NMDA receptor functions in health and disease: Old actor, new dimensions.

N-Methyl-D-aspartate ionotropic glutamate receptors (NMDARs) play key roles in synaptogenesis, synaptic maturation, long-term plasticity, neuronal network activity, and cognition. Mirroring this wide range of instrumental functions, abnormalities in NMDAR-mediated signaling have been associated with numerous neurological and psychiatric disorders. Thus, identifying the molecular mechanisms underpinning the physiological and pathological contributions of NMDAR has been a major area of investigation. Over the past decades, a large body of literature has flourished, revealing that the physiology of ionotropic glutamate receptors cannot be restricted to fluxing ions, and involves additional facets controlling synaptic transmissions in health and disease. Here, we review newly discovered dimensions of postsynaptic NMDAR signaling supporting neural plasticity and cognition, such as the nanoscale organization of NMDAR complexes, their activity-dependent redistributions, and non-ionotropic signaling capacities. We also discuss how dysregulations of these processes may directly contribute to NMDAR-dysfunction-related brain diseases.

Increased surface P2X4 receptor regulates anxiety and memory in P2X4 internalization-defective knock-in mice.

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.

CD8+ T Cell-Mediated Mechanisms Contribute to the Progression of Neurocognitive Impairment in Both Multiple Sclerosis and Alzheimer's Disease?

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.

CaMKIIß in Neuronal Development and Plasticity: An Emerging Candidate in Brain Diseases.

The calcium/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous and central player in Ca2+ signaling that is best known for its functions in the brain. In particular, the α isoform of CaMKII has been the subject of intense research and it has been established as a central regulator of neuronal plasticity. In contrast, little attention has been paid to CaMKIIß, the other predominant brain isoform that interacts directly with the actin cytoskeleton, and the functions of CaMKIIß in this organ remain largely unexplored. However, recently, the perturbation of CaMKIIß expression has been associated with multiple neuropsychiatric and neurodevelopmental diseases, highlighting CAMK2B as a gene of interest. Herein, after highlighting the main structural and expression differences between the α and ß isoforms, we will review the specific functions of CaMKIIß, as described so far, in neuronal development and plasticity, as well as its potential implication in brain diseases.

A novel role for CAMKIIß in the regulation of cortical neuron migration: implications for neurodevelopmental disorders.

Perturbation of CaMKIIß expression has been associated with multiple neuropsychiatric diseases, highlighting CaMKIIß as a gene of interest. Yet, in contrast to CaMKIIα, the specific functions of CaMKIIß 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 CaMKIIß is an important regulator of radial migration of projection neurons during cerebral cortex development. Knockdown of CaMKIIß causes accelerated migration of nascent pyramidal neurons, whereas overexpression of CaMKIIß inhibits migration, demonstrating that precise regulation of CaMKIIß expression is required for correct neuronal migration. More precisely, CaMKIIß 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 CaMKIIß and cofilin activities is necessary to ensure proper migration of cortical neurons. Thus, our findings define a novel isoform-specific function for CaMKIIß, demonstrating that CaMKIIß has a major biological function in the developing brain.

GluN2B Subunit Labeling with Fluorescent Probes and High-Resolution Live Imaging.

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.

Assessing recent and remote associative olfactory memory in rats using the social transmission of food preference paradigm.

Rats have the ability to learn about potential food sources by sampling their odors on the breath of conspecifics. Although this ethologically based social behavior has been transposed to the laboratory to probe nonspatial associative olfactory memory, only a few studies have taken full advantage of its unique features to examine the organization of recently and remotely acquired information. We provide a set of standardized procedures and technical refinements that are particularly useful in achieving this goal while minimizing confounding factors. These procedures, built upon a three-stage protocol (odor exposure, social interaction and preference test), are designed to optimize performance across variable retention delays, thus enabling the reliable assessment of recent and remote memory, and underlying processes, including encoding, consolidation, retrieval and forgetting. The different variants of the social transmission of food preference paradigm, which take a few days to several weeks to perform, make it an attractive and versatile tool that can be coupled to many applications in CNS research. The paradigm can be easily implemented in a typical rodent facility by personnel with standard animal behavioral expertise.

Soluble amyloid beta oligomers block the learning-induced increase in hippocampal sharp wave-ripple rate and impair spatial memory formation.

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 (Aßos) which, surprisingly, seem to spare the SWR dynamics during routine behavior. To unravel a potential effect of Aßos on SWRs in cognitively-challenged animals, we submitted vehicle- and Aßo-injected mice to spatial recognition memory testing. While capable of forming short-term recognition memory, Aß 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 Aß mice, indicating impaired hippocampal processing of spatial information. These results point to a crucial involvement of SWRs in spatial memory formation and identify the Aß-induced impairment in SWRs dynamics as a disruptive mechanism responsible for the spatial memory deficits associated with Alzheimer's disease.

Synthesis and in vitro characterisation of ifenprodil-based fluorescein conjugates as GluN1/GluN2B N-Methyl-D-aspartate receptor antagonists.

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.

Interaction between αCaMKII and GluN2B controls ERK-dependent plasticity.

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 ßCaMKII, 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.

Selective impairment of some forms of synaptic plasticity by oligomeric amyloid-ß peptide in the mouse hippocampus: implication of extrasynaptic NMDA receptors.

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-ß peptide (Aßo) 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 Aß on long-term potentiation is well documented, a more general understanding of Aß 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 Aßo 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 Aßo 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 Aßo has differential effects on these two receptors types. We conclude that the pool of extrasynaptic GluN2B-containing NMDARs is a major target of Aßo in the hippocampus. During high frequency stimulation, Aßo dramatically impairs long-term neuronal responses.

Confocal microscopy imaging of NR2B-containing NMDA receptors based on fluorescent ifenprodil-like conjugates.

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.

Moderately delayed post-insult treatment with normobaric hyperoxia reduces excitotoxin-induced neuronal degeneration but increases ischemia-induced brain damage.

BACKGROUND: The use and benefits of normobaric oxygen (NBO) in patients suffering acute ischemic stroke is still controversial. RESULTS: Here we show for the first time to the best of our knowledge that NBO reduces both NMDA-induced calcium influxes in vitro and NMDA-induced neuronal degeneration in vivo, but increases oxygen and glucose deprivation-induced cell injury in vitro and ischemia-induced brain damage produced by middle cerebral artery occlusion in vivo. CONCLUSIONS: Taken together, these results indicate that NBO reduces excitotoxin-induced calcium influx and subsequent neuronal degeneration but favors ischemia-induced brain damage and neuronal death. These findings highlight the complexity of the mechanisms involved by the use of NBO in patients suffering acute ischemic stroke.

Status of RASSF1A in uveal melanocytes and melanoma cells.

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 p27(Kip1) 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 p27(Kip1) expression in normal uveal melanocytes. Moreover, that the depletion of RASSF1A induced senescence-associated ß-galactosidase activity and increased p21(Cip1) 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.

Synthesis, evaluation and metabolic studies of radiotracers containing a 4-(4-[18F]-fluorobenzyl)piperidin-1-yl moiety for the PET imaging of NR2B NMDA receptors.

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.

Enriched housing reverses age-associated impairment of cognitive functions and tPA-dependent maturation of BDNF.

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.

Activation of extrasynaptic, but not synaptic, NMDA receptors modifies amyloid precursor protein expression pattern and increases amyloid-ß 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 ß-amyloid (Aß), and contributing to the initiation of Alzheimer's disease (AD). A linkage between excessive glutamate receptor activation and neuronal Aß 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 Aß. 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 Aß 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 Aß release. Altogether, these data suggest that a chronic activation of extrasynaptic NMDAR promotes amyloidogenic KPI-APP expression leading to neuronal Aß release, representing a causal risk factor for developing AD.

Aprotinin confers neuroprotection by reducing apoptotic cell death.

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 <5% astrocytes were obtained from fetal mice. Serum deprivation was initiated at 7 days by transferring the cultures, which are dependent on serum for survival, into growth medium lacking serum for 24 h. Neuronal cell death was assessed by phase-contrast cell counting after staining with 0.4% trypan blue dye. Aprotinin at a clinically relevant concentration of 100 KIU.mL(-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.

Copper-catalyzed amination of (bromophenyl)ethanolamine for a concise synthesis of aniline-containing analogues of NMDA NR2B antagonist ifenprodil.

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.