Olivier Nicole

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.

Neuronal viability is controlled by a functional relation between synaptic and extrasynaptic NMDA receptors

N‐methyl‐D‐aspartate receptors (NMDARs) are critical for synaptic plasticity that underlies learning and memory. But, they have also been described as a common source of neuronal damage during stroke and neurodegenerative diseases. Several studies have sug gested that cellular location of NMDARs (synaptic or extrasynaptic) is a key parameter controlling their effect on neuronal viability. The aim of the study was to understand the relation between these two pools of receptors and to determine their implication in both beneficial and/or deleterious events related to NMDAR activation. We demonstrated that selective extrasynap tic NMDAR activation, as well as NMDA bath applica tion, does not activate extracellular signal‐regulated kinase (ERK) pathways, but induces mitochondrial membrane potential breakdown and triggers cell body and dendrite damages, whereas synaptic NMDAR acti vation is innocuous and induces a sustained ERK acti vation. The functional dichotomy between these two NMDAR pools is tightly controlled by glutamate uptake systems. Finally, we demonstrated that the only clini cally approved NMDAR antagonist, memantine, prefer entially antagonizes extrasynaptic NMDARs. Together, these results suggest that extrasynaptic NMDAR activa tion contributes to excitotoxicity and that a selective targeting of the extrasynaptic NMDARs represents a promising therapeutic strategy for brain injuries.— Léveillé, F., El gaamouch, F., Gouix, E., Lecocq, M., Lobner, D., Nicole, O., Buisson, A. Neuronal viability is controlled by a functional relation between synaptic and extrasynaptic NMDA receptors. FASEB J. 22, 4258–4271 (2008)

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.

Up-regulation of a serine protease inhibitor in astrocytes mediates the neuroprotective activity of transforming growth factor β1

Serine proteases play a key role in the fundamental biology of the central nervous system (CNS), and recent data suggest their involvement in the pathophysiology of neurodegenerative diseases. Little is known about the physiological regulation of these proteases in the CNS. Among the multiple growth factors present in the brain, transforming growth factor β1 (TGF‐β1) has been described as an injury‐related growth factor. However, its beneficial or deleterious role remains unclear. In the present study, we investigated the influence of TGF‐β1 in apoptosis and necrosis, two mechanisms involved in ischemic neuronal death. We show that TGF‐β1 exerts a neuroprotective role restricted to necrosis induced by N‐methyl‐D‐aspartate. This effect is observable only in the obligatory presence of TGF‐β1‐responsive astrocytes. We demonstrate that this neuroprotective activity is mediated through an up‐regulation of a serine protease inhibitor (PAI‐1) in astrocytes. These results underline the involvement of serine proteases and extracellular matrix components such as the PAI‐1/t‐PA axis in the excitotoxic cascade. Moreover, regardless of the underlying mechanisms of t‐PA involvement in excitotoxic injury, our observations might warn against the use of tissular plasminogen activator as an alternative therapy for the treatment of hypoxic‐ischemic injury in the brain.—Buisson, A., Nicole, O., Docagne, F., Sartelet, H., MacKenzie, E. T., Vivien, D. Up‐regulation of a serine protease inhibitor in astrocytes mediates the neuroprotective activity of transforming growth factor β1. FASEB J. 12, 1683–1691 (1998)

Activation of Extrasynaptic, But Not Synaptic, NMDA Receptors Modifies Amyloid Precursor Protein Expression Pattern and Increases Amyloid-β Production

Calcium is a key mediator controlling essential neuronal functions depending on electrical activity. Altered neuronal calcium homeostasis affects metabolism of amyloid precursor protein (APP), leading to increased production of β-amyloid (Aβ), and contributing to the initiation of Alzheimers disease (AD). A linkage between excessive glutamate receptor activation and neuronal Aβ release was established, and recent reports suggest that synaptic and extrasynaptic NMDA receptor (NMDAR) activation may have distinct consequences in plasticity, gene regulation, and neuronal death. Here, we report for the first time that prolonged activation of extrasynaptic NMDAR, but not synaptic NMDAR, dramatically increased the neuronal production of Aβ. This effect was preceded by a shift from APP695 to Kunitz protease inhibitory domain (KPI) containing APPs (KPI-APPs), isoforms exhibiting an important amyloidogenic potential. Conversely, after synaptic NMDAR activation, we failed to detect any KPI-APP expression and neuronal Aβ production was not modified. Calcium imaging data showed that intracellular calcium concentration after extrasynaptic NMDAR stimulation was lower than after synaptic activation. This suggests distinct signaling pathways for each pool of receptors. We found that modification of neuronal APP expression pattern triggered by extrasynaptic NMDAR activation was regulated at an alternative splicing level involving calcium-/calmodulin-dependent protein kinase IV, but overall APP expression remained identical. Finally, memantine dose-dependently inhibited extrasynaptic NMDAR-induced KPI-APPs expression as well as neuronal Aβ release. Altogether, these data suggest that a chronic activation of extrasynaptic NMDAR promotes amyloidogenic KPI-APP expression leading to neuronal Aβ release, representing a causal risk factor for developing AD.

A Transforming Growth Factor-β Antagonist Unmasks the Neuroprotective Role of This Endogenous Cytokine in Excitotoxic and Ischemic Brain Injury

Various studies describe increased concentrations of transforming growth factor-β (TGF-β) in brain tissue after acute brain injury. However, the role of endogenously produced TGF-β after brain damage to the CNS remains to be clearly established. Here, the authors examine the influence of TGF-β produced after an episode of cerebral ischemia by injecting a soluble TGF-β type II receptor fused with the Fc region of a human immunoglobulin (TβRIIs-Fc). First, this molecular construct was characterized as a selective antagonist of TGF-β. Then, the authors tested its ability to reverse the effect of TGF-β 1 on excitotoxic cell death in murine cortical cell cultures. The addition of 1 μg/mL of TβRIIs-Fc to the exposure medium antagonized the neuroprotective activity of TGF-β 1 in N-methyl-D-aspartate (NMDA)-induced excitotoxic cell death. These results are consistent with the hypothesis that TGF-β 1 exerts a negative modulatory action on NMDA receptor-mediated excitotoxicity. To determine the role of TGF-β 1 produced in response to brain damage, the authors used a model of an excitotoxic lesion induced by the intrastriatal injection of 75 nmol of NMDA in the presence of 1.5 μg of TβRIIs-Fc. The intrastriatal injection of NMDA was demonstrated to induce an early upregulation of the expression of TGF-β 1 mRNA. Furthermore, when added to the excitotoxin, TβRIIs-Fc increased (by 2.2-fold, P < 0.05) the lesion size. These observations were strengthened by the fact that an intracortical injection of TβRIIs-Fc in rats subjected to a 30-minute reversible cerebral focal ischemia aggravated the volume of infarction. In the group injected with the TGF-β 1 antagonist, a 3.5-fold increase was measured in the infarction size (43.3 ± 9.5 versus 152.8 ± 46.3 mm3; P < 0.05). In conclusion, by antagonizing the influence of TGF-β in brain tissue subjected to excitotoxic or ischemic lesion, the authors markedly exacerbated the resulting extent of necrosis. These results suggest that, in response to such insults, brain tissue responds by the synthesis of a neuroprotective cytokine, TGF-β1, which is involved in the limitation of the extent of the injury. The pharmacologic potentiation of this endogenous defensive mechanism might represent an alternative and novel strategy for the therapy of hypoxic-ischemic cerebral injury.

Complement anaphylatoxin C3a is selectively protective against NMDA-induced neuronal cell death.

The anaphylatoxin C3a is a potent inflammatory polypeptide released at sites of complement activation. To test whether C3a might alter neuronal outcome following an ischemic insult, we determined the effects of purified human C3a on murine primary cortical cell cultures exposed to apoptotic or excitotoxic paradigms. C3a prevented neither serum deprivation-induced apoptotic neuronal death, nor AMPA/kainate-mediated excitotoxicity. However, in mixed cultures of neurons and astrocytes, C3a dose-dependently protected neurons against NMDA toxicity (47% neuroprotection using 100 nM C3a, p < 0.01, n = 12). The neuroprotective effect of C3a was observable only in the presence of astrocytes. These observations suggest that C3a is involved in excitotoxicity-mediated neuronal death through astrocyte stimulation and extend its role beyond immune functions.

Transforming growth factor-β1 as a regulator of the serpins/t-PA axis in cerebral ischemia

The tissue type plasminogen activator (t‐PA) is a serine protease that is involved in neuronal plasticity and cell death induced by excitotoxins and ischemia in the brain. t‐PA activity in the central nervous system is regulated through the activation of serine protease inhibitors (serpins) such as the plasminogen activator inhibitor (PAI‐1), the protease nexin‐1 (PN‐1), and neuroserpin (NSP). Recently we demonstrated in vitro that PAI‐1 produced by astrocytes mediates the neuroprotective effect of the transforming growth factor‐β1 (TGF‐β1) in NMDA‐induced neuronal cell death. To investigate whether serpins may be involved in neuronal cell death after cerebral ischemia, we determined, by using semiquantitative RT‐PCR and in situ hybridization, that focal cerebral ischemia in mice induced a dramatic overexpression of PAI‐1 without any effect on PN‐1, NSP, or t‐PA. Then we showed that although the expression of PAI‐1 is restricted to astrocytes, PN‐1, NSP, and t‐PA are expressed in both neurons and astrocytes. Moreover, by using semiquantitative RT‐PCR and Western blotting, we observed that only the expression of PAI‐1 was modulated by TGF‐β1 treatment via a TGF‐β‐inducible element contained in the PAI‐1 promoter (CAGAbox). Finally, we compared the specificity of TGF‐β 1 action with other members of the TGF‐β family by using luciferase reporter genes. These data show that TGF‐β and activin were able to induce the overexpression of PAI‐1 in astrocytes, but that bone morphogenetic proteins, glial cell line‐derived neutrophic factor, and neurturin did not. These results provide new insights into the regulation of the serpins/t‐PA axis and the mechanism by which TGF‐β may be neuroprotective.—Docagne, F., Nicole, O., Marti, H. H., MacKenzie, E. T., Buisson, A., Vivien, D. Transforming growth factor‐β1 as a regulator of the serpins/t‐PA axis in cerebral ischemia. FASEB J. 13, 1315–1324 (1999)

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.