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ATP signaling and surface P2X4 receptors are upregulated selectively in neurons and/or glia in various CNS disorders including anxiety, chronic pain, epilepsy, ischemia, and neurodegenerative diseases. However, the cell-specific functions of P2X4 in pathological contexts remain elusive. To elucidate P2X4 functions, we created a conditional transgenic knock-in P2X4 mouse line (Floxed P2X4mCherryIN) allowing the Cre activity-dependent genetic swapping of the internalization motif of P2X4 by the fluorescent mCherry protein to prevent constitutive endocytosis of P2X4. By combining molecular, cellular, electrophysiological, and behavioral approaches, we characterized two distinct knock-in mouse lines expressing noninternalized P2X4mCherryIN either exclusively in excitatory forebrain neurons or in all cells natively expressing P2X4. The genetic substitution of wild-type P2X4 by noninternalized P2X4mCherryIN in both knock-in mouse models did not alter the sparse distribution and subcellular localization of P2X4 but increased the number of P2X4 receptors at the surface of the targeted cells mimicking the pathological increased surface P2X4 state. Increased surface P2X4 density in the hippocampus of knock-in mice altered LTP and LTD plasticity phenomena at CA1 synapses without affecting basal excitatory transmission. Moreover, these cellular events translated into anxiolytic effects and deficits in spatial memory. Our results show that increased surface density of neuronal P2X4 contributes to synaptic deficits and alterations in anxiety and memory functions consistent with the implication of P2X4 in neuropsychiatric and neurodegenerative disorders. Furthermore, these conditional P2X4mCherryIN knock-in mice will allow exploring the cell-specific roles of P2X4 in various physiological and pathological contexts.

The kernel is the most safety- and security-critical component of many computer systems, as the most severe bugs lead to complete system crash or exploit. It is thus desirable to guarantee that a kernel is free from these bugs using formal methods, but the high cost and expertise required to do so are deterrent to wide applicability. We propose a method that can verify both absence of runtime errors (i.e. crashes) and absence of privilege escalation (i.e. exploits) in embedded kernels from their binary executables. The method can verify the kernel runtime independently from the application, at the expense of only a few lines of simple annotations. When given a specific application, the method can verify simple kernels without any human intervention. We demonstrate our method on two different use cases: we use our tool to help the development of a new embedded real-time kernel, and we verify an existing industrial real-time kernel executable with no modification. Results show that the method is fast, simple to use, and can prevent real errors and security vulnerabilities., Comment: Published in IEEE Real-Time and Embedded Technology and Applications Symposium (RTAS'21)

Neurocognitive impairment (NCI) is one of the most relevant clinical manifestations of multiple sclerosis (MS). The profile of NCI and the structural and functional changes in the brain structures relevant for cognition in MS share some similarities to those in Alzheimer's disease (AD), the most common cause of neurocognitive disorders. Additionally, despite clear etiopathological differences between MS and AD, an accumulation of effector/memory CD8+ T cells and CD8+ tissue-resident memory T (Trm) cells in cognitively relevant brain structures of MS/AD patients, and higher frequency of effector/memory CD8+ T cells re-expressing CD45RA (TEMRA) with high capacity to secrete cytotoxic molecules and proinflammatory cytokines in their blood, were found. Thus, an active pathogenetic role of CD8+ T cells in the progression of MS and AD may be assumed. In this mini-review, findings supporting the putative role of CD8+ T cells in the pathogenesis of MS and AD are displayed, and putative mechanisms underlying their pathogenetic action are discussed. A special effort was made to identify the gaps in the current knowledge about the role of CD8+ T cells in the development of NCI to “catalyze” translational research leading to new feasible therapeutic interventions.

Perturbation of CaMKIIβ expression has been associated with multiple neuropsychiatric diseases, highlighting CaMKIIβ as a gene of interest. Yet, in contrast to CaMKIIα, the specific functions of CaMKIIβ in the brain remain poorly explored. Here, we reveal a novel function for this CaMKII isoform in vivo during neuronal development. By using in utero electroporation, we show that CaMKIIβ is an important regulator of radial migration of projection neurons during cerebral cortex development. Knockdown of CaMKIIβ causes accelerated migration of nascent pyramidal neurons, whereas overexpression of CaMKIIβ inhibits migration, demonstrating that precise regulation of CaMKIIβ expression is required for correct neuronal migration. More precisely, CaMKIIβ controls the multipolar-bipolar transition in the intermediate zone and locomotion in the cortical plate through its actin-binding and -bundling activities. In addition, our data indicate that a fine-tuned balance between CaMKIIβ and cofilin activities is necessary to ensure proper migration of cortical neurons. Thus, our findings define a novel isoform-specific function for CaMKIIβ, demonstrating that CaMKIIβ has a major biological function in the developing brain.

ATP signaling and surface P2X4 receptors are upregulated selectively in neurons and/or glia in various CNS disorders including anxiety, chronic pain, epilepsy, ischemia, and neurodegenerative diseases. However, the cell-specific functions of P2X4 in pathological contexts remain elusive. To elucidate P2X4 functions, we created a conditional transgenic knock-in P2X4 mouse line (Floxed P2X4mCherryIN) allowing the Cre activity-dependent genetic swapping of the internalization motif of P2X4 by the fluorescent mCherry protein to prevent constitutive endocytosis of P2X4. By combining molecular, cellular, electrophysiological, and behavioral approaches, we characterized two distinct knock-in mouse lines expressing noninternalized P2X4mCherryIN either exclusively in excitatory forebrain neurons or in all cells natively expressing P2X4. The genetic substitution of wild-type P2X4 by noninternalized P2X4mCherryIN in both knock-in mouse models did not alter the sparse distribution and subcellular localization of P2X4 but increased the number of P2X4 receptors at the surface of the targeted cells mimicking the pathological increased surface P2X4 state. Increased surface P2X4 density in the hippocampus of knock-in mice altered LTP and LTD plasticity phenomena at CA1 synapses without affecting basal excitatory transmission. Moreover, these cellular events translated into anxiolytic effects and deficits in spatial memory. Our results show that increased surface density of neuronal P2X4 contributes to synaptic deficits and alterations in anxiety and memory functions consistent with the implication of P2X4 in neuropsychiatric and neurodegenerative disorders. Furthermore, these conditional P2X4mCherryIN knock-in mice will allow exploring the cell-specific roles of P2X4 in various physiological and pathological contexts.

Laser Scanning Confocal Microscopy (LSCM) imaging using an appropriate fluorescent probe enables the visualization of a molecular target with high resolution, and represents a method of choice for studying expression, subcellular location, and trafficking of receptors in living cells. The chemical, physical, and pharmacological properties of the probe remain essential. Here, we describe (1) the preparation of a specific probe for NMDAR GluN2B receptor by conjugation of fluorescein to an ifenprodil-based ligand, (2) an in vitro functional assay by calcium imaging for GluN2B binding and inhibition evaluation of the probe, and (3) the labeling and confocal imaging of GluN2B in DS-red labeled living cortical neurons.

International audience; Post-learning hippocampal sharp wave-ripples (SWRs) generated during slow wave sleep are thought to play a crucial role in memory formation. While in Alzheimer's disease, abnormal hippocampal oscillations have been reported, the functional contribution of SWRs to the typically observed spatial memory impairments remains unclear. These impairments have been related to degenerative synaptic changes produced by soluble amyloid beta oligomers (Aβos) which, surprisingly, seem to spare the SWR dynamics during routine behavior. To unravel a potential effect of Aβos on SWRs in cognitively-challenged animals, we submitted vehicle-and Aβo-injected mice to spatial recognition memory testing. While capable of forming short-term recognition memory, Aβ mice exhibited faster forgetting, suggesting successful encoding but an inability to adequately stabilize and/or retrieve previously acquired information. Without prior cognitive requirements, similar properties of SWRs were observed in both groups. In contrast, when cognitively challenged, the post-encoding and-recognition peaks in SWR occurrence observed in controls were abolished in Aβ mice, indicating impaired hippocampal processing of spatial information. These results point to a crucial involvement of SWRs in spatial memory formation and identify the Aβ-induced impairment in SWRs dynamics as a disruptive mechanism responsible for the spatial memory deficits associated with Alzheimer's disease. Information processing and memory formation in rodents have been reported to be accompanied by an array of hippocampal field potential oscillations that are important functionally. For instance, theta oscillations occur during active behavior and rapid eye movement (REM) sleep and have been suggested to provide the temporal frame for the encoding of information 1. Gamma oscillations triggered during exploratory behavior are thought to be involved in memory acquisition 2 and their synchronization contributes to successful execution of working memory 3. During slow wave sleep (SWS) that follows learning, hippocampal circuits consistently increase the occurrence rates of sharp wave-ripples (SWRs) which typically recur at 0.4 to 1 Hz 4,5. Importantly, upon occurrence of SWRs, ensembles of hippocampal place cells can replay in faster timescales their sequential activity triggered during a previous learning episode, suggesting an essential role for SWRs in driving memory consolidation processes and subsequent long-term stabilization of newly acquired spatial memory traces 6. When such SWRs are experimentally disrupted, it causes memory deficits in hippocampus-dependent memory tasks 7 , further suggesting that abnormal hippocampal rhythmic activity can interfere with hippocampal information processing, a dysfunctional pattern also observed in pathological conditions such as Alzheimer's disease 8 (AD). The cognitive impairments associated with AD are related to degenerative synaptic changes produced by the presence of soluble amyloid beta proteins (Aβ s) in vulnerable brain regions such as the hippocampus considered

INRange CONSORT flow diagram. (DOCX 191 kb)

International audience; Understanding how brief synaptic events can lead to sustained changes in synaptic structure and strength is a necessary step in solving the rules governing learning and memory. Activation of ERK1/2 (extracellular signal regulated protein kinase 1/2) plays a key role in the control of functional and structural synaptic plasticity. One of the triggering events that activates ERK1/2 cascade is an NMDA receptor (NMDAR)-dependent rise in free intracellular Ca(2+) concentration. However the mechanism by which a short-lasting rise in Ca(2+) concentration is transduced into long-lasting ERK1/2-dependent plasticity remains unknown. Here we demonstrate that although synaptic activation in mouse cultured cortical neurons induces intracellular Ca(2+) elevation via both GluN2A and GluN2B-containing NMDARs, only GluN2B-containing NMDAR activation leads to a long-lasting ERK1/2 phosphorylation. We show that αCaMKII, but not βCaMKII, is critically involved in this GluN2B-dependent activation of ERK1/2 signaling, through a direct interaction between GluN2B and αCaMKII. We then show that interfering with GluN2B/αCaMKII interaction prevents synaptic activity from inducing ERK-dependent increases in synaptic AMPA receptors and spine volume. Thus, in a developing circuit model, the brief activity of synaptic GluN2B-containing receptors and the interaction between GluN2B and αCaMKII have a role in long-term plasticity via the control of ERK1/2 signaling. Our findings suggest that the roles that these major molecular elements have in learning and memory may operate through a common pathway.

RASSF1A gene, found at the 3p21.3 locus, is a tumor suppressor gene frequently hypermethylated in human cancers. In this study, we report that compared with melanocytes in normal choroid, RASSF1A is downregulated in uveal melanoma samples and in uveal melanoma cell lines. LOH at 3p21.3 was detected in 50% of uveal melanoma. Moreover, methylation of the RASSF1A promoter was detected in 35 of 42 tumors (83%) and RASSF1A was also weakly expressed at the mRNA level. These data indicate that LOH at the RASSF1A locus or RASSF1A promoter methylation may partly account for the suppression of RASSF1A expression observed in uveal melanoma. Furthermore, following ectopic expression in three RASSF1A-deficient melanoma cell lines (OCM-1, Mel270, and 92.1), RASSF1A weakly reduces cell proliferation and anchorage-independent growth of uveal melanoma cells without effect on ERK1/2 activation, cyclin D1 and p27Kip1 expression. This study explored biological functions and underlying mechanisms of RASSF1A in the ERK1/2 pathway in normal uveal melanocytes. We showed that siRNA-mediated depletion of RASSF1A increased ERK1/2 activation, cyclin D1 expression, and also decreased p27Kip1 expression in normal uveal melanocytes. Moreover, that the depletion of RASSF1A induced senescence-associated β-galactosidase activity and increased p21Cip1 expression suggests that RASSF1A plays a role in the escape of cellular senescence in normal uveal melanocytes. Interestingly, we found that RASSF1A was epigenetically inactivated in long-term culture of uveal melanocytes. Taken together, these data show that depletion of RASSF1A could be an early event observed during senescence of normal uveal melanocytes and that additional alterations are acquired during malignant transformation to uveal melanoma. Mol Cancer Res; 9(9); 1187–98. ©2011 AACR.

Although tissue type plasminogen activator (tPA) and brain derived neurotrophic factor (BDNF) have been extensively described to influence brain outcomes in a number of disorders, their roles during physiological aging are poorly investigated. In the present study, we investigated whether maintenance of mice in different environmental conditions could influence age-associated changes in hippocampal tPA expression and BDNF maturation in relation with modifications of their cognitive performances. Our data indicate that maintenance in enriched housing led to a reversal of age-associated decrease in expression of hippocampal tPA. A subsequent increase in the level of mature BDNF and an improvement in emotional and spatial memories were observed. Taken together, these data suggest that the tPA-BDNF axis could play a critical role in the control of cognitive functions influenced both by the age and housing conditions.

ObjectiveAprotinin is used in cardiac surgery for its anti-inflammatory and hemostatic benefits. Recent reports describe the neuroprotective effects of other serine protease inhibitors via reduced excitotoxic cell death, a common pathway causing cytotoxic edema induced in various neuropathologic conditions. The purpose of this study was to investigate whether aprotinin directly protects against glutamatergic excitotoxicity in cell cultures.MethodsMixed cortical cultures containing neuronal and glial cells were prepared from fetal mice at 13 to 15 days' gestation and plated on a layer of confluent astrocytes from 1- to 3-day-old postnatal pups. Near-pure neuronal culture containing less than 5% astrocytes was obtained from the same gestational stage and plated in multiwell vessels previously coated with poly-D-lysine and laminin. Both cultures were used at 12 to 14 days in vitro. Slowly triggered excitotoxicity was induced at 37°C by 24-hour exposure to 12.5 μM N-methyl-D-aspartate or 50 μM kainate. Neuronal death was quantified by measuring the release of lactate dehydrogenase from damaged cells into the bathing medium. Data were analyzed by analysis of variance with post hoc Bonferroni comparisons.ResultsAprotinin at a clinically relevant concentration of 100 KIU/mL significantly reduced N-methyl-D-aspartate–induced neuronal death in both pure and mixed cultures (P < .001). Aprotinin also reduced neuronal death induced by kainate from 36% to 23% in mixed cortical culture (P = .008) and from 40% to 27% in near-pure culture (P = .015), indicating that the neuroprotective effects of aprotinin are mediated directly through neurons.ConclusionAprotinin provides direct neuroprotection against glutamatergic excitotoxicity as demonstrated by reduced neuronal death in near-pure neuronal cell culture. Additional studies are needed to evaluate the potential of aprotinin to reduce neurologic injury in patients at high risk of cerebral injury, including those undergoing circulatory arrest.

Background and Purpose— Whereas the effects of chronic arterial hypertension on the cerebral vasculature have been widely studied, its effects on brain tissue have been studied less so. Here we examined if spontaneously hypertensive rats (SHRs) or the normotensive control Wistar Kyoto rats (WKYs) made hypertensive by renal artery stenosis (R-WKYs) are vulnerable to an excitotoxic brain lesion provoked by an overactivation of glutamate receptors. Methods— Lesion volumes were quantified by histology in WKYs and SHRs subjected to striatal administration of N -methyl- d -aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA). The expression of AMPA receptors subunits and calcium/calmodulin kinase-IIα was analyzed by real-time polymerase chain reaction and Western blot. Results— NMDA (50 and 75 nmol) induced similar lesions in both SHRs (10±2 mm 3 and 16±4 mm 3 , respectively) and WKYs (11±2 mm 3 and 19±7 mm 3 , respectively). However, AMPA-induced (2.5 and 5 nmol) lesions were significantly greater in 14-week-old SHRs (14±3 mm 3 and 20±5 mm 3 , respectively) than WKYs (4±2 mm 3 , P 3 , P 3 vs 6±3 mm 3 ; P 3 and 5±4 mm 3 , respectively) compared with WKYs. Striatal levels of AMPA receptors subunits, GluR1 and GluR2, were not different between SHRs and WKYs. However, SHRs displayed an increase in phosphorylated form of GluR1 at Ser-831 ( P P P Conclusions— These findings show that an increase in phosphorylated GluR1, which increases AMPA receptor conductance, may be involved in the vulnerability of SHRs to AMPA.

La necrose et l’apoptose sont les deux processus majeurs de la neurodegenerescence post-ischemique au sein du systeme nerveux central. Neanmoins, la thrombolyse, grâce a l’utilisation de la serine protease, t-PA, reste aujourd’hui le seul traitement des accidents vasculaires cerebraux admis chez l’Homme. Ces dernieres annees, une cytokine appelee Trans- forming growth factor-β1 (TGF-β1) a ete montree comme etant fortement sur-exprimee au sein du systeme nerveux central en reponse a un stress ischemique. Des etudes recentes ont montre un net effet neuroprotecteur de cette cytokine dans un modele de mort neuronale ischemique in vivo. In vitro, nous avons pu montrer que cet effet neuroprotecteur du TGF-P contre la mort neuronale excitotoxique etait lie a une augmentation d’expression de l'inhibiteur de l’activateur du plasminogene (PAI-1) dans les astrocytes en reponse au TGF-P, en inhibant l’effet poten- tialisateur de l’activateur tissulaire du plasminogene (t-PA) sur la mort neuronale excitotoxique. Ainsi, l’ensemble de ces observation suggere fortement que la voie de signalisation du TGF-β1 ou les genes modules par cette cytokine au sein du systeme nerveux central puissent constituer des cibles moleculaires interessantes pour le developpement de nouvelles strategies therapeutiques des accidents vasculaires cerebraux chez l’Homme.

1. 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.

The intravenous injection of the serine protease, tissue-type plasminogen activator (t-PA), has shownto 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-β1 and focused on the transduction pathway involved in this effect. We demonstrated that PAI-1, produced by astrocytes, mediates the neuroprotective activity of TGF-β1 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-β-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.

The glial cell line-derived neurotrophic factor (GDNF) is first characterized for its trophic activity on dopaminergic neurons. Recent data suggested that GDNF could modulate the neuronal death induced by ischemia. The purpose of this study was to characterize the influence of GDNF on cultured cortical neurons subjected to two paradigms of injury (necrosis and apoptosis) that have been identified during cerebral ischemia and to determine the molecular mechanisms involved. First, we demonstrated that both neurons and astrocytes express the mRNA and the protein for GDNF and its receptor complex (GFRα-1 and c-Ret). Next, we showed that the application of recombinant human GDNF to cortical neurons and astrocytes induces the activation of the MAP kinase (MAPK) pathway, as visualized by an increase in the phosphorylated forms of extracellular signal-regulated kinases (ERKs). Thereafter, we demonstrated that GDNF fails to prevent apoptotic neuronal death but selectively attenuates slowly triggered NMDA-induced excitotoxic neuronal death via a direct effect on cortical neurons. To further characterize the neuroprotective mechanisms of GDNF against NMDA-mediated neuronal death, we showed that a pretreatment with GDNF reduces NMDA-induced calcium influx. This effect likely results from a reduction of NMDA receptor activity rather than an enhanced buffering or extrusion capacity for calcium. Finally, we also demonstrated that an ERKs activation pathway is necessary for GDNF-mediated reduction of the NMDA-induced calcium response. Together, these results describe a novel mechanism by which the activation of MAPK induced by GDNF modulates NMDA receptor activity, a mechanism that could be responsible for the neuroprotective effect of GDNF in acute brain injury.

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.

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 astrocyt...

International audience; GluN2B-containing NMDA receptors are involved in many important physiological functions and play a pivotal role in mediating pain as well as in several neurodegenerative disorders. We aimed to develop fluorescent probes to target the GluN2B subunit selectively in order to allow better understanding of the relationships between receptor localisation and physiological importance. Ifenprodil, known as the GluNR2B antagonist of reference, was chosen as the template for the elaboration of probes. We had previously reported a fluorescein conjugate that was shown (by confocal microscopy imaging of DS-red-labelled cortical neurons) to bind specifically to GluN2B. To elaborate this probe, we explored the influence of both the nature and the attachment point of the spacer between the fluorophore and the parent compound, ifenprodil. We performed chemical modifications of ifenprodil at the benzylic position and on the phenol ring by introducing secondary amine or amide functions and evaluated alkyl chains from two to 20 bonds either including or not including secondary amide functions as spacers. The previously developed probe was found to display the greatest activity in the inhibition of NMDA-induced Ca(2+) influx by calcium imaging experiments on HEK293 cells transfected with the cDNA encoding for GluN1-1A and GluN2B. Further investigations revealed that this probe had a neuroprotective effect equivalent to that of ifenprodil in a standard test for neurotoxicity. Despite effects of lesser amplitude with these probes relative to ifenprodil, we demonstrated that they displaced [(3) H]ifenprodil in mouse brain slices in a similar manner.

Alzheimer's disease is characterized by the loss of memory and synaptic damage. Evidence is accumulating for a causal role of soluble oligomeric species of amyloid-β peptide (Aβo) in the impairment of synaptic plasticity and cognition but the precise mechanisms underlying these effects are still not clear. Synaptic plasticity such as long-term potentiation is thought to underlie learning and memory. While the effect of Aβ on long-term potentiation is well documented, a more general understanding of Aβ action on various aspects of plasticity involving synaptic and extrasynaptic receptors and the nature of the mechanisms involved in its effects are lacking. Using a combination of electrophysiological and biochemical techniques in mouse hippocampal slices, we show here that Aβo drastically affects synaptic plasticities induced by high stimulation frequencies through the involvement of extrasynaptic glutamate receptors. Experiments on hippocampal slices as well as on cultured cortical neurons show that Aβo potentiates extrasynaptic NMDA receptors-mediated responses. Pharmacological characterization indicates that GluN2B-containing NMDARs are involved in these responses. When synaptic and extrasynaptic glutamate receptor-mediated effects are dissociated using cortical neurons in culture, it appears that Aβo has differential effects on these two receptors types. We conclude that the pool of extrasynaptic GluN2B-containing NMDARs is a major target of Aβo in the hippocampus. During high frequency stimulation, Aβo dramatically impairs long-term neuronal responses.

We describe the synthesis and pharmacological characterization of a first generation of ifenprodil conjugates 4–7 as fluorescent probes for the confocal microscopy imaging of the NR2B-containing NMDA receptor. The fluorescein conjugate 6 displayed a moderate affinity for NMDAR but a high selectivity for the NR2B subunit and its NTD. Fluorescence imaging of DS-red labeled cortical neurons showed an exact colocalization of the probe 6 with small protrusions along the dendrites related to a specific binding on NR2B-containing NMDARs.

In this study, novel specific PET radioligands containing the 4-(4-fluorobenzyl)piperidine moiety and selectively antagonistic for the NR2B subunit containing NMDA receptors were developed. Two antagonists, RGH-896 (1a) and 4-(4-fluorobenzyl)piperidinyl-1-methyl-2-benzimidazol-5-ol (2a), belonging to two different structural families, were radiolabeled by an aromatic nucleophilic radiofluorination followed by a reduction of the para-position carbonyl function. Radiotracers [18F]1a, [18F]2a or the pattern 4-(4-[18F]-fluorobenzyl)piperidine ([18F]6) demonstrated an identical in vivo behavior with high accumulation of radioactivity in bone and cartilage which would suggest a radiodefluorination of the radiotracers. The identification of metabolites from 6 by LC-MS-MS confirmed the significant degree of defluorination as a result of the in vivo hydroxylation in the benzyl ring. In conclusion, [18F]1a or [18F]2a are not suitable for imaging the NR2B NMDA receptors due to their poor brain penetration. We also argue for a cautious use of the radiolabeled pattern, 4-(4-[18F]-fluorobenzyl)piperidine, to develop PET radiotracers.

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 Alzheimer's 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.

Aprotinin has been used in pediatric cardiac surgery for its antiinflammatory and hemostatic benefits. We have reported that aprotinin has a direct cellular neuroprotective effect through reduction of excitotoxicity. The purpose of this study was to investigate whether aprotinin is neuroprotective against apoptotic cell death. Near-pure neuronal cultures containing −1 significantly reduced apoptotic neuronal cell death from 84.4% to 51.8%. This result suggests that aprotinin has the potential to reduce brain injury resulting from apoptotic cell death induced by an ischemic insult. Additional studies are needed to evaluate the potential of aprotinin to reduce neurological injury in patients at high risk of cerebral injury, including those undergoing circulatory arrest.

An operationally simple and concise synthesis of anilinoethanolamines, as NMDA NR2B receptor antagonist ifenprodil analogues, was developed via a copper-catalyzed amination of the corresponding bromoarene. Coupling was achieved with linear primary alkylamines, alpha,omega-diamines, hexanolamine and benzophenone imine, as well as with aqueous ammonia, in good yields using CuI and N,N-diethylsalicylamide, 2,4-pentadione or 2-acetylcyclohexanone as catalytic systems. Amination with ethylene diamine was efficient even in the absence of an additive ligand, whereas no reaction occurred with ethanolamine whatever the conditions used. The anilinoethanolamines were evaluated as NR2B receptor antagonists in a functional inhibition assay. Aminoethylanilines displayed inhibition effects close to that of ifenprodil.

Objective Ibuprofen has been shown to reduce cerebral ischemic injury, such as may occur after deep hypothermic circulatory arrest. We investigated whether ibuprofen has direct protective effects against excitotoxic neuronal injury, as may be seen after cerebral ischemia, by using a cell culture model. Methods Mixed cortical cultures containing neuronal and glial cells were prepared from fetal mice at 13 to 15 days gestation, plated on a layer of confluent astrocytes from 1- to 3-day-old postnatal pups. Near-pure neuronal cultures containing less than 5% astrocytes were obtained from mice of the same gestational stage. Slowly triggered excitotoxic injury was induced at 37°C by 24-hour exposure to 12.5 μmol/L N-methyl-D-aspartate or 50 μmol/L kainate. Neuronal death was quantified by release of lactate dehydrogenase from damaged cells. Data were analyzed using 1-way analysis of variance with Tukey post hoc multiple comparisons. Results In mixed cultures, ibuprofen concentrations of 25 μg/mL, 50 μg/mL, and 100 μg/mL all significantly reduced N-methyl-D-aspartate–induced neuronal cell death from 74.5% to 56.1%, 38.7%, and 12.3%, respectively, revealing a strong dose response ( P near-pure cultures, ibuprofen at a concentration of 25 μg/mL failed to protect neurons, indicating that the neuroprotective effects of ibuprofen require interaction with glial cells. Furthermore, ibuprofen at 100 μg/mL was not protective against neuronal cell death induced by kainate exitotoxicity in near-pure culture but was effective in mixed cultures. Conclusion Ibuprofen provides neuroprotection through glial cells against excitotoxic neuronal injury caused by glutamatergic excitotoxicity after cerebral ischemia as demonstrated by reduced neuronal cell death in mixed cell cultures. Further studies are needed to evaluate the potential of ibuprofen to reduce neurologic injury in patients experiencing an hypoxic/ischemic insult.

Evidence have accumulated that reverse glutamate uptake plays a key role in the pathophysiology of cerebral ischemia. Here, we investigated the effects of glial glutamate transporter dysfunction on neuronal survival using the substrate inhibitor of glutamate transporters, L-trans-pyrrolidine,2-4,dicarboxylate (PDC), that partly mimics reverse glutamate uptake. On mice primary cortical co-cultures of neurons and astrocytes, PDC treatment triggered an elevation of extracellular glutamate concentration, induced neuronal calcium influx and a massive NMDA receptor (NMDAR) mediated-neuronal death without having any direct agonist activity on NMDARs. We investigated the NMDAR subpopulation activated by PDC-induced glutamate release. PDC application led to the activation of both subtypes of NMDARs but the presence of astrocytes was required to activate NMDARs located extra-synaptically. Extrasynaptic NMDAR activation was also confirmed by the loss of neuronal mitochondrial membrane potential and the inhibition of pro-survival p-ERK signalling pathway. These data suggest that reverse glial glutamate uptake may trigger neuronal death through preferential activation of extrasynaptic NMDAR-related pathways.

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.

Bone-marrow-derived mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after neurodegenerative diseases. Recently, several studies suggested that oxygen-dependent gene expression is of crucial importance in governing the essential steps of neurogenesis such as cell proliferation, survival and differentiation. In this context, we analysed the effect of the HIF-1 (hypoxia inducible factor-1) activation-mimicking agent CoCl2 on MSCs. CoCl2 treatment increased the expression of the anti-proliferative gene BTG2/PC3 and decreased cyclin D1 expression. Expression of HIF-1α and its target genes EPO, VEGF and p21 was also upregulated. These changes were followed by inhibition of cell proliferation and morphological changes resulting in neuron-like cells, which had increased neuronal marker expression and responded to neurotransmitters. Echinomycin, a molecule inhibiting HIF-1 DNA-binding activity, blocked the CoCl2 effect on MSCs. Additionally, by using Y-27632, we demonstrated that Rho kinase (ROCK) inhibition potentiated CoCl2-induced MSC differentiation in particular into dopaminergic neuron-like cells as attested by its effect on tyrosine hydroxylase expression. Altogether, these results support the ability of MSCs to differentiate into neuron-like cells in response to CoCl2, an effect that might act, in part, through HIF-1 activation and cell-cycle arrest, and which is potentiated by inhibition of ROCK.

Receptors for the serine protease thrombin and for lysophospholipids are coupled to G proteins and control a wide range of cellular functions, including mitogenesis. Activators of these receptors are present in blood, and can enter the brain during central nervous system (CNS) injury. Reactive astrogliosis, a prominent component of CNS injury with potentially harmful consequences, may involve proliferation of astrocytes. In this study, we have examined the expression and activation of protease activated receptors (PARs), lysophosphatidic acid (LPA) receptors, and sphingosine-1-phosphate (S1P) receptors on murine astrocytes. We show that activation of these three receptor classes can lead to astrogliosis in vivo and proliferation of astrocytes in vitro. Cultured murine cortical astrocytes express mRNA for multiple receptor subtypes of PAR (PAR-1-4), LPA (LPA-1-3) and S1P (S1P-1, -3, -4, and -5) receptors. Comparison of the intracellular signaling pathways of glial PAR-1, LPA, and S1P receptors indicates that each receptor class activates multiple downstream signaling pathways, including Gq/11-directed inositol lipid/Ca2+ signaling, Gi/o activation of mitogen-activated protein kinases (MAPK) (extracellular signal-regulated kinase 1/2 and stress activated protein kinase/c-jun N-terminal kinase, but not p38), and activation of Rho pathways. Furthermore, activation of these different receptor classes can differentially regulate two transcription factor pathways, serum response element and nuclear factor of activated T cells. Blockade of Gi/o signaling with pertussis toxin, MAPK activation with 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene (U0126), or Rho kinase signaling with R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide (Y27632) can markedly reduce the proliferative response of glial cells to PAR-1, LPA, or S1P receptor activation, suggesting that each of these pathways is important in coupling of receptor activation to glial proliferation.

1. 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-beta1 (TGF-beta1) 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-beta1 against ischemia-induced neuronal death. In vitro, TGF-beta1 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-beta1 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-beta signaling or TGF-beta1-modulated genes could be good targets for the development of new therapeutic strategies for stroke in man.

Necrosis and apoptosis are the two fundamental hallmarks of neuronal death in stroke. Nevertheless, thrombolysis, by means of the recombinant serine protease t-PA, remains until now the only approved treatment of stroke in man. Over the last years, the cytokine termed Transforming Growth Factor-beta 1 (TGF-beta 1) has been found to be strongly up regulated in the central nervous system following ischemia-induced brain damage. Recent studies have shown a neuroprotective activity of TGF-beta 1 against ischemia-induced neuronal death. In vitro, TGF-beta 1 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. Altogether, these observations suggest that either TGF-beta signaling or TGF-beta 1-modulated genes could be good targets for the development of new therapeutic strategies for stroke in man.

There has been an increasing interest in recent years in the evaluation of the neuronal and glial responses to ischemic insult. Some cytokines, including transforming growth factor-β (TGF-β), that are overexpressed after experimental stroke in rodents are thought to be implicated in the neuronal processes that lead to necrosis. Thus, such cytokines could predict tissue fate after stroke in humans, although data are currently sparse for gyrencephalic species. The current study addressed the expression pattern of TGF-β1 in a nonhuman primate model of middle cerebral artery occlusion. Focal permanent ischemia was induced for 1 or 7 days in 6 baboons and the following investigations were undertaken: cerebral oxygen metabolism (CMRO2) positron emission tomography studies, magnetic resonance imaging, postmortem histology, and reverse transcription-polymerase chain reaction. The aim of the current study was to correlate the expression of TGF-β1 to the underlying metabolic and histologic state of the threatened cerebral parenchyma. The authors evidenced increased TGF-β1 mRNA levels (up to 25-fold) in those regions displaying a moderate (20% to 49%) reduction in CMRO2. The current findings suggest that the greatly enhanced expression of TGF-β1 in the penumbral zones that surround tissue destined to infarction may represent a robust index of potentially salvageable brain. The current investigation, in the nonhuman primate, strengthens the authors' hypothesis, derived from rodent models, that TGF-β1 may be involved in the physiopathology of human stroke.

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.