Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

Morphing cholinesterase inhibitor amiridine into multipotent drugs for the treatment of Alzheimer's disease.

The search for novel drugs to address the medical needs of Alzheimer's disease (AD) is an ongoing process relying on the discovery of disease-modifying agents. Given the complexity of the disease, such an aim can be pursued by developing so-called multi-target directed ligands (MTDLs) that will impact the disease pathophysiology more comprehensively. Herewith, we contemplated the therapeutic efficacy of an amiridine drug acting as a cholinesterase inhibitor by converting it into a novel class of novel MTDLs. Applying the linking approach, we have paired amiridine as a core building block with memantine/adamantylamine, trolox, and substituted benzothiazole moieties to generate novel MTDLs endowed with additional properties like N-methyl-d-aspartate (NMDA) receptor affinity, antioxidant capacity, and anti-amyloid properties, respectively. The top-ranked amiridine-based compound 5d was also inspected by in silico to reveal the butyrylcholinesterase binding differences with its close structural analogue 5b. Our study provides insight into the discovery of novel amiridine-based drugs by broadening their target-engaged profile from cholinesterase inhibitors towards MTDLs with potential implications in AD therapy.

Phenoxytacrine derivatives: Low-toxicity neuroprotectants exerting affinity to ifenprodil-binding site and cholinesterase inhibition.

Tacrine (THA), a long withdrawn drug, is still a popular scaffold used in medicinal chemistry, mainly for its good reactivity and multi-targeted effect. However, THA-associated hepatotoxicity is still an issue and must be considered in drug discovery based on the THA scaffold. Following our previously identified hit compound 7-phenoxytacrine (7-PhO-THA), we systematically explored the chemical space with 30 novel derivatives, with a focus on low hepatotoxicity, anticholinesterase action, and antagonism at the GluN1/GluN2B subtype of the NMDA receptor. Applying the down-selection process based on in vitro and in vivo pharmacokinetic data, two candidates, I-52 and II-52, selective GluN1/GluN2B inhibitors thanks to the interaction with the ifenprodil-binding site, have entered in vivo pharmacodynamic studies. Finally, compound I-52, showing only minor affinity to AChE, was identified as a lead candidate with favorable behavioral and neuroprotective effects using open-field and prepulse inhibition tests, along with scopolamine-based behavioral and NMDA-induced hippocampal lesion models. Our data show that compound I-52 exhibits low toxicity often associated with NMDA receptor ligands, and low hepatotoxicity, often related to THA-based compounds.

Structure-Guided Design of N-Methylpropargylamino-Quinazoline Derivatives as Multipotent Agents for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a complex disease with an unknown etiology. Available treatments, limited to cholinesterase inhibitors and N-methyl-d-aspartate receptor (NMDAR) antagonists, provide symptomatic relief only. As single-target therapies have not proven effective, rational specific-targeted combination into a single molecule represents a more promising approach for treating AD, and is expected to yield greater benefits in alleviating symptoms and slowing disease progression. In the present study, we designed, synthesized, and biologically evaluated 24 novel N-methylpropargylamino-quinazoline derivatives. Initially, compounds were thoroughly inspected by in silico techniques determining their oral and CNS availabilities. We tested, in vitro, the compounds' effects on cholinesterases and monoamine oxidase A/B (MAO-A/B), as well as their impacts on NMDAR antagonism, dehydrogenase activity, and glutathione levels. In addition, we inspected selected compounds for their cytotoxicity on undifferentiated and differentiated neuroblastoma SH-SY5Y cells. We collectively highlighted II-6h as the best candidate endowed with a selective MAO-B inhibition profile, NMDAR antagonism, an acceptable cytotoxicity profile, and the potential to permeate through BBB. The structure-guided drug design strategy applied in this study imposed a novel concept for rational drug discovery and enhances our understanding on the development of novel therapeutic agents for treating AD.

Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes.

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.

The pathogenic N650K variant in the GluN1 subunit regulates the trafficking, conductance, and pharmacological properties of NMDA receptors.

N-methyl-D-aspartate receptors (NMDARs) play an essential role in excitatory neurotransmission in the mammalian brain, and their physiological importance is underscored by the large number of pathogenic mutations that have been identified in the receptor's GluN subunits and associated with a wide range of diseases and disorders. Here, we characterized the functional and pharmacological effects of the pathogenic N650K variant in the GluN1 subunit, which is associated with developmental delay and seizures. Our microscopy experiments showed that when expressed in HEK293 cells (from ATCC®), the GluN1-N650K subunit increases the surface expression of both GluN1/GluN2A and GluN1/GluN2B receptors, but not GluN1/GluN3A receptors, consistent with increased surface expression of the GluN1-N650K subunit expressed in hippocampal neurons (from embryonic day 18 of Wistar rats of both sexes). Using electrophysiology, we found that the GluN1-N650K variant increases the potency of GluN1/GluN2A receptors to both glutamate and glycine but decreases the receptor's conductance and open probability. In addition, the GluN1-N650K subunit does not form functional GluN1/GluN2B receptors but does form fully functional GluN1/GluN3A receptors. Moreover, in the presence of extracellular Mg2+, GluN1-N650K/GluN2A receptors have a similar and increased response to ketamine and memantine, respectively, while the effect of both drugs had markedly slower onset and offset compared to wild-type GluN1/GluN2A receptors. Finally, we found that expressing the GluN1-N650K subunit in hippocampal neurons reduces excitotoxicity, and memantine shows promising neuroprotective effects in neurons expressing either wild-type GluN1 or the GluN1-N650K subunit. This study provides the functional and pharmacological characterization of NMDARs containing the GluN1-N650K variant.

Follistatin-like 1 and its paralogs in heart development and cardiovascular disease.

Cardiovascular diseases (CVDs) are a group of disorders affecting the heart and blood vessels and a leading cause of death worldwide. Thus, there is a need to identify new cardiokines that may protect the heart from damage as reported in GBD 2017 Causes of Death Collaborators (2018) (The Lancet 392:1736-1788). Follistatin-like 1 (FSTL1) is a cardiokine that is highly expressed in the heart and released to the serum after cardiac injury where it is associated with CVD and predicts poor outcome. The action of FSTL1 likely depends not only on the tissue source but also post-translation modifications that are target tissue- and cell-specific. Animal studies examining the effect of FSTL1 in various models of heart disease have exploded over the past 15 years and primarily report a protective effect spanning from inhibiting inflammation via transforming growth factor, preventing remodeling and fibrosis to promoting angiogenesis and hypertrophy. A better understanding of FSTL1 and its homologs is needed to determine whether this protein could be a useful novel biomarker to predict poor outcome and death and whether it has therapeutic potential. The aim of this review is to provide a comprehensive description of the literature for this family of proteins in order to better understand their role in normal physiology and CVD.

Pregnane-based steroids are novel positive NMDA receptor modulators that may compensate for the effect of loss-of-function disease-associated GRIN mutations.

BACKGROUND AND PURPOSE: N-methyl-D-aspartate receptors (NMDARs) play a critical role in synaptic plasticity, and mutations in human genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. Compounds with a positive allosteric effect are thought to compensate for reduced receptor function. EXPERIMENTAL APPROACH: We have used whole-cell patch-clamp electrophysiology on recombinant rat NMDARs and human variants found in individuals with neuropsychiatric disorders, in combination with in silico modelling, to explore the site of action of novel epipregnanolone-based NMDAR modulators. KEY RESULTS: Analysis of the action of 4-(20-oxo-5ß-pregnan-3ß-yl) butanoic acid (EPA-But) at the NMDAR indicates that the effect of this steroid with a "bent" structure is different from that of cholesterol and oxysterols and shares a disuse-dependent mechanism of NMDAR potentiation with the "planar" steroid 20-oxo-pregn-5-en-3ß-yl sulfate (PE-S). The potentiating effects of EPA-But and PE-S are additive. Alanine scan mutagenesis identified residues that reduce the potentiating effect of EPA-But. No correlation was found between the effects of EPA-But and PE-S at mutated receptors that were less sensitive to either steroid. The relative degree of potentiation induced by the two steroids also differed in human NMDARs carrying rare variants of hGluN1 or hGluN2B subunits found in individuals with neuropsychiatric disorders, including intellectual disability, epilepsy, developmental delay, and autism spectrum disorder. CONCLUSION AND IMPLICATIONS: Our results show novel sites of action for pregnanolones at the NMDAR and provide an opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with glutamatergic system hypofunction.

Variants in the MIPEP gene presenting with complex neurological phenotype without cardiomyopathy, impair OXPHOS protein maturation and lead to a reduced OXPHOS abundance in patient cells.

Most mitochondrial proteins are synthesized in the cytosol and targeted to mitochondria via N-terminal mitochondrial targeting signals (MTS) that are proteolytically removed upon import. Sometimes, MTS removal is followed by a cleavage of an octapeptide by the mitochondrial intermediate peptidase (MIP), encoded by the MIPEP gene. Previously, MIPEP variants were linked to four cases of multisystemic disorder presenting with cardiomyopathy, developmental delay, hypotonia and infantile lethality. We report here a patient carrying compound heterozygous MIPEP variants-one was not previously linked to mitochondrial disease-who did not have cardiomyopathy and who is alive at the age of 20 years. This patient had developmental delay, global hypotonia, mild optic neuropathy and mild ataxia. Functional characterization of patient fibroblasts and HEK293FT cells carrying MIPEP hypomorphic alleles demonstrated that deficient MIP activity was linked to impaired post-import processing of subunits from four of the five OXPHOS complexes and decreased abundance and activity of some of these complexes in human cells possibly underlying the development of mitochondrial disease. Thus, our work expands the genetic and clinical spectrum of MIPEP-linked disease and establishes MIP as an important regulator of OXPHOS biogenesis and function in human cells.

Familial occurrence of skeletal developmental anomalies as a reflection of biological relationships in a genealogically documented Central European sample (19th to 20th centuries).

Skeletal developmental anomalies (SDA) are a subject of constant interest across scientific disciplines, but still mostly as isolates and curiosities. The aim of this study was to find out to what extent the occurrence of SDA reflects documented biological relationships. The skeletal remains of 34 individuals with known genealogical data were available, members of one family over four generations (19th to 20th centuries, Bohemia, Czech Republic), including some inbred individuals. The occurrence of 89 SDA was assessed on the basis of scopic morphological evaluation and X-ray and CT examinations. The degree of similarity between individuals was calculated using a "similarity coefficient" (SC). A linear model was used to test the relationship between positive values of the SC and the relatedness of biologically related individuals. Simultaneously, based on population frequencies of the evaluated anomalies, those that could be considered familial were recorded. A statistically significant relationship between morphological similarity and the biological distance between individuals was found. The greatest similarity was found among close relatives such as parents and children, siblings, or grandparents and grandchildren. The effect of increased consanguinity on the occurrence of anomalies was not confirmed, however. Seventeen SDA shared by closely related individuals were found in the sample, supporting the documented family relationships among them. Eleven of these were selected as possibly familial, but only five were statistically significant: an elongated styloid process, a cervical block vertebrae (arch, facet joints), hamate hamulus aplasia, anteater nose sign, and incomplete fusion of the S1 spinous process. There were also 28 cases of individual occurrences of 17 different SDA, without connection to the documented relationships between individuals.

Validation and Update of the Lémann Index to Measure Cumulative Structural Bowel Damage in Crohn's Disease.

BACKGROUND & AIMS: The Lémann Index is a tool measuring cumulative structural bowel damage in Crohn's disease (CD). We reported on its validation and updating. METHODS: This was an international, multicenter, prospective, cross-sectional observational study. At each center, 10 inclusions, stratified by CD duration and location, were planned. For each patient, the digestive tract was divided into 4 organs, upper tract, small bowel, colon/rectum, anus, and subsequently into segments, explored systematically by magnetic resonance imaging and by endoscopies in relation to disease location. For each segment, investigators retrieved information on previous surgical procedures, identified predefined strictures and penetrating lesions of maximal severity (grades 1-3) at each organ investigational method (gastroenterologist and radiologist for magnetic resonance imaging), provided segmental damage evaluation ranging from 0.0 to 10.0 (complete resection). Organ resection-free cumulative damage evaluation was then calculated from the sum of segmental damages. Then investigators provided a 0-10 global damage evaluation from the 4-organ standardized cumulative damage evaluations. Simple linear regressions of investigator damage evaluations on their corresponding Lémann Index were studied, as well as calibration plots. Finally, updated Lémann Index was derived through multiple linear mixed models applied to combined development and validation samples. RESULTS: In 15 centers, 134 patients were included. Correlation coefficients between investigator damage evaluations and Lémann Indexes were >0.80. When analyzing data in 272 patients from both samples and 27 centers, the unbiased correlation estimates were 0.89, 0,97, 0,94, 0.81, and 0.91 for the 4 organs and globally, and stable when applied to one sample or the other. CONCLUSIONS: The updated Lémann Index is a well-established index to assess cumulative bowel damage in CD that can be used in epidemiological studies and disease modification trials.

The Extracellular Domains of GluN Subunits Play an Essential Role in Processing NMDA Receptors in the ER.

N-methyl-D-aspartate receptors (NMDARs) belong to a family of ionotropic glutamate receptors that play essential roles in excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). Functional NMDARs consist of heterotetramers comprised of GluN1, GluN2A-D, and/or GluN3A-B subunits, each of which contains four membrane domains (M1 through M4), an intracellular C-terminal domain, a large extracellular N-terminal domain composed of the amino-terminal domain and the S1 segment of the ligand-binding domain (LBD), and an extracellular loop between M3 and M4, which contains the S2 segment of the LBD. Both the number and type of NMDARs expressed at the cell surface are regulated at several levels, including their translation and posttranslational maturation in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, lateral diffusion in the plasma membrane, and internalization and degradation. This review focuses on the roles played by the extracellular regions of GluN subunits in ER processing. Specifically, we discuss the presence of ER retention signals, the integrity of the LBD, and critical N-glycosylated sites and disulfide bridges within the NMDAR subunits, each of these steps must pass quality control in the ER in order to ensure that only correctly assembled NMDARs are released from the ER for subsequent processing and trafficking to the surface. Finally, we discuss the effect of pathogenic missense mutations within the extracellular domains of GluN subunits with respect to ER processing of NMDARs.

Structure-activity relationships of dually-acting acetylcholinesterase inhibitors derived from tacrine on N-methyl-d-Aspartate receptors.

Tacrine is a classic drug whose efficacy against neurodegenerative diseases is still shrouded in mystery. It seems that besides its inhibitory effect on cholinesterases, the clinical benefit is co-determined by NMDAR-antagonizing activity. Our previous data showed that the direct inhibitory effect of tacrine, as well as its 7-methoxy derivative (7-MEOTA), is ensured via a "foot-in-the-door" open-channel blockage, and that interestingly both tacrine and 7-MEOTA are slightly more potent at the GluN1/GluN2A receptors when compared with the GluN1/GluN2B receptors. Here, we report that in a series of 30 novel tacrine derivatives, designed for assessment of structure-activity relationship, blocking efficacy differs among different compounds and receptors using electrophysiology with HEK293 cells expressing the defined types of NMDARs. Selected compounds (4 and 5) potently inhibited both GluN1/GluN2A and GluN1/GluN2B receptors; other compounds (7 and 23) more effectively inhibited the GluN1/GluN2B receptors; or the GluN1/GluN2A receptors (21 and 28). QSAR study revealed statistically significant model for the data obtained for inhibition of GluN1/Glu2B at -60 mV expressed as IC50 values, and for relative inhibition of GluN1/Glu2A at +40 mV caused by a concentration of 100 µM. The models can be utilized for a ligand-based virtual screening to detect potential candidates for inhibition of GluN1/Glu2A and/or GluN1/Glu2B subtypes. Using in vivo experiments in rats we observed that unlike MK-801, the tested novel compounds did not induce hyperlocomotion in open field, and also did not impair prepulse inhibition of startle response, suggesting minimal induction of psychotomimetic side effects. We conclude that tacrine derivatives are promising compounds since they are centrally available subtype-specific inhibitors of the NMDARs without detrimental behavioral side-effects.

Specific pathogenic mutations in the M3 domain of the GluN1 subunit regulate the surface delivery and pharmacological sensitivity of NMDA receptors.

N-methyl-d-aspartate receptors (NMDARs) play an essential role in regulating glutamatergic neurotransmission. Recently, pathogenic missense mutations were identified in genes encoding NMDAR subunits; however, their effect on NMDAR activity is often poorly understood. Here, we examined whether three previously identified pathogenic mutations (M641I, A645S, and Y647S) in the M3 domain of the GluN1 subunit affect the receptor's surface delivery, agonist sensitivity, Mg2+ block, and/or inhibition by the FDA-approved NMDAR blocker memantine. When expressed in HEK293 cells, we found reduced surface expression of GluN1-M641I/GluN2A, GluN1-Y647S/GluN2A, and GluN1-Y647S/GluN2B receptors; other mutation-bearing NMDAR combinations, including GluN1/GluN3A receptors, were expressed at normal surface levels. When expressed in rat hippocampal neurons, we consistently found reduced surface expression of the GluN1-M641I and GluN1-Y647S subunits when compared with wild-type GluN1 subunit. At the functional level, we found that GluN1-M641I/GluN2 and GluN1-A645S/GluN2 receptors expressed in HEK293 cells have wild-type EC50 values for both glutamate and glycine; in contrast, GluN1-Y647S/GluN2 receptors do not produce glutamate-induced currents. In the presence of a physiological concentration of Mg2+, we found that GluN1-M641I/GluN2 receptors have a lower memantine IC50 and slower offset kinetics, whereas GluN1-A645S/GluN2 receptors have a higher memantine IC50 and faster offset kinetics when compared to wild-type receptors. Finally, we found that memantine was the most neuroprotective in hippocampal neurons expressing GluN1-M641I subunits, followed by neurons expressing wild-type GluN1 and then GluN1-A645S subunits in an NMDA-induced excitotoxicity assay. These results indicate that specific pathogenic mutations in the M3 domain of the GluN1 subunit differentially affect the trafficking and functional properties of NMDARs.

7-phenoxytacrine is a dually acting drug with neuroprotective efficacy in vivo.

N-methyl-D-aspartaterecepro receptor (NMDARs) are a subclass of glutamate receptors, which play an essential role in excitatory neurotransmission, but their excessive overactivation by glutamate leads to excitotoxicity. NMDARs are hence a valid pharmacological target for the treatment of neurodegenerative disorders; however, novel drugs targeting NMDARs are often associated with specific psychotic side effects and abuse potential. Motivated by currently available treatment against neurodegenerative diseases involving the inhibitors of acetylcholinesterase (AChE) and NMDARs, administered also in combination, we developed a dually-acting compound 7-phenoxytacrine (7-PhO-THA) and evaluated its neuropsychopharmacological and drug-like properties for potential therapeutic use. Indeed, we have confirmed the dual potency of 7-PhO-THA, i.e. potent and balanced inhibition of both AChE and NMDARs. We discovered that it selectively inhibits the GluN1/GluN2B subtype of NMDARs via an ifenprodil-binding site, in addition to its voltage-dependent inhibitory effect at both GluN1/GluN2A and GluN1/GluN2B subtypes of NMDARs. Furthermore, whereas NMDA-induced lesion of the dorsal hippocampus confirmed potent anti-excitotoxic and neuroprotective efficacy, behavioral observations showed also a cholinergic component manifesting mainly in decreased hyperlocomotion. From the point of view of behavioral side effects, 7-PhO-THA managed to avoid these, notably those analogous to symptoms of schizophrenia. Thus, CNS availability and the overall behavioral profile are promising for subsequent investigation of therapeutic use.

Pursuing the Complexity of Alzheimer's Disease: Discovery of Fluoren-9-Amines as Selective Butyrylcholinesterase Inhibitors and N-Methyl-d-Aspartate Receptor Antagonists.

Alzheimer's disease (AD) is a complex disorder with unknown etiology. Currently, only symptomatic therapy of AD is available, comprising cholinesterase inhibitors and N-methyl-d-aspartate (NMDA) receptor antagonists. Drugs targeting only one pathological condition have generated only limited efficacy. Thus, combining two or more therapeutic interventions into one molecule is believed to provide higher benefit for the treatment of AD. In the presented study, we designed, synthesized, and biologically evaluated 15 novel fluoren-9-amine derivatives. The in silico prediction suggested both the oral availability and permeation through the blood-brain barrier (BBB). An initial assessment of the biological profile included determination of the cholinesterase inhibition and NMDA receptor antagonism at the GluN1/GluN2A and GluN1/GluN2B subunits, along with a low cytotoxicity profile in the CHO-K1 cell line. Interestingly, compounds revealed a selective butyrylcholinesterase (BChE) inhibition pattern with antagonistic activity on the NMDARs. Their interaction with butyrylcholinesterase was elucidated by studying enzyme kinetics for compound 3c in tandem with the in silico docking simulation. The docking study showed the interaction of the tricyclic core of new derivatives with Trp82 within the anionic site of the enzyme in a similar way as the template drug tacrine. From the kinetic analysis, it is apparent that 3c is a competitive inhibitor of BChE.

The pathogenic S688Y mutation in the ligand-binding domain of the GluN1 subunit regulates the properties of NMDA receptors.

Although numerous pathogenic mutations have been identified in various subunits of N-methyl-D-aspartate receptors (NMDARs), ionotropic glutamate receptors that are central to glutamatergic neurotransmission, the functional effects of these mutations are often unknown. Here, we combined in silico modelling with microscopy, biochemistry, and electrophysiology in cultured HEK293 cells and hippocampal neurons to examine how the pathogenic missense mutation S688Y in the GluN1 NMDAR subunit affects receptor function and trafficking. We found that the S688Y mutation significantly increases the EC50 of both glycine and D-serine in GluN1/GluN2A and GluN1/GluN2B receptors, and significantly slows desensitisation of GluN1/GluN3A receptors. Moreover, the S688Y mutation reduces the surface expression of GluN3A-containing NMDARs in cultured hippocampal neurons, but does not affect the trafficking of GluN2-containing receptors. Finally, we found that the S688Y mutation reduces Ca2+ influx through NMDARs and reduces NMDA-induced excitotoxicity in cultured hippocampal neurons. These findings provide key insights into the molecular mechanisms that underlie the regulation of NMDAR subtypes containing pathogenic mutations.

N-linked glycosylation of the mGlu7 receptor regulates the forward trafficking and transsynaptic interaction with Elfn1.

Metabotropic glutamate receptor 7 (mGlu7) regulates neurotransmitter release at the presynaptic active zone in the mammalian brain. The regulation of mGlu7 trafficking into and out of the plasma membrane by binding proteins within the C-terminal region of mGlu7 governs the bidirectional synaptic plasticity. However, the functional importance of the extracellular domain of mGlu7 has not yet been characterized. N-glycosylation is an abundant posttranslational modification that plays crucial roles in protein folding and forward trafficking, but the role of N-glycosylation in mGlu7 function remains unknown. In this study, we find that mGlu7 is N-glycosylated at four asparagine residues in heterologous cells and rat cultured neurons. We demonstrate that N-glycosylation is essential for forward transport and surface expression of mGlu7. Deglycosylated mGlu7 is retained in the ER, obstructing expression on the cell surface, and is degraded through the autophagolysosomal degradation pathway. In addition, we identify the binding domain of mGlu7 to Elfn1, a transsynaptic adhesion protein. We find that N-glycosylation of mGlu7 promotes its interaction with Elfn1, thereby enabling proper localization and stable surface expression of mGlu7 at the presynaptic active zone. These findings provide evidence that N-glycans act to modulate the surface expression, stability, and function of mGlu7.

Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission.

Cholesterol is a structural component of cellular membranes particularly enriched in synapses but its role in synaptic transmission remains poorly understood. We used rat hippocampal cultures and their acute cholesterol depletion by methyl-ß-cyclodextrin as a tool to describe the physiological role of cholesterol in glutamatergic synaptic transmission. Cholesterol proved to be a key molecule for the function of synapses as its depletion resulted in a significant reduction of both NMDA receptor (NMDAR) and AMPA/kainate receptor-mediated evoked excitatory postsynaptic currents (eEPSCs), by 94% and 72%, respectively. We identified two presynaptic and two postsynaptic steps of synaptic transmission which are modulated by cholesterol and explain together the above-mentioned reduction of eEPSCs. In the postsynapse, we show that physiological levels of cholesterol are important for maintaining the normal probability of opening of NMDARs and for keeping NMDARs localized in synapses. In the presynapse, our results favour the hypothesis of a role of cholesterol in the propagation of axonal action potentials. Finally, cholesterol is a negative modulator of spontaneous presynaptic glutamate release. Our study identifies cholesterol as an important endogenous regulator of synaptic transmission and provides insight into molecular mechanisms underlying the neurological manifestation of diseases associated with impaired cholesterol synthesis or decomposition.

Structural features in the glycine-binding sites of the GluN1 and GluN3A subunits regulate the surface delivery of NMDA receptors.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an essential role in mediating excitatory neurotransmission in the mammalian central nervous system (CNS). Functional NMDARs are tetramers composed of GluN1, GluN2A-D, and/or GluN3A-B subunits, giving rise to a wide variety of NMDAR subtypes with unique functional properties. Here, we examined the surface delivery and functional properties of NMDARs containing mutations in the glycine-binding sites in GluN1 and GluN3A subunits expressed in mammalian cell lines and primary rat hippocampal neurons. We found that the structural features of the glycine-binding sites in both GluN1 and GluN3A subunits are correlated with receptor forward trafficking to the cell surface. In addition, we found that a potentially clinically relevant mutation in the glycine-binding site of the human GluN3A subunit significantly reduces surface delivery of NMDARs. Taken together, these findings provide novel insight into how NMDARs are regulated by their glycine-binding sites and may provide important information regarding the role of NMDARs in both physiological and pathophysiological processes in the mammalian CNS.