanced in various chronic or acute central nervous system disorders. However, the significance of IL-6 production in such neuropathologic states remains controversial. The present study investigated the role of IL-6 after cerebral ischemia. First, the authors showed that focal cerebral ischemia in rats early up-regulated the expression of IL-6 mRNA, without affecting the transcription of its receptors (IL-6Rα: and gp130). Similarly, the striatal injection of N-methyl-d-aspartate (NMDA) in rats, a paradigm of excitotoxic injury, activated the expression of IL-6 mRNA. The involvement of glutamatergic receptor activation was further investigated by incubating cortical neurons with NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). NMDA and ionomycin (a calcium ionophore) up-regulated IL-6 mRNA, suggesting that neurons may produce IL-6 in response to the calcium influx mediated through NMDA receptors. The potential role of IL-6 during ischemic/excitotoxic insults was then studied by testing the effect of IL-6 against apoptotic or excitotoxic challenges in cortical cultures. IL-6 did not prevent serum deprivation- or staurosporine-induced apoptotic neuronal death, or AMPA/kainate-mediated excitotoxicity. However, in both mixed and pure neuronal cultures, IL-6 dose-dependently protected neurons against NMDA toxicity. This effect was blocked by a competitive inhibitor of IL-6. Overall, the results suggest that the up-regulation of IL-6 induced by cerebral ischemia could represent an endogenous neuroprotective mechanism against NMDA receptor-mediated injury.

Transforming Growth Factor-β1 Potentiates Amyloid-β Generation in Astrocytes and in Transgenic Mice

Accumulation of the amyloid-β peptide (Aβ) in the brain is crucial for development of Alzheimers disease. Expression of transforming growth factor-β1 (TGF-β1), an immunosuppressive cytokine, has been correlated in vivowith Aβ accumulation in transgenic mice and recently with Aβ clearance by activated microglia. Here, we demonstrate that TGF-β1 drives the production of Aβ40/42 by astrocytes leading to Aβ production in TGF-β1 transgenic mice. First, TGF-β1 induces the overexpression of the amyloid precursor protein (APP) in astrocytes but not in neurons, involving a highly conserved TGF-β1-responsive element in the 5′-untranslated region (+54/+74) of the APP promoter. Second, we demonstrated an increased release of soluble APP-β which led to TGF-β1-induced Aβ generation in both murine and human astrocytes. These results demonstrate that TGF-β1 potentiates Aβ production in human astrocytes and may enhance the formation of plaques burden in the brain of Alzheimers disease patients.

Transforming Growth Factor-β and Ischemic Brain Injury

Abstract1. Necrosis and apoptosis are the two fundamental hallmarks of neuronal death in stroke. Nevertheless, thrombolysis, by using the recombinant serine protease t-PA, remains until now the only approved treatment of stroke in man.2. Over the last years, the cytokine termed Transforming Growth Factor-β1 (TGF-β1) has been found to be strongly up-regulated in the central nervous system following ischemia-induced brain damage.3. Recent studies have shown a neuroprotective activity of TGF-β1 against ischemia-induced neuronal death. In vitro, TGF-β1 protects neurons against excitotoxicity by inhibiting the t-PA-potentiated NMDA-induced neuronal death through a mechanism involving the up-regulation of the type-1 plasminogen activator inhibitor (PAI-1) in astrocytes.4. In addition, TGF-β1 has been recently characterized as an antiapoptotic factor in a model of staurosporine-induced neuronal death through a mechanism involving activation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and a concomitant increase phosphorylation of the antiapoptotic protein Bad.5. Altogether, these observations suggest that either TGF-β signaling or TGF-β1-modulated genes could be good targets for the development of new therapeutic strategies for stroke in man.

Smad3-Dependent Induction of Plasminogen Activator Inhibitor-1 in Astrocytes Mediates Neuroprotective Activity of Transforming Growth Factor-β1 against NMDA-Induced Necrosis

The intravenous injection of the serine protease, tissue-type plasminogen activator (t-PA), has shown to benefit stroke patients by promoting early reperfusion. However, it has recently been suggested that t-PA activity, in the cerebral parenchyma, may also potentiate excitotoxic neuronal death. The present study has dealt with the role of the t-PA inhibitor, PAI-1, in the neuroprotective activity of the cytokine TGF-beta1 and focused on the transduction pathway involved in this effect. We demonstrated that PAI-1, produced by astrocytes, mediates the neuroprotective activity of TGF-beta 1 against N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity. This t-PA inhibitor, PAI-1, protected neurons against NMDA-induced neuronal death by modulating the NMDA-evoked calcium influx. Finally, we showed that the activation of the Smad3-dependent transduction pathway mediates the TGF-beta-induced up-regulation of PAI-1 and subsequent neuroprotection. Overall, this study underlines the critical role of the t-PA/PAI-1 axis in the regulation of glutamatergic neurotransmission.

Sp1 and Smad transcription factors co-operate to mediate TGF-β-dependent activation of amyloid-β precursor protein gene transcription

Abnormal deposition of Abeta (amyloid-beta peptide) is one of the hallmarks of AD (Alzheimers disease). This peptide results from the processing and cleavage of its precursor protein, APP (amyloid-beta precursor protein). We have demonstrated previously that TGF-beta (transforming growth factor-beta), which is overexpressed in AD patients, is capable of enhancing the synthesis of APP by astrocytes by a transcriptional mechanism leading to the accumulation of Abeta. In the present study, we aimed at further characterization of the molecular mechanisms sustaining this TGF-beta-dependent transcriptional activity. We report the following findings: first, TGF-beta is capable of inducing the transcriptional activity of a reporter gene construct corresponding to the +54/+74 region of the APP promoter, named APP(TRE) (APP TGF-beta-responsive element); secondly, although this effect is mediated by a transduction pathway involving Smad3 (signalling mother against decapentaplegic peptide 3) and Smad4, Smad2 or other Smads failed to induce the activity of APP(TRE). We also observed that the APP(TRE) sequence not only responds to the Smad3 transcription factor, but also the Sp1 (signal protein 1) transcription factor co-operates with Smads to potentiate the TGF-beta-dependent activation of APP. TGF-beta signalling induces the formation of nuclear complexes composed of Sp1, Smad3 and Smad4. Overall, the present study gives new insights for a better understanding of the fine molecular mechanisms occurring at the transcriptional level and regulating TGF-beta-dependent transcription. In the context of AD, our results provide additional evidence for a key role for TGF-beta in the regulation of Abeta production.

Transforming growth factor alpha-induced expression of type 1 plasminogen activator inhibitor in astrocytes rescues neurons from excitotoxicity.

Although transforming growth factor (TGF)‐α, a member of the epidermal growth factor (EGF) family, has been shown to protect neurons against excitotoxic and ischemic brain injuries, its mechanism of action remains unknown. In the present study, we used in vitro models of apoptotic or necrotic paradigms demonstrating that TGF‐α rescues neurons from N‐methyl‐d‐aspartate (NMDA)‐induced excitotoxic death, with the obligatory presence of astrocytes. Because neuronal tissue‐type plasminogen activator (t‐PA) release was shown to potentiate NMDA‐induced excitotoxicity, we observed that TGF‐α treatment reduced NMDA‐induced increase of t‐PA activity in mixed cultures of neurons and astrocytes. In addition, we showed that although TGF‐α induces activation of the extracellular signalregulated kinases (ERKs) in astrocytes, it failed to activate p42/p44 in neurons. Finally, we showed that TGF‐α, by an ERK‐dependent mechanism, stimulates the astrocytic expression of PAI‐1, a t‐PA inhibitor, which mediates the neuroprotective activity of TGF‐α against NMDA‐mediated excitotoxic neuronal death. Taken together, we indicate that TGF‐α rescues neurons from NMDA‐induced excitotoxicity in mixed cultures through inhibition of t‐PA activity, involving PAI‐1 overexpression by an ERK‐dependent pathway in astrocytes.

Differential regulation of type I and type II interleukin-1 receptors in focal brain inflammation

Most pathologies of the brain have an inflammatory component, associated with the release of cytokines such as interleukin‐1β (IL‐1β) from resident and infiltrating cells. The IL‐1 type I receptor (IL‐1RI) initiates a signalling cascade but the type II receptor (IL‐1RII) acts as a decoy receptor. Here we have investigated the expression of IL‐1β, IL‐1RI and IL‐1RII in distinct inflammatory lesions in the rat brain. IL‐1β was injected into the brain to generate an inflammatory lesion in the absence of neuronal cell death whereas neuronal death was specifically induced by the microinjection of N‐methyl‐d‐aspartate (NMDA). Using TaqMan RT‐PCR and ELISA, we observed elevated de novo IL‐1β synthesis 2 h after the intracerebral microinjection of IL‐1β; this de novo IL‐1β remained elevated 24 h later. There was a concomitant increase in IL‐1RI mRNA but a much greater increase in IL‐1RII mRNA. Immunostaining revealed that IL‐1RII was expressed on brain endothelial cells and on infiltrating neutrophils. In contrast, although IL‐1β and IL‐1RI were elevated to similar levels in response to NMDA challenge, the response was delayed and IL‐1RII mRNA expression was unchanged. The lesion‐specific expression of IL‐1 receptors suggests that the receptors are differentially regulated in a manner not directly related to the endogenous level of IL‐1 in the CNS.

Mechanisms of glutamate toxicity in multiple sclerosis: biomarker and therapeutic opportunities

Research advances support the idea that excessive activation of the glutamatergic pathway plays an important part in the pathophysiology of multiple sclerosis. Beyond the well established direct toxic effects on neurons, additional sites of glutamate-induced cell damage have been described, including effects in oligodendrocytes, astrocytes, endothelial cells, and immune cells. Such toxic effects could provide a link between various pathological aspects of multiple sclerosis, such as axonal damage, oligodendrocyte cell death, demyelination, autoimmunity, and blood-brain barrier dysfunction. Understanding of the mechanisms underlying glutamate toxicity in multiple sclerosis could help in the development of new approaches for diagnosis, treatment, and follow-up in patients with this debilitating disease. While several clinical trials of glutamatergic modulators have had disappointing results, our growing understanding suggests that there is reason to remain optimistic about the therapeutic potential of these drugs.

The plasminogen activation system in neuroinflammation.

The plasminogen activation (PA) system consists in a group of proteases and protease inhibitors regulating the activation of the zymogen plasminogen into its proteolytically active form, plasmin. Here, we give an update of the current knowledge about the role of the PA system on different aspects of neuroinflammation. These include modification in blood-brain barrier integrity, leukocyte diapedesis, removal of fibrin deposits in nervous tissues, microglial activation and neutrophil functions. Furthermore, we focus on the molecular mechanisms (some of them independent of plasmin generation and even of proteolysis) and target receptors responsible for these effects. The description of these mechanisms of action may help designing new therapeutic strategies targeting the expression, activity and molecular mediators of the PA system in neurological disorders involving neuroinflammatory processes. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.