GluN2B-specific NMDAR positive allosteric modulation reverses cognitive and behavioral abnormalities in Mecp2 and Disc1 transgenic mice

Among glutamate-gated channels, NMDA receptors produce currents that subside with unusually slow kinetics, and this feature is essential to the physiology of central excitatory synapses. Relative to the homologous AMPA and kainate receptors, NMDA receptors have additional intersubunit contacts in the ligand binding domain that occur at both conserved and non-conserved sites. We examined GluN1/GluN2A single-channel currents with kinetic analyses and modeling to probe these class-specific intersubunit interactions for their role in glutamate binding and receptor gating. We found that substitutions that eliminate such interactions at non-conserved sites reduced stationary gating, accelerated deactivation, and imparted sensitivity to aniracetam, an AMPA receptor-selective positive modulator. Abolishing unique contacts at conserved sites also reduced stationary gating and accelerated deactivation. These results show that contacts specific to NMDA receptors, which brace the heterodimer interface within the ligand binding domain, stabilize actively gating receptor conformations and result in longer bursts and slower deactivations. They support the view that the strength of the heterodimer interface modulates gating in both NMDA and non-NMDA receptors and that unique interactions at this interface are responsible in part for basic differences between the kinetics of NMDA and non-NMDA currents at glutamatergic synapses.

IntroductionSulforaphane (SFN) has been found to alleviate complications linked with several diseases by regulating gut microbiota (GM), while the effect of GM on SFN for autism spectrum disorders (ASD) has not been studied. Therefore, this study aimed to investigate the relationship between the effects of SFN on childhood ASD and GM through animal model and human studies.MethodsWe evaluated the therapeutic effects of SFN on maternal immune activation (MIA) induced ASD-like rat model and pediatric autism patients using three-chamber social test and OSU Autism Rating Scale-DSM-IV (OARS-4), respectively, with parallel GM analysis using 16SrRNA sequencing.ResultsSFN significantly improved the sniffing times of ASD-like rats in the three-chamber test. For human participants, the average verbal or non-verbal communication (OSU-CO) scores of SFN group had changed significantly at the 12-wk endpoint. SFN was safe and no serious side effects after taking. GM changes were similar for both ASD-like rats and ASD patients, such as consistent changes in order Bacillales, family Staphylococcaceae and genus Staphylococcus. Although the gut microbiota composition was significantly altered in SFN-treated ASD-like rats, the alteration of GM was not evident in ASD patients after 12 weeks of SFN treatment. However, in the network analysis, we found 25 taxa correlated with rats' social behavior, 8 of which were associated with SFN treatment in ASD-like rats, For ASD patients, we found 35 GM abundance alterations correlated with improvements in ASD symptoms after SFN treatment. Moreover, family Pasteurellaceae and genus Haemophilus were found to be associated with SFN administration in the network analyses in both ASD-like rats and ASD patients.DiscussionThese findings suggest that SFN could provide a novel avenue for preventing and treating ASD, and its therapeutic effects might be related to gut microbiota.

Reduced N-methyl D-aspartate (NMDA) receptor function is hypothesized to contribute to the pathophysiology of schizophrenia. In order to model chronic and developmental NMDA receptor hypofunction, a mouse line was developed that expresses low levels of the NMDA R1 (NR1) subunit of the NMDA receptor. These mice show increased acoustic startle reactivity and deficits in prepulse inhibition (PPI) of acoustic startle. The present study tested the hypothesis that these altered acoustic startle responses in the NR1 hypomorphic (NR1-/-) mice would be affected by antipsychotic drug treatment. Mice were injected with drugs 30 min before assessment of acoustic startle responses with and without prepulse stimuli. Haloperidol (0.5 or 1.0 mg/kg) did not reduce the increased startle reactivity in the NR1-/- mice, but did increase PPI in both the mutant and wild type mice. Clozapine (3 mg/kg) and quetiapine (20 mg/kg) reduced startle magnitude and increased PPI in both the wild type and mutant mice. The antidepressant drug imipramine (10 and 20 mg/kg) had minimal effects on startle amplitude in NR1-/- or wild type mice. However, for the 20-mg/kg dose of imipramine, a significant increase in PPI was observed in the wild type animals, but not in the mutant mice. The results demonstrate that PPI can be increased in a mouse model of chronic NMDA receptor hypofunction by typical and atypical antipsychotic drugs. The similar effects of typical and atypical antipsychotic drugs to increase PPI in the wild type and mutant mice indicates that the assessment of behavior of the NR1 hypomorphic mice in the PPI paradigm offers no advantage over the wild type controls for identifying new clozapine-like drugs.

Despite ample evidence supporting the N-methyl-D-aspartate receptor (NMDAR) hypofunction hypothesis of schizophrenia, progress in the development of effective therapeutics based on this hypothesis has been limited. Facilitation of NMDA receptor function by co-agonists (D-serine or glycine) only partially alleviates the symptoms in schizophrenia; other means to facilitate NMDA receptors are required. NMDA receptor sub-types differ in their subunit composition, with varied GluN2 subunits (GluN2A-GluN2D) imparting different physiological, biochemical and pharmacological properties. CIQ is a positive allosteric modulator that is selective for GluN2C/GluN2D-containing NMDA receptors (Mullasseril et al.). The effect of systemic administration of CIQ was tested on impairment in prepulse inhibition (PPI), hyperlocomotion and stereotypy induced by i.p. administration of MK-801 and methamphetamine. The effect of CIQ was also tested on MK-801-induced impairment in working memory in Y-maze spontaneous alternation test. We found that systemic administration of CIQ (20 mg·kg⁻¹, i.p.) in mice reversed MK-801 (0.15 mg·kg⁻¹, i.p.)-induced, but not methamphetamine (3 mg·kg⁻¹, i.p.)-induced, deficit in PPI. MK-801 increased the startle amplitude to pulse alone, which was not reversed by CIQ. In contrast, methamphetamine reduced the startle amplitude to pulse alone, which was reversed by CIQ. CIQ also partially attenuated MK-801- and methamphetamine-induced hyperlocomotion and stereotyped behaviours. Additionally, CIQ reversed the MK-801-induced working memory deficit in spontaneous alternation in a Y-maze. Together, these results suggest that facilitation of GluN2C/GluN2D-containing receptors may serve as an important therapeutic strategy for treating positive and cognitive symptoms in schizophrenia.

Association Among Gut Microbes, Intestinal Physiology, and Autism

The mGluR5 antagonist MPEP, but not the mGluR2/3 agonist LY314582, augments PCP effects on prepulse inhibition and locomotor activity

Pyrroloquinoline quinone improves abnormal functional development of whisker-mediated tactile perception and social behaviors caused by neonatal whisker trimming

Environmental enrichment modulates affiliative and aggressive social behaviour in the neuroligin-3 R451C mouse model of autism spectrum disorder

Differential regulation of STP, LTP and LTD by structurally diverse NMDA receptor subunit-specific positive allosteric modulators

High glycine levels are associated with prepulse inhibition deficits in chronic schizophrenia patients

GluN1 antibody causes behavioral deficits in prepulse inhibition and memory through CaMKIIβ signaling

N-methyl-D-aspartate receptor (NMDAR) serves for both high cortical function and fundamental CNS mechanisms. NMDAR-mediated neurotransmission is the molecular engine for CNS development and plasticity; it is also the molecular underpin of learning, memory and cognition. Due to its critical role in CNS function, either over- or under-activation of NMDAR-mediated neurotransmission contributes significantly to the development of CNS disorders. In this issue of Current Pharmaceutical Design, the authors discuss the involvement of NMDARmediated neurotransmission in a variety of CNS disorders including schizophrenia [1], cognitive deficits in schizophrenia [2, 3], depression [4], aging [5], mild cognitive impairment and Alzheimer’s dementia [6], attention deficit hyperactivity disorder [7], frontal lobe synaptic plasticity [8] as well as autism spectrum disorder [9]. NMDAR distinguish itself in two ways: first, it is both ligand-gated and voltage-dependent; second, it requires co-activation by two ligands: glutamate or aspartate and either D-serine or glycine. The experience-dependent learning originates from these two critical coincidental mechanisms. First, the activation of non-NMDA glutamate receptor will relieve the magnesium blockade and allow the opening of NMDAR channel ionophore and calcium influx. This “coincidence” mechanism between NMDA and non-NMDA receptors provides a transduction and transformation of excitatory input from multiple modalities of stimulation through non-NMDARs into the molecular machinery of NMDAR that mediates complex CNS behaviors. In this regard, NMDAR serves as a high order integrator to summate the signals from the EPSP carried by the non-NMDARs. Second, D-serine and glycine, as obligatory co-agonists, provide another dimension of dynamics in the neocortex. D-serine and the racemic enzyme converting L-serine to D-serine are enriched in the corticolimbic regions [10]. D-serine plays a key role in NMDAR activation for high order cognitive functions, while glycine’s localization is much less specific, which is also enriched in the brain stem and spinal cord other than the forebrain. There are a variety of approaches to regulate NMDAR-mediated neurotransmission; not only by the electrophysiological and molecular coincidental mechanisms mentioned above, NMDAR-mediated neurotransmission also has multiple regulatory mechanisms. As in the aminergic or GABAergic system, the regulation can happen at the level of precursor and neurotransmitter synthesis and release, or termination of action by uptake or catabolism. Parallel to the aminergic or GABAergic systems (Table 1), administration of D-serine or glycine, can activation NMDAR to improve cognitive and psychotic symptoms as tryptophan loading can facilitate the synthesis of serotonin and improves the depressive symptoms. Blocking the high efficient uptake site of glycine transporter-1 (GlyT-1) in the forebrain [11] potentiates NMDA function similar to selective serotonin uptake inhibitor’s (SSRI’s) action to raise serotonin tone. However, D-serine appears not to have a high efficient uptake site; instead, there are low affinity exchangers alanine serine cysteine transporter-1 (ASC-1) and -2 which physiological role is unclear [12]. Therefore, D-serine regulation may provide a tone that slower in time scale than the high efficient mechanism like GlyT-1. The tonicclonic coordination between D-serine and glycine can provide another dimension of complexity, and thus possibilities of regulation.The occupation of the co-agonist site is obligatory for the activation of NMDAR. However, the presence of both glycine and D-serine is not essential since both co-agonists are full agonists with strong potency. These two co-agonists not only provide redundancy for the activation of NMDAR, it also provides an unique opportunities to regulate NMDAR [13]. Although glycine and D-serine have similar potency, the anatomic specificity favors D-serine. Other than their different anatomical localization at the gross regional level, their microanatomical and physiological functions are also different; synaptic NMDAR 2A subunit-containing and extrasynaptic 2B-containing NMDARs have different co-agonists: D-serine for synaptic NMDARs and glycine for extrasynaptic NMDARs [13]. In addition, it has been found that synaptic and extrasynaptic NMDARs have opposing effects in determining the fate of neurons; the mechanisms of cell destruction or cell survival in response to the activation of NMDAR depend in part on calcium and its route of entry, and more significantly on the subunit composition and localization of the NMDARs. Overall, the synaptic NMDAR activation is involved in neuroprotection, the stimulation of extrasynaptic NMDARs, triggers cell destruction pathways and may play a key role in the neurodegeneration associated with excitotoxicity. The multi-dimensional complexity of the physiology and pathology of NMDAR can be best exampled by these two co-agonists. Although the microscopic availability of the co-agonists matches the preferential affinity of synaptic NMDARs for D-serine and extrasynaptic NMDARs for glycine, this dichotomy is not universal. For example, long-term potentiation rely on synaptic NMDARs, but both glycine and D-serine can be involved [12]. Conversely, long-term depression requires both synaptic and extrasynaptic receptors. While the initial thought that Dserine originates from astrocytes, recent evidences indicate D-serine is also neuronal in origin [10]. Neuronal D-serine is required for NMDAR-dependent, long-term potentiation at the hippocampal CA1-CA3 synapses and proper synapse formation in the cerebral cortex. However, glycine is present on both forebrain and hindbrain, for both inhibitory and excitatory neurotransmission. Based upon the prediction that enhancement of NMDA function will improve the pathological state induced by NMDAR antagonists like phencyclidine and ketamine, glycine, a full agonist, was the first to be tested in schizophrenia [14]. However, glycine has poor efficacy and requires large amount of administration (>= 60 grams/day) to have a modest effect [15]. We first proposed D-cycloserine, a partial agonist, would be a better NMDA agent than glycine due to its CNS bioavailability. We found D-cycloserine offered an inverted-U dose-response curve consistent with its partial agonist activity in a dose-finding trial of schizophrenia [16]. To facilitate higher NMDA activation, we further conducted trials with full agonists, D-serine [17] and D-alanine [18]. As we predicted, the full agonists were able to elicit a higher level of NMDA activation and clinical improvement than the partial agonist. The dose-response of NMDA activation by the treatment of full agonist was later supported by an open-label trial, indicating 60 or 120 mg/kg D-serine has better efficacy than 30 mg/kg in symptom reduction and cognitive improvement [19]. We further hypothesized that the facilitation of NMDAR activation can be achieved by blocking the reuptake of the agonist, either glycine or D-serine. However, no high affinity uptake site for D-serine has been identified, therefore we focused on the GlyT-1 which is enriched in corticolimbic region, unlike GlyT-2 which is not present in the corticolimbic region. We also predicted, the competitive antagonist will elicit a safer pharmacological profile than the noncompetitive antagonist, for the concern that the high affinity blockade of the GlyT-1 may overactivate NMDAR, particularly when extrasynaptic NMDAR that mediates toxicity is involved. In addition, strong inhibition by noncompetitive antagonism can induce endocytosis [20]. The prototype GlyT-1 inhibitor we applied, sarcosine, is a naturally occurring amino acid, discovered at high concentration in tissues including CNS. Sarcocine’s efficacy had been proved in several small scale double blind, placebo controlled studies [21-25]. Supporting the advantage of competitive vs. noncompetitive GlyT-1 antagonist, all noncompetitive antagonists had failed the development so far, including bitopertin, which gave a weak signal at the Phase II study and did not meet its endpoints of the improvement of negative symptoms in two Phase III trials [26]. Infact, bitopertin provides a inverted-U dose-response, which also discourage the therapeutic approach of noncompetitive antagonism [27]. In addition to its efficacy in the main symptom domains of schizophrenia like positive, negative and cognitive symptoms, the depressive symptoms are also improved by NMDA enhancement treatments [23]. To determine whether the antidepressant effect is primary or secondary to the improvement of other symptom domains, we has conducted both rodent behavior studies and a trial of sarcosine treatment in major depression. In which, sarcosine treatment not only elicits an antidepressant-like behavior profile in both acute and chronic stress model of depression, but also reach a much higher remission rate than a standard SSRI treatment in major depression [28]. It had been well known that magnesium infusion can quickly relieve migraine and eclampsia, likely due to its blockade of NMDAR. Given the recent findings that NMDAR antagonists can improve the symptoms of depression, the mechanistic question was raised why both NMDA enhancement and blockade can improve the symptoms of depression [4]. It is possible that both treatments share a final common target, like m-TOR or BDNF, through some unidentified intermediate mechanism. However, NMDAR agonist and antagonist have different time scales in improving the depressive symptoms; NMDAR antagonist elicits an almost immediate effect, which is much faster than agonist treatment. At the same time, the underline molecular mechanism of NMDAR antagonists is unclear given that the proposed BDNF activation and synaptogenesis will take days to weeks to develop, while ketamine’s effect is immediate. The efficacy of NMDA treatment is not limited to schizophrenia and depression. The efficacy had been shown in improving the symptoms of dementia and obsessive compulsive disorder (OCD) by sarcosine treatment [29], which is consistent with the involvement of glutamatergic neurotransmission in dementia and memory and the circuitry of OCD. NMDA neurotransmission is ubiquitous and involved in many fundamental function of CNS including psychosis, cognition, rewarding, motor, etc. Its modulation can certainly offer beneficial outcome in the symptoms involving these circuitries. In the hindsight, though NMDA-enhancement treatment is particularly relevant to schizophrenia given that the NMDAR antagonists generate “schizophrenia-like” symptoms, it is not surprising that the treatment is also beneficial for a variety of CNS disorders. While looking back the development of aminergic and GABAergic treatments, I saw the history of glutamatergic treatments, developed in the past two to three decades, could follow a similar path. All three lines of treatments can involve neurotransmitter and its precursor (chloroziapoxide, tryptophan), agonist/antagonist (L-dopa, chlopromazine), uptake blocker (imipramine, fluoxetine, bupropion), catabolism inhibitor (iproniazid, selegiline) (Table 1). In analogy to the aminergic and GABAergic treatments, I saw the missing NMDA treatment options of: first, the neurotransmitter uptake inhibition by GlyT-1 inhibitor; second, NMDAR antagonists; third, the inhibition of the D-amino acid oxidase (DAAO), which metabolize D-serine [30]. The treatment of DAAO inhibition is analogous to monoamin oxidase inhibitors (MAOI), which upregulates monoamine for CNS disorders like depression and Parkinson disease. In another word, DAAO inhibitors are similar to MAOI in raising the tone of neurotransmitter of interest, by inhibiting the catabolism enzyme. The legendary biochemist, Sir Hans Adolf Krebs discovered D-amino acid deaminase and considered the enzyme “in search of function” [31]. Almost eighty years later, the enzyme, now known as DAAO, has gained attention as a critical regulator of CNS neurotransmission. Its main substrate, D-serine is a critically important co-agonist of the NMDAR. We recently demonstrated that sodium benzoate, as a DAAO inhibitor, can substantially improve the symptoms and neurocognition of schizophrenia, presumably enhance NMDA function by raising D-serine level [32]. In mild cognitive impairment (MCI), sodium benzoate also improves the cognition and function [33]. In late 19 century, the planet Neptune was mathematically predicted before it was directly observed; working from Le Verrier's calculations, telescopic observations of Neptune was confirming afterwards. In developing the NMDA treatment for CNS disorder, we predicted DAAO inhibition could be the last missing approach in regulating the grand regulator of CNS, NMDAR-mediated neurotransmission. To search for novel DAAO inhibitors, one of the articles in this issue discuss an innovative informatics method to determine the potential activity of DAAO inhibitors in this new frontier of CNS drug development [34]. Keywords: NMDA, D-serine, glycine transporter, D-amino acid oxidase.

N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors important for synaptic plasticity, memory, and neuropsychiatric health. NMDAR hypofunction contributes to multiple disorders, including anti-NMDAR encephalitis (NMDARE), an autoimmune disease of the CNS associated with GluN1 antibody-mediated NMDAR internalization. Here we characterize the functional/pharmacological consequences of exposure to CSF from female human NMDARE patients on NMDAR function, and we characterize the effects of intervention with recently described positive allosteric modulators (PAMs) of NMDARs. Incubation (48 h) of rat hippocampal neurons of both sexes in confirmed NMDARE patient CSF, but not control CSF, attenuated NMDA-induced current. Residual NMDAR function was characterized by lack of change in channel open probability, indiscriminate loss of synaptic and extrasynaptic NMDARs, and indiscriminate loss of GluN2B-containing and GluN2B-lacking NMDARs. NMDARs tagged with N-terminal pHluorin fluorescence demonstrated loss of surface receptors. Thus, function of residual NMDARs following CSF exposure was indistinguishable from baseline, and deficits appear wholly accounted for by receptor loss. Coapplication of CSF and PAMs of NMDARs (SGE-301 or SGE-550, oxysterol-mimetic) for 24 h restored NMDAR function following 24 h incubation in patient CSF. Curiously, restoration of NMDAR function was observed despite washout of PAMs before electrophysiological recordings. Subsequent experiments suggested that residual allosteric potentiation of NMDAR function explained the persistent rescue. Further studies of the pathogenesis of NMDARE and intervention with PAMs may inform new treatments for NMDARE and other disorders associated with NMDAR hypofunction.SIGNIFICANCE STATEMENT Anti-N-methyl-d-aspartate receptor encephalitis (NMDARE) is increasingly recognized as an important cause of sudden-onset psychosis and other neuropsychiatric symptoms. Current treatment leaves unmet medical need. Here we demonstrate cellular evidence that newly identified positive allosteric modulators of NMDAR function may be a viable therapeutic strategy.

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder with both genetic and environmental contribution risk factors. Our previous study has demonstrated that DK-I-56-1 (DK), a positive allosteric modulator (PAM) selective to the α6 subunit-containing GABAA receptors (α6GABAARs) can prevent the development of ASD-like core symptoms in prenatal valproic acid-exposure juvenile rats (accompanied presentation), representing the impact of environmental factors on ASD.Aims & ObjectivesIn this study, we utilized the Fmr1 knockout (KO) (Fmr1 KO) mouse model, a preclinical ASD model representing the genetic impact in the pathogenesis of ASD, for screening potential therapy, to validate our hypothesis that α6GABAARs can enhance GABAergic transmission in granule cells (GCs) and restore Purkinje cell (PC) activity in the cerebellum and then mitigate ASD-like symptoms.MethodThe Fmr1 gene is an X-linked risk gene of ASD. We thus employed male hemizygotes (Fmr1-/y), as well as female hemizygotes (Fmr1-/+) and female homozygotes (Fmr1 -/-), using their wildtype littermates of the same sex as the Control groups. Juvenile mice at 5-6 weeks of age received the three-chamber social interaction test to assess their sociability and social preference. DK (10 mg/kg) or vehicle (Veh) was i.p. administered five days and 30 min before the three-chamber test on the tested day.ResultsResults showed a gene-dose effect where male hemizygous Fmr1 KO mice exhibited more severe deficits in sociability than female Fmr1 KO mice. Among females, homozygous Fmr1 KO mice showed greater impairments in both social preference and sociability than hemizygous counterparts. DK treatment (10 mg/kg, i.p.) pretreatment rescued social impairments across genotypes and sex.Discussion & ConclusionsThese therapeutic effects are consistent with DK’s action on α6GABAARs in the cerebellum. With favorable pharmacokinetics, including good oral bioavailability, a 13-hour half-life, and no sedative or abuse potential, DK is a promising candidate for treating ASD.1

Mutations in NLGN4X have been identified in individuals with autism spectrum disorders and other neurodevelopmental disorders. A previous study reported that adult male mice lacking neuroligin4 (Nlgn4) displayed social approach deficits in the three-chambered test, altered aggressive behaviors and reduced ultrasonic vocalizations. To replicate and extend these findings, independent comprehensive analyses of autism-relevant behavioral phenotypes were conducted in later generations of the same line of Nlgn4 mutant mice at the National Institute of Mental Health in Bethesda, MD, USA and at the Institut Pasteur in Paris, France. Adult social approach was normal in all three genotypes of Nlgn4 mice tested at both sites. Reciprocal social interactions in juveniles were similarly normal across genotypes. No genotype differences were detected in ultrasonic vocalizations in pups separated from the nest or in adults during reciprocal social interactions. Anxiety-like behaviors, self-grooming, rotarod and open field exploration did not differ across genotypes, and measures of developmental milestones and general health were normal. Our findings indicate an absence of autism-relevant behavioral phenotypes in subsequent generations of Nlgn4 mice tested at two locations. Testing environment and methods differed from the original study in some aspects, although the presence of normal sociability was seen in all genotypes when methods taken from Jamain et al. (2008) were used. The divergent results obtained from this study indicate that phenotypes may not be replicable across breeding generations, and highlight the significant roles of environmental, generational and/or procedural factors on behavioral phenotypes.