NMDA Receptor Signaling Research Articles

Temporal manipulation of Cdkl5 reveals essential postdevelopmental functions and reversible CDKL5 deficiency disorder–related deficits

CDKL5 deficiency disorder (CDD) is an early onset, neurodevelopmental syndrome associated with pathogenic variants in the X-linked gene encoding cyclin-dependent kinase-like 5 (CDKL5). CDKL5 has been implicated in neuronal synapse maturation, yet its postdevelopmental necessity and the reversibility of CDD-associated impairments remain unknown. We temporally manipulated endogenous Cdkl5 expression in male mice and found that postdevelopmental loss of CDKL5 disrupts numerous behavioral domains, hippocampal circuit communication, and dendritic spine morphology, demonstrating an indispensable role for CDKL5 in the adult brain. Accordingly, restoration of Cdkl5 after the early stages of brain development using a conditional rescue mouse model ameliorated CDD-related behavioral impairments and aberrant NMDA receptor signaling. These findings highlight the requirement of CDKL5 beyond early development, underscore the potential for disease reversal in CDD, and suggest that a broad therapeutic time window exists for potential treatment of CDD-related deficits.

Regulation of Phosphorylated State of NMDA Receptor by STEP61 Phosphatase after Mild-Traumatic Brain Injury: Role of Oxidative Stress.

Traumatic Brain Injury (TBI) mediates neuronal death through several events involving many molecular pathways, including the glutamate-mediated excitotoxicity for excessive stimulation of N-methyl-D-aspartate receptors (NMDARs), producing activation of death signaling pathways. However, the contribution of NMDARs (distribution and signaling-associated to the distribution) remains incompletely understood. We propose a critical role of STEP61 (Striatal-Enriched protein tyrosine phosphatase) in TBI; this phosphatase regulates the dephosphorylated state of the GluN2B subunit through two pathways: by direct dephosphorylation of tyrosine-1472 and indirectly via dephosphorylation and inactivation of Fyn kinase. We previously demonstrated oxidative stress’s contribution to NMDAR signaling and distribution using SOD2+/− mice such a model. We performed TBI protocol using a controlled frontal impact device using C57BL/6 mice and SOD2+/− animals. After TBI, we found alterations in cognitive performance, NMDAR-dependent synaptic function (decreased synaptic form of NMDARs and decreased synaptic current NMDAR-dependent), and increased STEP61 activity. These changes are reduced partially with the STEP61-inhibitor TC-2153 treatment in mice subjected to TBI protocol. This study contributes with evidence about the role of STEP61 in the neuropathological progression after TBI and also the alteration in their activity, such as an early biomarker of synaptic damage in traumatic lesions.

Saracatinib Fails to Reduce Alcohol-Seeking and Consumption in Mice and Human Participants.

More effective treatments to reduce pathological alcohol drinking are needed. The glutamatergic system and the NMDA receptor (NMDAR), in particular, are implicated in behavioral and molecular consequences of chronic alcohol use, making the NMDAR a promising target for novel pharmacotherapeutics. Ethanol exposure upregulates Fyn, a protein tyrosine kinase that indirectly modulates NMDAR signaling by phosphorylating the NR2B subunit. The Src/Fyn kinase inhibitor saracatinib (AZD0530) reduces ethanol self-administration and enhances extinction of goal-directed ethanol-seeking in mice. However, less is known regarding how saracatinib affects habitual ethanol-seeking. Moreover, no prior studies have assessed the effects of Src/Fyn kinase inhibitors on alcohol-seeking or consumption in human participants. Here, we tested the effects of saracatinib on alcohol consumption and craving/seeking in two species, including the first trial of an Src/Fyn kinase inhibitor to reduce drinking in humans. Eighteen male C57BL/6NCrl mice underwent operant conditioning on a variable interval schedule to induce habitual responding for 10% ethanol/0.1% saccharin. Next, mice received 5 mg/kg saracatinib or vehicle 2 h or 30 min prior to contingency degradation to measure habitual responding. In the human study, 50 non-treatment seeking human participants who drank heavily and met DSM-IV criteria for alcohol abuse or dependence were randomized to receive 125 mg/day saracatinib (n = 33) or placebo (n = 17). Alcohol Drinking Paradigms (ADP) were completed in a controlled research setting: before and after 7–8 days of treatment. Each ADP involved consumption of a priming drink of alcohol (0.03 mg%) followed by ad libitum access (3 h) to 12 additional drinks (0.015 g%); the number of drinks consumed and craving (Alcohol Urge Questionnaire) were recorded. In mice, saracatinib did not affect habitual ethanol seeking or consumption at either time point. In human participants, no significant effects of saracatinib on alcohol craving or consumption were identified. These results in mice and humans suggest that Fyn kinase inhibition using saracatinib, at the doses tested here, may not reduce alcohol consumption or craving/seeking among those habitually consuming alcohol, in contrast to reports of positive effects of saracatinib in individuals that seek ethanol in a goal-directed manner. Nevertheless, future studies should confirm these negative findings using additional doses and schedules of saracatinib administration.

Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

A dysfunction of the glutamatergic transmission, especially of the NMDA receptor (NMDAR), constitutes one of the main biological substrate of psychotic disorders, such as schizophrenia. The NMDAR signaling hypofunction, through genetic and/or environmental insults, would cause a neurodevelopmental myriad of molecular, cellular, and network alterations that persist throughout life. Yet, the mechanisms underpinning NMDAR dysfunctions remain elusive. Here, we compared the membrane trafficking of NMDAR in three gold-standard models of schizophrenia, i.e., patient’s cerebrospinal fluids, genetic manipulations of susceptibility genes, and prenatal developmental alterations. Using a combination of single nanoparticle tracking, electrophysiological, biochemical, and behavioral approaches in rodents, we identified that the NMDAR trafficking in hippocampal neurons was consistently altered in all these different models. Artificial manipulations of the NMDAR surface dynamics with competing ligands or antibody-induced receptor cross-link in the developing rat brain were sufficient to regulate the adult acoustic startle reflex and compensate for an early pathological challenge. Collectively, we show that the NMDAR trafficking is markedly altered in all clinically relevant models of psychosis, opening new avenues of therapeutical strategies.

The Effect of Monosodium Glutamate on Neuronal Signaling Molecules in the Hippocampus and the Neuroprotective Effects of Omega-3 Fatty Acids.

Monosodium glutamate (MSG) is a flavoring substance added to many ready-to-eat foods and has known neurotoxic effects. This study was performed in order to examine the potential toxic effect of MSG on neurons in various regions of the hippocampus in prepubertal rats. It also investigated the protective effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on brain-derived neurotropic factor (BDNF), n-methyl-d-aspartate receptor (NMDA-R), and neuropeptide-Y (NPY) expression in the brain, using immunohistochemical and biochemical methods. Six female prepubertal Wistar albino rats were used in each group. Group 1, the control group, received 0.9% saline solution subcutaneously (sc) on days 1, 3, 5, 7, and 9. Group 2 received 4 mg/g MSG sc on days 1, 3, 5, 7, and 9. Group 3 received MSG + EPA (4 mg/g sc on days 1, 3, 5, 7, and 9. Oral 300 mg/kg for 9 d), while Group 4 received MSG + DHA (4 mg/g sc on days 1, 3, 5, 7, and 9 and 300 mg/kg orally for 9 d, respectively). At the end of the ninth day the hippocampal regions of the brain were removed and either fixed for immunohistochemical staining or stored at -80 °C for biochemical parameter investigation. BDNF, NMDA-R, and NPY expression results were evaluated using immunohistochemistry and an enzyme-linked immunosorbent assay. According to our findings, neurons in the control group hippocampal CA1 and DG regions exhibited strong BDNF, NPY, and NMDA-R reactions, while an expression in both regions decreased in the MSG group (p < 0.00). However, in the MSG-EPA and MSG-DHA groups, BDNF, NPY, and NMDA-R immunoreactions in neurons in the same region were similar to those of the control group (p = 0.00). No significant difference was observed in terms of expression in hippocampal neurons between the MSG-EPA and MSG-DHA groups (p > 0.00). In conclusion, since MSG caused a decrease in BDNF, NMDA-R, and NPY neural signaling molecules in the CA1 and DG regions of the hippocampus of prepubertal rats compared to the control group, care is required over the consumption of MSG, since it may affect memory-related neurons in these age groups. In addition, we concluded that the use of omega-3 fatty acids such as EPA and DHA in addition to MSG may protect against the neurotoxic effects of MSG.

Dopamine System, NMDA Receptor and EGF Family Expressions in Brain Structures of Bl6 and 129Sv Strains Displaying Different Behavioral Adaptation.

C57BL/6NTac (Bl6) and 129S6/SvEvTac (129Sv) mice display different coping strategies in stressful conditions. Our aim was to evaluate biomarkers related to different adaptation strategies in the brain of male 129Sv and Bl6 mice. We focused on signaling pathways related to the dopamine (DA) system, N-methyl-D-aspartate (NMDA) receptor and epidermal growth factor (EGF) family, shown as the key players in behavioral adaptation. Mice from Bl6 and 129Sv lines were divided into either home cage controls (HCC group) or exposed to repeated motility testing and treated with saline for 11 days (RMT group). Distinct stress responses were reflected in severe body weight loss in 129Sv and the increased exploratory behavior in Bl6 mice. Besides that, amphetamine caused significantly stronger motor stimulation in Bl6. Together with the results from gene expression (particularly Maob), this study supports higher baseline activity of DA system in Bl6. Interestingly, the adaptation is reflected with opposite changes of DA markers in dorsal and ventral striatum. In forebrain, stress increased the gene expressions of Egf-Erbb1 and Nrg1/Nrg2-Erbb4 pathways more clearly in 129Sv, whereas the corresponding proteins were significantly elevated in Bl6. We suggest that not only inhibited activity of the DA system, but also reduced activity of EGF family and NMDA receptor signaling underlies higher susceptibility to stress in 129Sv. Altogether, this study underlines the better suitability of 129Sv for modelling neuropsychiatric disorders than Bl6.

Synaptic NMDA receptor signalling controls R-type calcium channel recruitment.

Regulation of extracellular Ca2+ influx by neuronal activity is a key mechanism underlying synaptic plasticity. At the neuronal synapse, activity-dependent Ca2+ entry involves N-methyl-D-aspartate receptors (NMDARs) and voltage-gated calcium channels (VGCCs); the relationship between NMDARs and VGCCs, however, is poorly understood. Here, we report that neuronal activity rapidly (1h) regulates recruitment of R-type VGCCs in hippocampal neurons through synaptic NMDAR signalling. This finding reveals a link between two key neuronal signalling pathways, suggesting a feedback mode for regulation of synaptic Ca2+ signalling.

CYFIP1 Dosages Exhibit Divergent Behavioral Impact via Diametric Regulation of NMDA Receptor Complex Translation in Mouse Models of Psychiatric Disorders

BackgroundGene dosage imbalance caused by copy number variations (CNVs) is a prominent contributor to brain disorders. In particular, 15q11.2 CNV duplications and deletions have been associated with autism spectrum disorder and schizophrenia, respectively. The mechanism underlying these diametric contributions remains unclear. MethodsWe established both loss-of-function and gain-of-function mouse models of Cyfip1, one of four genes within 15q11.2 CNVs. To assess the functional consequences of altered CYFIP1 levels, we performed systematic investigations on behavioral, electrophysiological, and biochemical phenotypes in both mouse models. In addition, we utilized RNA immunoprecipitation sequencing (RIP-seq) analysis to reveal molecular targets of CYFIP1 in vivo. ResultsCyfip1 loss-of-function and gain-of function mouse models exhibited distinct and shared behavioral abnormalities related to autism spectrum disorder and schizophrenia. RIP-seq analysis identified messenger RNA targets of CYFIP1 in vivo, including postsynaptic NMDA receptor (NMDAR) complex components. In addition, these mouse models showed diametric changes in levels of postsynaptic NMDAR complex components at synapses because of dysregulated protein translation, resulting in bidirectional alteration of NMDAR-mediated signaling. Importantly, pharmacological balancing of NMDAR signaling in these mouse models with diametric Cyfip1 dosages rescues behavioral abnormalities. ConclusionsCYFIP1 regulates protein translation of NMDAR and associated complex components at synapses to maintain normal synaptic functions and behaviors. Our integrated analyses provide insight into how gene dosage imbalance caused by CNVs may contribute to divergent neuropsychiatric disorders.

NMDAR in cultured astrocytes: Flux‐independent pH sensor and flux‐dependent regulator of mitochondria and plasma membrane‐mitochondria bridging

Glutamate N-methyl-D-aspartate (NMDA) receptor (NMDAR) is critical for neu-rotransmission as a Ca2+ channel. Nonetheless, flux-independent signaling has also been demonstrated. Astrocytes express NMDAR distinct from its neuronal counter-part, but cultured astrocytes have no electrophysiological response to NMDA. We recently demonstrated that in cultured astrocytes, NMDA at pH6 (NMDA/pH6) act-ing through the NMDAR elicits flux-independent Ca2+ release from the Endoplasmic Reticulum (ER) and depletes mitochondrial membrane potential (mΔΨ). Here we show that Ca2+ release is due to pH6 sensing by NMDAR, whereas mΔΨ deple-tion requires both: pH6 and flux-dependent NMDAR signaling. Plasma membrane (PM) NMDAR guard a non-random distribution relative to the ER and mitochon-dria. Also, NMDA/pH6 induces ER stress, endocytosis, PM electrical capacitance reduction, mitochondria-ER, and -nuclear contacts. Strikingly, it also produces the formation of PM invaginations near mitochondria along with structures referred to here as PM-mitochondrial bridges (PM-m-br). These and earlier data strongly sug-gest PM-mitochondria communication. As proof of the concept of mass transfer, we found that NMDA/pH6 provoked mitochondria labeling by the PM dye FM-4-64FX. NMDA/pH6 caused PM depolarization, cell acidification, and Ca2+ release from most mitochondria. Finally, the MCU and microtubules were not involved in mΔΨ depletion, while actin cytoskeleton was partially involved. These findings demon-strate that NMDAR has concomitant flux-independent and flux-dependent actions in cultured astrocytes. This work has been recently accepted for publication in The FASEB J.

Chronically altered NMDAR signaling in epilepsy mediates comorbid depression

Depression is the most common psychiatric comorbidity of epilepsy. However, the molecular pathways underlying this association remain unclear. The NMDA receptor (NMDAR) may play a role in this association, as its downstream signaling has been shown to undergo long-term changes following excitotoxic neuronal damage. To study this pathway, we used an animal model of fluoxetine-resistant epilepsy-associated depression (EAD). We determined the molecular changes associated with the development of depressive symptoms and examined their response to various combinations of fluoxetine and a selective neuronal nitric oxide synthase inhibitor, 7-nitroindazole (NI). Depressive symptoms were determined using the forced swim test. Furthermore, expression and phosphorylation levels of markers in the ERK/CREB/ELK1/BDNF/cFOS pathway were measured to determine the molecular changes associated with these symptoms. Finally, oxidative stress markers were measured to more clearly determine the individual contributions of each treatment. While chronic fluoxetine (Flxc) and NI were ineffective alone, their combination had a statistically significant synergistic effect in reducing depressive symptoms. The development of depressive symptoms in epileptic rats was associated with the downregulation of ERK2 expression and ELK1 and CREB phosphorylation. These changes were exactly reversed upon Flxc + NI treatment, which led to increased BDNF and cFOS expression as well. Interestingly, ERK1 did not seem to play a role in these experiments. NI seemed to have augmented Flxc’s antidepressant activity by reducing oxidative stress. Our findings suggest NMDAR signaling alterations are a major contributor to EAD development and a potential target for treating conditions associated with underlying excitotoxic neuronal damage.

Kinetic Model Distinguishes Subanesthetic Doses of Ketamine Block on Single Channel N-Methyl-D-Aspartate Receptors from Effects of Protons

Ketamine, one of the most commonly used anesthetics world-wide, is also FDA-approved to treat major depressive disorder, when infused at sub anesthetic doses (0.3 to 0.5 mg/kg IV bolus). In these conditions, plasma levels of ketamine peak at 0.85 µM, and decay to 0.2 µM, within 230 minutes. In vitro, ketamine blocks NMDA receptor (NMDAR) currents, which are major contributors to excitatory post synaptic currents in the central nervous system, with a reported range of IC50, 0.7 - 50 µM. Moreover, this effect varies with pH due to both reduced activity of protonated NMDAR (IC50, 7.2), and increased inhibitory potency of protonated ketamine (pKa, 7.5). To ascertain the mechanisms by which low-dose ketamine treatment affects NMDAR signaling in physiologically relevant conditions, we examined in HEK293 cells macroscopic and single-molecule GluN1a/GluN2A currents, in the presence of several concentrations of ketamine, at several pH values. We found that when applied to steady-state whole-cell currents, ketamine inhibition varied strongly (IC50, 0.8 - 2.2 µM) with pH in physiologic ranges (pH, 6.8 - 7.6), being most potent at pH 7.2 (IC50, 0.5 µM). When applied onto individual channels at this pH (0.05 - 1 µM), we found that 0.1 µM ketamine halved the open probability (Po) of NMDARs. As reported previously, ketamine had negligible effects on mean open times, such that the decrease in Po was solely due to an increase in closed times. These data allowed detailed kinetic modeling of NMDAR activity in the presence of ketamine, to estimate channel gating rates. Upon further validation, these models will help to anticipate the effects of ketamine on synaptic NMDAR currents, a necessary step in ascertaining the mechanism by which ketamine exerts its antidepressant effects in patients.

The Role of D-Amino Acids in Alzheimer's Disease.

Alzheimer's disease (AD), the main cause of dementia worldwide, is characterized by a complex and multifactorial etiology. In large part, excitatory neurotransmission in the central nervous system is mediated by glutamate and its receptors are involved in synaptic plasticity. The N-methyl-D-aspartate (NMDA) receptors, which require the agonist glutamate and a coagonist such as glycine or the D-enantiomer of serine for activation, play a main role here. A second D-amino acid, D-aspartate, acts as agonist of NMDA receptors. D-amino acids, present in low amounts in nature and long considered to be of bacterial origin, have distinctive functions in mammals. In recent years, alterations in physiological levels of various D-amino acids have been linked to various pathological states, ranging from chronic kidney disease to neurological disorders. Actually, the level of NMDA receptor signaling must be balanced to promote neuronal survival and prevent neurodegeneration: this signaling in AD is affected mainly by glutamate availability and modulation of the receptor's functions. Here, we report the experimental findings linking D-serine and D-aspartate, through NMDA receptor modulation, to AD and cognitive functions. Interestingly, AD progression has been also associated with the enzymes related to D-amino acid metabolism as well as with glucose and serine metabolism. Furthermore, the D-serine and D-/total serine ratio in serum have been recently proposed as biomarkers of AD progression. A greater understanding of the role of D-amino acids in excitotoxicity related to the pathogenesis of AD will facilitate novel therapeutic treatments to cure the disease and improve life expectancy.

Non-ionotropic NMDA receptor signaling gates bidirectional structural plasticity of dendritic spines

SUMMARYExperience-dependent refinement of neuronal connections is critically important for brain development and learning. Here, we show that ion-flow-independent NMDA receptor (NMDAR) signaling is required for the long-term dendritic spine growth that is a vital component of brain circuit plasticity. We find that inhibition of p38 mitogen-activated protein kinase (p38 MAPK), which is downstream of non-ionotropic NMDAR signaling in long-term depression (LTD) and spine shrinkage, blocks long-term potentiation (LTP)-induced spine growth but not LTP. We hypothesize that non-ionotropic NMDAR signaling drives the cytoskeletal changes that support bidirectional spine structural plasticity. Indeed, we find that key signaling components downstream of non-ionotropic NMDAR function in LTD-induced spine shrinkage are also necessary for LTP-induced spine growth. Furthermore, NMDAR conformational signaling with coincident Ca2+ influx is sufficient to drive CaMKII-dependent long-term spine growth, even when Ca2+ is artificially driven through voltage-gated Ca2+ channels. Our results support a model in which non-ionotropic NMDAR signaling gates the bidirectional spine structural changes vital for brain plasticity.

Effects of datumetine on hippocampal NMDAR activity.

The usage (abuse) of Datura metel is becoming increasingly worrisome among the Nigerian populace especially among the youth considering its side effects such as hallucination. This work was designed to identify the phytochemicals in datura plant that potentially interact with NMDAR as it affects the electrical and memory activities of the brain. Ligand-protein interaction was assessed using autodock vina to identify phytochemicals that can interact with NMDAR. Datumetine was found to have the best interaction fit with NMDAR at both allosteric and orthosteric binding sites. Furthermore, using electrophysiological, behavioural and western blotting techniques, it was observed that the administration of datumetine positively modulates the NMDAR current by prolonging burst duration and interspike interval, induces seizures in C57BL/6 mice. Acute exposure leads to memory deficit on NOR and Y-maze test while immunoblotting results showed increased expression of GluN1 and CamKIIα while pCamKIIα-T286, CREB and BDNF were downregulated. The results showed that the memory deficit seen in datura intoxication is possibly the effects of datumetine on NMDAR.

NMDAR in cultured astrocytes: Flux-independent pH sensor and flux-dependent regulator of mitochondria and plasma membrane-mitochondria bridging.

Glutamate N-methyl-D-aspartate (NMDA) receptor (NMDAR) is critical for neurotransmission as a Ca2+ channel. Nonetheless, flux-independent signaling has also been demonstrated. Astrocytes express NMDAR distinct from its neuronal counterpart, but cultured astrocytes have no electrophysiological response to NMDA. We recently demonstrated that in cultured astrocytes, NMDA at pH6 (NMDA/pH6) acting through the NMDAR elicits flux-independent Ca2+ release from the Endoplasmic Reticulum (ER) and depletes mitochondrial membrane potential (mΔΨ). Here we show that Ca2+ release is due to pH6 sensing by NMDAR, whereas mΔΨ depletion requires both: pH6 and flux-dependent NMDAR signaling. Plasma membrane (PM) NMDAR guard a non-random distribution relative to the ER and mitochondria. Also, NMDA/pH6 induces ER stress, endocytosis, PM electrical capacitance reduction, mitochondria-ER, and -nuclear contacts. Strikingly, it also produces the formation of PM invaginations near mitochondria along with structures referred to here as PM-mitochondrial bridges (PM-m-br). These and earlier data strongly suggest PM-mitochondria communication. As proof of the concept of mass transfer, we found that NMDA/pH6 provoked mitochondria labeling by the PM dye FM-4-64FX. NMDA/pH6 caused PM depolarization, cell acidification, and Ca2+ release from most mitochondria. Finally, the MCU and microtubules were not involved in mΔΨ depletion, while actin cytoskeleton was partially involved. These findings demonstrate that NMDAR has concomitant flux-independent and flux-dependent actions in cultured astrocytes.

A microscopy-based small molecule screen in primary neurons reveals neuroprotective properties of the FDA-approved anti-viral drug Elvitegravir

Glutamate toxicity is a pathomechanism that contributes to neuronal cell death in a wide range of acute and chronic neurodegenerative and neuroinflammatory diseases. Activation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor and breakdown of the mitochondrial membrane potential are key events during glutamate toxicity. Due to its manifold functions in nervous system physiology, however, the NMDA receptor is not well suited as a drug target. To identify novel compounds that act downstream of toxic NMDA receptor signaling and can protect mitochondria from glutamate toxicity, we developed a cell viability screening assay in primary mouse cortical neurons. In a proof-of-principle screen we tested 146 natural products and 424 FDA-approved drugs for their ability to protect neurons against NMDA-induced cell death. We confirmed several known neuroprotective drugs that include Dutasteride, Enalapril, Finasteride, Haloperidol, and Oxybutynin, and we identified neuroprotective properties of Elvitegravir. Using live imaging of tetramethylrhodamine ethyl ester-labelled primary cortical neurons, we found that Elvitegravir, Dutasteride, and Oxybutynin attenuated the NMDA-induced breakdown of the mitochondrial membrane potential. Patch clamp electrophysiological recordings in NMDA receptor-expressing HEK293 cell lines and primary mouse hippocampal neurons revealed that Elvitegravir does not act at the NMDA receptor and does not affect the function of glutamatergic synapses. In summary, we have developed a cost-effective and easy-to-implement screening assay in primary neurons and identified Elvitegravir as a neuro- and mitoprotective drug that acts downstream of the NMDA receptor.

Superoxide and Non-ionotropic Signaling in Neuronal Excitotoxicity.

Excitotoxicity is classically attributed to Ca2+ influx through NMDA receptors (NMDAr), leading to production of nitric oxide by neuronal nitric oxide synthase and superoxide by mitochondria, which react to form highly cytotoxic peroxynitrite. More recent observations warrant revision of the classic view and help to explain some otherwise puzzling aspects of excitotoxic cell injury. Studies using pharmacological and genetic approaches show that superoxide produced by NMDAr activation originates primarily from NADPH oxidase rather than from mitochondria. As NADPH oxidase is localized to the plasma membrane, this also provides an explanation for the extracellular release of superoxide and cell-to-cell “spread” of excitotoxic injury observed in vitro and in vivo. The signaling pathway linking NMDAr to NADPH oxidase involves Ca2+ influx, phosphoinositol-3-kinase, and protein kinase Cζ, and interventions at any of these steps can prevent superoxide production and excitotoxic injury. Ca2+ influx specifically through NMDAr is normally required to induce excitotoxicity, through a mechanism presumed to involve privileged Ca2+ access to local signaling domains. However, experiments using selective blockade of the NMDAr ion channel and artificial reconstitution of Ca2+ by other routes indicate that the special effects of NMDAr activation are attributable instead to concurrent non-ionotropic NMDAr signaling by agonist binding to NMDAr. The non-ionotropic signaling driving NADPH oxidase activation is mediated in part by phosphoinositol-3-kinase binding to the C-terminal domain of GluN2B receptor subunits. These more recently identified aspects of excitotoxicity expand our appreciation of the complexity of excitotoxic processes and suggest novel approaches for limiting neuronal injury.

Hypoxia-inducible factors and obstructive sleep apnea.

Intermittent hypoxia (IH) is a hallmark manifestation of obstructive sleep apnea (OSA), a widespread disorder of breathing. This Review focuses on the role of hypoxia-inducible factors (HIFs) in hypertension, type 2 diabetes (T2D), and cognitive decline in experimental models of IH patterned after O2 profiles seen in OSA. IH increases HIF-1α and decreases HIF-2α protein levels. Dysregulated HIFs increase reactive oxygen species (ROS) through HIF-1-dependent activation of pro-oxidant enzyme genes in addition to reduced transcription of antioxidant genes by HIF-2. ROS in turn activate chemoreflex and suppress baroreflex, thereby stimulating the sympathetic nervous system and causing hypertension. We also discuss how increased ROS generation by HIF-1 contributes to IH-induced insulin resistance and T2D as well as disrupted NMDA receptor signaling in the hippocampus, resulting in cognitive decline.

Consequences of NMDA receptor deficiency can be rescued in the adult brain

N-methyl-D-aspartate receptors (NMDARs) are required to shape activity-dependent connections in the developing and adult brain. Impaired NMDAR signalling through genetic or environmental insults causes a constellation of neurodevelopmental disorders that manifest as intellectual disability, epilepsy, autism, or schizophrenia. It is not clear whether the developmental impacts of NMDAR dysfunction can be overcome by interventions in adulthood. This question is paramount for neurodevelopmental disorders arising from mutations that occur in the GRIN genes, which encode NMDAR subunits, and the broader set of mutations that disrupt NMDAR function. We developed a mouse model where a congenital loss-of-function allele of Grin1 can be restored to wild type by gene editing with Cre recombinase. Rescue of NMDARs in adult mice yields surprisingly robust improvements in cognitive functions, including those that are refractory to treatment with current medications. These results suggest that neurodevelopmental disorders arising from NMDAR deficiency can be effectively treated in adults.

DLG2 variants in patients with pubertal disorders

PurposeImpaired function of gonadotropin-releasing hormone (GnRH) neurons can cause a phenotypic spectrum ranging from delayed puberty to isolated hypogonadotropic hypogonadism (IHH). We sought to identify a new genetic etiology for these conditions. MethodsExome sequencing was performed in an extended family with autosomal dominant, markedly delayed puberty. The effects of the variant were studied in a GnRH neuronal cell line. Variants in the same gene were sought in a large cohort of individuals with IHH. ResultsWe identified a rare missense variant (F900V) in DLG2 (which encodes PSD-93) that cosegregated with the delayed puberty. The variant decreased GnRH expression in vitro. PSD-93 is an anchoring protein of NMDA receptors, a type of glutamate receptor that has been implicated in the control of puberty in laboratory animals. The F900V variant impaired the interaction between PSD-93 and a known binding partner, Fyn, which phosphorylates NMDA receptors. Variants in DLG2 that also decreased GnRH expression were identified in three unrelated families with IHH. ConclusionThe findings indicate that variants in DLG2/PSD-93 cause autosomal dominant delayed puberty and may also contribute to IHH. The findings also suggest that the pathogenesis involves impaired NMDA receptor signaling and consequently decreased GnRH secretion.