Carine Ali

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.

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.

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.

NMDA Receptor Activation Inhibits α-Secretase and Promotes Neuronal Amyloid-β Production

Acute brain injuries have been identified as a risk factor for developing Alzheimers disease (AD). Because glutamate plays a pivotal role in these pathologies, we studied the influence of glutamate receptor activation on amyloid-β (Aβ) production in primary cultures of cortical neurons. We found that sublethal NMDA receptor activation increased the production and secretion of Aβ. This effect was preceded by an increased expression of neuronal Kunitz protease inhibitory domain (KPI) containing amyloid-β precursor protein (KPI-APP) followed by a shift from α-secretase to β-secretase-mediated APP processing. This shift is a result of the inhibition of the α-secretase candidate tumor necrosis factor-α converting enzyme (TACE) when associated with neuronal KPI-APPs. This KPI-APP/TACE interaction was also present in AD brains. Thus, our findings reveal a cellular mechanism linking NMDA receptor activation to neuronal Aβ secretion. These results suggest that even mild deregulation of the glutamatergic neurotransmission may increase Aβ production and represent a causal risk factor for developing AD.

Tissue-Type Plasminogen Activator Crosses the Intact Blood-Brain Barrier by Low-Density Lipoprotein Receptor–Related Protein-Mediated Transcytosis

Background—Accumulating evidence demonstrates a critical involvement of tissue-type plasminogen activator (tPA) in pathological and physiological brain conditions. Determining whether and how vascular tPA can cross the blood-brain barrier (BBB) to enter the brain is thus important, not only during stroke but also in physiological conditions. Methods and Results—In the present work, we provide evidence in vivo that intravenous injection of tPA increases NMDA-induced striatal lesion in the absence of BBB leakage. Accordingly, we show that tPA crosses the BBB both after excitotoxic lesion and in control conditions. Indeed, vascular injected tPA can be detected within the brain parenchyma and in the cerebrospinal fluid. By using an in vitro model of BBB, we have confirmed that tPA can cross the intact BBB. Its passage was blocked at 4°C, was saturable, and was independent of its proteolytic activity. We have shown that tPA crosses the BBB by transcytosis, mediated by a member of the LDL receptor–related protein family. Conclusions—We demonstrate that blood-derived tPA can reach the brain parenchyma without alteration of the BBB. The molecular mechanism of the passage of tPA from blood to brain described here could represent an interesting target to improve thrombolysis in stroke

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.

Arginine 260 of the Amino-terminal Domain of NR1 Subunit Is Critical for Tissue-type Plasminogen Activator-mediated Enhancement of N-Methyl-D-aspartate Receptor Signaling

Tissue-type plasminogen activator (tPA) has been involved in both physiological and pathological glutamatergic-dependent processes, such as synaptic plasticity, seizure, trauma, and stroke. In a previous study, we have shown that the proteolytic activity of tPA enhances the N-methyl-d-aspartate (NMDA) receptor-mediated signaling in neurons (Nicole, O., Docagne, F., Ali, C., Margaill, I., Carmeliet, P., MacKenzie, E. T., Vivien, D., and Buisson, A. (2001) Nat. Med. 7, 59–64). Here, we show that tPA forms a direct complex with the amino-terminal domain (ATD) of the NR1 subunit of the NMDA receptor and cleaves this subunit at the arginine 260. Furthermore, point mutation analyses show that arginine 260 is necessary for both tPA-induced cleavage of the ATD of NR1 and tPA-induced potentiation of NMDA receptor signaling. Thus, tPA is the first binding protein described so far to interact with the ATD of NR1 and to modulate the NMDA receptor function.

Neurobehavioral and Immunological Consequences of Prenatal Immune Activation in Rats. Influence of Antipsychotics

Increasing evidence suggests that pre- or perinatal events that influence the immune system contribute to the development of behavioral or neuropsychiatric disorders. For instance, exposure of pregnant rats to the bacterial endotoxin lipopolysaccharide (LPS) disrupts sensorimotor information processing, as assessed by the prepulse inhibition test (PPI), and also the immune function in adult offspring, which might be of particular relevance as regards schizophrenia. However, the consequences of maternal LPS exposure during pregnancy on synaptic functioning in adult offspring and, more importantly, the therapeutic opportunity to re-establish PPI and immune function have still to be demonstrated. In this work, we analyzed the consequences of prenatal LPS exposure on dopaminergic neurotransmission and presynaptic markers in adult brain areas related to PPI circuitry. In addition, we tested whether oral treatment with the typical antipsychotic drug haloperidol (HAL) could reinstate PPI performances and cytokine serum levels in six-month-old male rats with prenatal LPS exposure. Both sensory information processing deficits and immune anomalies induced by prenatal exposure to LPS were accompanied by changes in dopaminergic neurotransmission and synaptophysin expression. It is important to note that PPI disruption and serum increases in cytokines induced by prenatal LPS exposure were both reversed by HAL. Taken together, these results demonstrate the critical influence of prenatal immune events on the functioning of adult nervous and immune systems, in association with the putative role of the immune system in the development of behavior relevant to schizophrenia.

Activation of microglial N-methyl-D-aspartate receptors triggers inflammation and neuronal cell death in the developing and mature brain

Activated microglia play a central role in the inflammatory and excitotoxic component of various acute and chronic neurological disorders. However, the mechanisms leading to their activation in the latter context are poorly understood, particularly the involvement of N‐methyl‐D‐aspartate receptors (NMDARs), which are critical for excitotoxicity in neurons. We hypothesized that microglia express functional NMDARs and that their activation would trigger neuronal cell death in the brain by modulating inflammation.

Excitotoxicity in a chronic model of multiple sclerosis: Neuroprotective effects of cannabinoids through CB1 and CB2 receptor activation

Inflammation, autoimmune response, demyelination and axonal damage are thought to participate in the pathogenesis of multiple sclerosis (MS). Understanding whether axonal damage causes or originates from demyelination is a crucial issue. Excitotoxic processes may be responsible for white matter and axonal damage. Experimental and clinical studies indicate that cannabinoids could prove efficient in the treatment of MS. Using a chronic model of MS in mice, we show here that clinical signs and axonal damage in the spinal cord were reduced by the AMPA antagonist, NBQX. Amelioration of symptomatology by the synthetic cannabinoid HU210 was also accompanied by a reduction of axonal damage in this model. Moreover, HU210 reduced AMPA-induced excitotoxicity both in vivo and in vitro through the obligatory activation of both CB1 and CB2 cannabinoid receptors. Together, these data underline the implication of excitotoxic processes in demyelinating pathologies such as MS and the potential therapeutic properties of cannabinoids.