Tau-S214 Phosphorylation Inhibits Fyn Kinase Interaction and Increases the Decay Time of NMDAR-mediated Current.

Fyn kinase SH3 domain interaction with PXXP motif in the Tau protein is implicated in AD pathology and is central to NMDAR function. Among seven PXXP motifs localized in proline-rich domain of Tau protein, tandem 5th and 6th PXXP motifs are critical to Fyn-SH3 domain interaction. Here, we report the crystal structure of Fyn-SH3 -Tau (207-221) peptide consisting of 5th and 6th PXXP motif complex to 1.01 Å resolution. Among five AD-specific phosphorylation sites encompassing the 5th and 6th PXXP motifs, only S214 residue showed interaction with SH3 domain. Biophysical studies showed that Tau (207-221) with S214-phosphorylation (pS214) inhibits its interaction with Fyn-SH3 domain. The individual administration of Tau (207-221) with/without pS214 peptides to a single neuron increased the decay time of evoked NMDA current response. Recordings of spontaneous NMDA EPSCs at +40 mV indicate an increase in frequency and amplitude of events for the Tau (207-221) peptide. Conversely, the Tau (207-221) with pS214 peptide exhibited a noteworthy amplitude increase alongside a prolonged decay time. These outcomes underscore the distinctive modalities of action associated with each peptide in the study. Overall, this study provides insights into how Tau (207-221) with/without pS214 affects the molecular framework of NMDAR signaling, indicating its involvement in Tau-related pathogenesis.

Structure-Based Virtual Screening to Identify Negative Allosteric Modulators of NMDA.

BACKGROUND: NMDA (N-methyl-D-aspartate) receptor is one of the ionotropic receptor subtypes of glutamate, the most abundant excitatory neurotransmitter in the human brain. Besides physiological roles in learning and memory, neuronal plasticity and somatosensory function NMDAR overstimulation are also implicated in a pathophysiological mechanism of 'excitotoxicity.' In this study, an allosteric site has been focused on to design inhibitors of the most abundant form of this receptor of utility in many acute (stroke, traumatic brain injury) and chronic neurodegenerative diseases such as Parkinson's disease, Huntington's, Alzheimer's, and others. METHODS: In order to target this specific site at the interdimer interface of the ligand-binding domain of GluN2A-containing NMDA-Rs, blood-brain barrier-permeable potentially therapeutic compounds, as opposed to only pharmacological tools currently available, were sought. Pharmacophorebased virtual screening, docking, computational ADME prediction techniques, and MD simulation studies were used. RESULTS: Proceeding through the in-silico methodology, the study was successful at reaching 5 compounds from ChEMBL Database, which were predicted to be potential NMDA inhibitor drugs. CONCLUSION: The products of the study are compounds that have been validated through pharmacophore and score-based screening and MD simulation techniques to be allosterically inhibiting NMDA receptors and with favorable pharmacokinetic profiles. They are likely to be therapeutic agents ready for in-vitro and in-vivo testing.

Correlated conformational dynamics of the human GluN1-GluN2A type N-methyl-D-aspartate (NMDA) receptor.

N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels found in the nerve cell membranes. As a result of overexcitation of NMDARs, neuronal death occurs and may lead to diseases such as epilepsy, stroke, Alzheimer's disease, and Parkinson's disease. In this study, human GluN1- GluN2A type NMDAR structure is modeled based on the X-ray structure of the Xenopus laevis template and missing loops are added by ab-initio loop modeling. The final structure is chosen according to two different model assessment scores. To be able to observe the structural changes upon ligand binding, glycine and glutamate molecules are docked into the corresponding binding sites of the receptor. Subsequently, molecular dynamics simulations of 1.3 µs are performed for both apo and ligand-bound structures. Structural parameters, which have been considered to show functionally important changes in previous NMDAR studies, are monitored as conformational rulers to understand the dynamics of the conformational changes. Moreover, principal component analysis (PCA) is performed for the equilibrated part of the simulations. From these analyses, the differences in between apo and ligand-bound simulations can be summarized as the following: The girdle right at the beginning of the pore loop, which connects M2 and M3 helices of the ion channel, partially opens. Ligands act like an adhesive for the ligand-binding domain (LBD) by keeping the bi-lobed structure together and consequently this is reflected to the overall dynamics of the protein as an increased correlation of the LBD with especially the amino-terminal domain (ATD) of the protein.

Bioavailability and Neuroprotectivity of 3-(3, 4-dimethoxy phenyl)-1-4 (methoxy phenyl) prop-2-en-1-one against Schizophrenia: an in silico approach.

Schizophrenia is a major debilitating disorder worldwide. Schizophrenia is a result of multi-gene mutation and psycho-social factors. Mutated amino acid sequences in genes of DOPA such as TH, DDC, DBH, VMAT2, and NMDA (SET-1) have been implicated as major factors causing schizophrenia. In addition mutations in genes other than the DOPA genes such as RGS4, NRG1, COMT, AKT1 and DTNBP1 (SET 2) have also been implicated in the pathogenesis of schizophrenia. Several medicinal herbs and their bioactive constituents have been reported to be involved in ameliorating different neurological disorders including schizophrenia. The present study is mainly focused to study the effect of bioactive compound isolated from the celastrus panuculatus on DOPA and other related genes of schizophrenia using in silico approach. Moledular docking study was carriedout aginast all the selected targets with the lingds i.e. compound and clozapine using the autodock vina 4.0 module implemented in Pyrx 2010.12. The 3 D structures of genes of intrest were retrieved from the protein data bank (PDB). The bioavailability and pharmacological properties of the ligands were determined using OSIRIS server. The novelty of the compound was determined based on fitness, docking and bioavailability score. From the results it is observed that, the compoud has exhibited best dock score against all the selected targets than the clozapie except DBH and VMAT2 in SET-1 targets of DOPA genes. Where as the compound has shown best pharmacokinetic and biologicl property score than the clozapine. Hence, the compound can be considered for further in vitro and in vivo studies to determine the therapeutic efficacy and drug candidacy of the compound in future.

NMDA Antagonists for Treatment-Resistant Depression.

Fifteen to thirty percent of patients with major depressive disorder do not respond to antidepressants that target the monoaminergic systems. NMDA antagonists are currently being actively investigated as a treatment for these patients. Ketamine is the most widely studied of the compounds. A brief infusion of a low dose of this agent produces rapid improvement in depressive symptoms that lasts for several days. The improvement occurs after the agent has produced its well characterized psychotomimetic and cognitive side effects. Multiple infusions of the agent (e.g., 2-3× per week for several weeks) provide relief from depressive symptoms, but the symptoms reoccur once the treatment has been stopped. A 96-h infusion of a higher dose using add-on clonidine to mitigate the psychotomimetic effects appears to also provide relief and resulted in about 40% of the subjects still having a good response 8 weeks after the infusion. As this was a pilot study, additional work is needed to confirm and extend this finding. Nitrous oxide also has had positive results. Of the other investigational agents, CERC-301 and rapastinel remain in clinical development. When careful monitoring of neuropsychiatric symptoms has been conducted, these agents all produce similar side effects in the same dose range, indicating that NMDA receptor blockade produces both the wanted and unwanted effects. Research is still needed to determine the appropriate dose, schedule, and ways to mitigate against unwanted side effects of NMDA receptor blockade. These hurdles need to be overcome before ketamine and similar agents can be prescribed routinely to patients.

Cellulose exopolysaccharide from sugarcane molasses as a suitable substrate for 2D and 3D neuron and astrocyte primary cultures.

Bacteria-synthesized polysaccharides have attracted interest for biomedical applications as promising biomaterials to be used as implants and scaffolds. The present study tested the hypothesis that cellulose exopolysaccharide (CEC) produced from sugarcane molasses of low cost and adequate purity would be suitable as a template for 2D and 3D neuron and/or astrocyte primary cultures, considering its low toxicity. CEC biocompatibility in these primary cultures was evaluated with respect to cell viability, adhesion, growth and cell function (calcium imaging). Polystyrene or Matrigel® matrix were used as comparative controls. We demonstrated that the properties of this CEC in the 2D or 3D configurations are suitable for differentiation of cortical astrocytes and neurons in single or mixed cultures. No toxicity was detected in neurons that showed NMDA-induced Ca2+ influx. Unlike other polysaccharides of bacterial synthesis, the CEC was efficient as a support even in the absence of surface conjugation with extracellular matrix proteins, maintaining physiological characteristics of cultured neural cells. These observations open up the perspective for development of a novel 3D biofunctional scaffold produced from bacterial cellulose and obtained from renewable sources whose residues are not pollutants. Its low cost and possibility to be manufactured in scale are also suitable for potential applications in regenerative medicine.

Immunohistochemical localization of ionotropic glutamate receptors in the rat red nucleus.

In this study, we aimed to determine the presence as well as the diverse distribution of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor subunits in the rat red nucleus. Using adult Sprague-Dawley rats as the experimental animals, immunohistochemistry was performed on 30 µm thick coronal brain sections with antibodies against α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (GluA1-4), kainate (GluK1, GluK2/3, and GluK5), and NMDA (GluN1 and GluN2A) receptor subunits. The results showed that all ionotropic glutamate receptor subunits are expressed in the red nucleus. Specific staining was localized in the neuron bodies and processes. However, the pattern of immunoreactivity and the number of labeled neurons changed depending on the type of ionotropic glutamate receptor subunits and the localization of neurons in the red nucleus. The neurons localized in the magnocellular part of the red nucleus were particularly immunopositive for GluA2, GluA4, GluK2/3, GluK5, GluN1, and GluN2A receptor proteins. In the parvocellular part of the red nucleus, ionotropic glutamate receptor subunit immunoreactivity of variable intensity (lightly to moderately stained) was detected in the neurons. These results suggest that red nucleus neurons in rat heterogeneously express ionotropic glutamate receptor subunits to form functional receptor channels. In addition, the likelihood of the coexpression of different subunits in the same subgroup of neurons suggests the formation of receptor channels with diverse structure by way of different subunit combination, and the possibility of various neuronal functions through these channels in the red nucleus.

Transformation of ranitidine during water chlorination and ozonation: Moiety-specific reaction kinetics and elimination efficiency of NDMA formation potential.

Ranitidine can produce high yields of N-nitrosodimethylamine (NDMA) upon chloramination and its presence in water resources is a concern for water utilities using chloramine disinfection. This study assessed the efficiency of water chlorination and ozonation in transforming ranitidine and eliminating its NDMA formation potential (NDMA-FP) by determining moiety-specific reaction kinetics, stoichiometric factors, and elimination levels in real water matrices. Despite the fact that chlorine reacts rapidly with the acetamidine and thioether moieties of ranitidine (k>10(8)M(-1)s(-1) at pH 7), the NDMA-FP decreases significantly only when chlorine reacts with the less reactive tertiary amine (k=3×10(3)M(-1)s(-1) at pH 7) or furan moiety (k=81M(-1)s(-1) at pH 7). Ozone reacts rapidly with all four moieties of ranitidine (k=1.5×10(5)-1.6×10(6)M(-1)s(-1) at pH 7) and its reaction with the tertiary amine or furan moiety leads to complete elimination of the NDMA-FP. Treatments of ranitidine-spiked real water samples have shown that ozonation can efficiently deactivate ranitidine in water and wastewater treatment, while chlorination can be efficient for water containing low concentration of ammonia. This result can be applied to the other structurally similar, potent NDMA precursors.

Functional Comparison of Neuronal Cells Differentiated from Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Different Oxygen and Medium Conditions.

Because neurons are difficult to obtain from humans, generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells, we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation, glutamate receptor function, and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2, resulting in high responsiveness of neural cells to glutamate, N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ( S)-3,5-dihydroxyphenylglycine (an agonist for mGluR1/5), as revealed by calcium imaging assays. NMDA receptors, AMPA receptors, mGluR1, and mGluR5 were functionally validated by using the specific antagonists MK-801, NBQX, JNJ16259685, and 2-methyl-6-(phenylethynyl)-pyridine, respectively. Multielectrode array analysis showed that spontaneous firing occurred earlier in cells cultured in 2% O2 than in 20% O2. Optimization of O2 tension and culture medium for neural differentiation of hiPSCs can efficiently generate physiologically relevant cells for screening systems.

Synaptic mechanisms shaping delay-tuned combination-sensitivity in the auditory thalamus of mustached bats.

For the processing of target-distance information, delay-tuned auditory neurons of the mustached bat show facilitative responses to a combination of signal elements of a biosonar pulse-echo pair with a specific echo delay. They are initially produced in the inferior colliculus by facilitative responses based on the coincidence of the rebound response following glycinergic inhibition to the first harmonic of the pulse and a short-latency response to the 2nd-4th harmonics of its echo. Here, we report that further facilitative responses to pulse-echo pairs of thalamic delay-tuned neurons are mediated by glutamate receptors (NMDA and non-NMDA receptors), and that GABAergic inhibition shortens the duration of facilitative responses mediated by NMDA-receptors, without changing the delay tuning of thalamic delay-tuned neurons. Different from collicular delay-tuned neurons, thalamic ones respond much more to pulse-echo pairs than individual signal elements. The neural mechanisms involved in shaping thalamic delay-tuning support a model of hierarchical signal processing in the auditory system.

Two N-glycosylation Sites in the GluN1 Subunit Are Essential for Releasing N-methyl-d-aspartate (NMDA) Receptors from the Endoplasmic Reticulum.

NMDA receptors (NMDARs) comprise a subclass of neurotransmitter receptors whose surface expression is regulated at multiple levels, including processing in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, internalization, recycling, and degradation. With respect to early processing, NMDARs are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications, including phosphorylation and palmitoylation. However, the role of N-glycosylation, one of the most common posttranslational modifications, in regulating NMDAR processing has not been studied in detail. Using biochemistry, confocal and electron microscopy, and electrophysiology in conjunction with a lentivirus-based molecular replacement strategy, we found that NMDARs are released from the ER only when two asparagine residues in the GluN1 subunit (Asn-203 and Asn-368) are N-glycosylated. Although the GluN2A and GluN2B subunits are also N-glycosylated, their N-glycosylation sites do not appear to be essential for surface delivery of NMDARs. Furthermore, we found that removing N-glycans from native NMDARs altered the receptor affinity for glutamate. Our results suggest a novel mechanism by which neurons ensure that postsynaptic membranes contain sufficient numbers of functional NMDARs.

Mechanisms of left-right coordination in mammalian locomotor pattern generation circuits: a mathematical modeling view.

The locomotor gait in limbed animals is defined by the left-right leg coordination and locomotor speed. Coordination between left and right neural activities in the spinal cord controlling left and right legs is provided by commissural interneurons (CINs). Several CIN types have been genetically identified, including the excitatory V3 and excitatory and inhibitory V0 types. Recent studies demonstrated that genetic elimination of all V0 CINs caused switching from a normal left-right alternating activity to a left-right synchronized "hopping" pattern. Furthermore, ablation of only the inhibitory V0 CINs (V0D subtype) resulted in a lack of left-right alternation at low locomotor frequencies and retaining this alternation at high frequencies, whereas selective ablation of the excitatory V0 neurons (V0V subtype) maintained the left-right alternation at low frequencies and switched to a hopping pattern at high frequencies. To analyze these findings, we developed a simplified mathematical model of neural circuits consisting of four pacemaker neurons representing left and right, flexor and extensor rhythm-generating centers interacting via commissural pathways representing V3, V0D, and V0V CINs. The locomotor frequency was controlled by a parameter defining the excitation of neurons and commissural pathways mimicking the effects of N-methyl-D-aspartate on locomotor frequency in isolated rodent spinal cord preparations. The model demonstrated a typical left-right alternating pattern under control conditions, switching to a hopping activity at any frequency after removing both V0 connections, a synchronized pattern at low frequencies with alternation at high frequencies after removing only V0D connections, and an alternating pattern at low frequencies with hopping at high frequencies after removing only V0V connections. We used bifurcation theory and fast-slow decomposition methods to analyze network behavior in the above regimes and transitions between them. The model reproduced, and suggested explanation for, a series of experimental phenomena and generated predictions available for experimental testing.

Toward molecular mechanism of xenon anesthesia: a link to studies of xenon complexes with small aromatic molecules.

The present study illustrates the steps toward understanding molecular mechanism of xenon anesthesia by focusing on a link to the structures and spectra of intermolecular complexes of xenon with small aromatic molecules. A primary cause of xenon anesthesia is attributed to inhibition of N-methyl-D-aspartate (NMDA) receptors by an unknown mechanism. Following the results of quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) calculations we report plausible xenon action sites in the ligand binding domain of the NMDA receptor, which are due to interaction of xenon atoms with aromatic amino-acid residues. We rely in these calculations on computational protocols adjusted in combined experimental and theoretical studies of intermolecular complexes of xenon with phenol. Successful reproduction of vibrational shifts in molecular species upon complexation with xenon measured in low-temperature matrices allowed us to select a proper functional form in density functional theory (DFT) approach for use in QM subsystems, as well as to calibrate force field parameters for MD simulations. The results of molecular modeling show that xenon atoms can compete with agonists for a place in the corresponding protein cavity, thus indicating their active role in anesthetic action.

Deranged NMDAergic cortico-subthalamic transmission underlies parkinsonian motor deficits.

Parkinson's disease (PD) is the most prevalent hypokinetic movement disorder, and symptomatic PD pathogenesis has been ascribed to imbalances between the direct and indirect pathways in the basal ganglia circuitry. Here, we applied glutamate receptor blockers to the subthalamic nucleus (STN) of parkinsonian rats and evaluated locomotor behaviors via single-unit and local-field recordings. Using this model, we found that inhibition of NMDAergic cortico-subthalamic transmission ameliorates parkinsonian motor deficits without eliciting any vivid turning behavior and abolishes electrophysiological abnormalities, including excessive subthalamic bursts, cortico-subthalamic synchronization, and in situ beta synchronization in both the motor cortex and STN. Premotor cortex stimulation revealed that cortico-subthalamic transmission is deranged in PD and directly responsible for the excessive stimulation-dependent bursts and time-locked spikes in the STN, explaining the genesis of PD-associated pathological bursts and synchronization, respectively. Moreover, application of a dopaminergic agent via a microinfusion cannula localized the therapeutic effect to the STN, without correcting striatal dopamine deficiency. Finally, optogenetic overactivation and synchronization of cortico-subthalamic transmission alone sufficiently and instantaneously induced parkinsonian-associated locomotor dysfunction in normal mice. In addition to the classic theory emphasizing the direct-indirect pathways, our data suggest that deranged cortico-subthalamic transmission via the NMDA receptor also plays a central role in the pathophysiology of parkinsonian motor deficits.

The crystal structure of the adenylation enzyme VinN reveals a unique ß-amino acid recognition mechanism.

Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the ß-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of ß-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp(230) residue is used in the recognition of the ß-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a ß-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a ß-amino acid and provides new mechanistic insights into the selective recognition of ß-amino acids in this family of enzymes.

Enantioselective and rapid Rh-catalyzed arylation of N-tosyl- and N-nosylaldimines in methanol.

Enantiomerically enriched tosyl-protected diarylmethylamines were rapidly prepared by the asymmetric addition of arylboronic acids to N-tosylaldimines under mild conditions in the presence of a catalyst prepared in situ from Rh(I) and a chiral diene ligand. This methodology offers access to diarylmethylamines in good yields with excellent chiral purity at room temperature using MeOH as a solvent and NEt3 as a base. Its synthetic utility was demonstrated by the preparation of (S)-1-phenyl-1,2,3,4-tetrahydroisoquinoline (14), an antagonist of the N-methyl-d-aspartate (NMDA) receptor.

Mechanisms underlying subunit independence in pyramidal neuron dendrites.

Pyramidal neuron (PN) dendrites compartmentalize voltage signals and can generate local spikes, which has led to the proposal that their dendrites act as independent computational subunits within a multilayered processing scheme. However, when a PN is strongly activated, back-propagating action potentials (bAPs) sweeping outward from the soma synchronize dendritic membrane potentials many times per second. How PN dendrites maintain the independence of their voltage-dependent computations, despite these repeated voltage resets, remains unknown. Using a detailed compartmental model of a layer 5 PN, and an improved method for quantifying subunit independence that incorporates a more accurate model of dendritic integration, we first established that the output of each dendrite can be almost perfectly predicted by the intensity and spatial configuration of its own synaptic inputs, and is nearly invariant to the rate of bAP-mediated "cross-talk" from other dendrites over a 100-fold range. Then, through an analysis of conductance, voltage, and current waveforms within the model cell, we identify three biophysical mechanisms that together help make independent dendritic computation possible in a firing neuron, suggesting that a major subtype of neocortical neuron has been optimized for layered, compartmentalized processing under in-vivo-like spiking conditions.

Discovery of novel (1S)-(-)-verbenone derivatives with anti-oxidant and anti-ischemic effects.

A series of novel (1S)-(-)-verbenone derivatives was synthesized bearing a 4-styryl scaffold. The synthesized compounds were tested for their anti-oxidant, anti-excitotoxic, and anti-ischemic activities. These derivatives significantly reduced oxygen-glucose deprivation-induced neuronal injury and N-methyl-D-aspartic acid-evoked excitotoxicity in cortical neurons. Furthermore, compound 3f was identified as a potent anti-ischemic agent in an in vitro ischemic model, potentially due to the inhibition of N-methyl-D-aspartic acid-evoked excitotoxicity and oxidative/nitrosative stress.

A bionics chemical synapse.

Implementation of the current mode CMOS circuit for chemical synapses (AMPA and NMDA receptors) with dynamic change of glutamate as the neurotransmitter input is presented in this paper. Additionally, circuit realisation for receptor GABA(A) and GABA(B) with an electrical signal which symbolises γ-Aminobutyric Acid (GABA) perturbation is introduced. The chemical sensor for glutamate sensing is the modified ISFET with enzyme (glutamate oxidase) immobilisation. The measured results from these biomimetics chemical synapse circuits closely match with the simulation result from the mathematical model. The total power consumption of the whole chip (four chemical synapse circuits and all auxiliary circuits) is 168.3 µW. The total chip area is 3 mm(2) in 0.35-µm AMS CMOS technology.