L-serine treatment in patients with GRIN-related encephalopathy: a phase 2A, non-randomized study.

GRIN-related disorders are rare developmental encephalopathies with variable manifestations and limited therapeutic options. Here, we present the first non-randomized, open-label, single-arm trial (NCT04646447) designed to evaluate the tolerability and efficacy of L-serine in children with GRIN genetic variants leading to loss-of-function. In this phase 2A trial, patients aged 2-18 years with GRIN loss-of-function pathogenic variants received L-serine for 52 weeks. Primary end points included safety and efficacy by measuring changes in the Vineland Adaptive Behavior Scales, Bayley Scales, age-appropriate Wechsler Scales, Gross Motor Function-88, Sleep Disturbance Scale for Children, Pediatric Quality of Life Inventory, Child Behavior Checklist and the Caregiver-Teacher Report Form following 12 months of treatment. Secondary outcomes included seizure frequency and intensity reduction and EEG improvement. Assessments were performed 3 months and 1 day before starting treatment and 1, 3, 6 and 12 months after beginning the supplement. Twenty-four participants were enrolled (13 males/11 females, mean age 9.8 years, SD 4.8), 23 of whom completed the study. Patients had GRIN2B, GRIN1 and GRIN2A variants (12, 6 and 5 cases, respectively). Their clinical phenotypes showed 91% had intellectual disability (61% severe), 83% had behavioural problems, 78% had movement disorders and 58% had epilepsy. Based on the Vineland Adaptive Behavior Composite standard scores, nine children were classified as mildly impaired (cut-off score > 55), whereas 14 were assigned to the clinically severe group. An improvement was detected in the Daily Living Skills domain (P = 0035) from the Vineland Scales within the mild group. Expressive (P = 0.005), Personal (P = 0.003), Community (P = 0.009), Interpersonal (P = 0.005) and Fine Motor (P = 0.031) subdomains improved for the whole cohort, although improvement was mostly found in the mild group. The Growth Scale Values in the Cognitive subdomain of the Bayley-III Scale showed a significant improvement in the severe group (P = 0.016), with a mean increase of 21.6 points. L-serine treatment was associated with significant improvement in the median Gross Motor Function-88 total score (P = 0.002) and the mean Pediatric Quality of Life total score (P = 0.00068), regardless of severity. L-serine normalized the EEG pattern in five children and the frequency of seizures in one clinically affected child. One patient discontinued treatment due to irritability and insomnia. The trial provides evidence that L-serine is a safe treatment for children with GRIN loss-of-function variants, having the potential to improve adaptive behaviour, motor function and quality of life, with a better response to the treatment in mild phenotypes.

Disease-associated GRIN protein truncating variants trigger NMDA receptor loss-of-function.

De novo GRIN variants, encoding for the ionotropic glutamate NMDA receptor subunits, have been recently associated with GRIN-related disorders, a group of rare paediatric encephalopathies. Current investigational and clinical efforts are focused to functionally stratify GRIN variants, towards precision therapies of this primary disturbance of glutamatergic transmission that affects neuronal function and brain. In the present study, we aimed to comprehensively delineate the functional outcomes and clinical phenotypes of GRIN protein truncating variants (PTVs)-accounting for ~20% of disease-associated GRIN variants-hypothetically provoking NMDAR hypofunctionality. To tackle this question, we created a comprehensive GRIN PTVs variants database compiling a cohort of nine individuals harbouring GRIN PTVs, together with previously identified variants, to build-up an extensive GRIN PTVs repertoire composed of 293 unique variants. Genotype-phenotype correlation studies were conducted, followed by cell-based assays of selected paradigmatic GRIN PTVs and their functional annotation. Genetic and clinical phenotypes meta-analysis revealed that heterozygous GRIN1, GRIN2C, GRIN2D, GRIN3A and GRIN3B PTVs are non-pathogenic. In contrast, heterozygous GRIN2A and GRIN2B PTVs are associated with specific neurological clinical phenotypes in a subunit- and domain-dependent manner. Mechanistically, cell-based assays showed that paradigmatic pathogenic GRIN2A and GRIN2B PTVs result on a decrease of NMDAR surface expression and NMDAR-mediated currents, ultimately leading to NMDAR functional haploinsufficiency. Overall, these findings contribute to delineate GRIN PTVs genotype-phenotype association and GRIN variants stratification. Functional studies showed that GRIN2A and GRIN2B pathogenic PTVs trigger NMDAR hypofunctionality, and thus accelerate therapeutic decisions for this neurodevelopmental condition.

Metabolic characterization of neurogenetic disorders involving glutamatergic neurotransmission.

The study of inborn errors of neurotransmission has been mostly focused on monoamine disorders, GABAergic and glycinergic defects. The study of the glutamatergic synapse using the same approach than classic neurotransmitter disorders is challenging due to the lack of biomarkers in the CSF. A metabolomic approach can provide both insight into their molecular basis and outline novel therapeutic alternatives. We have performed a semi-targeted metabolomic analysis on CSF samples from 25 patients with neurogenetic disorders with an important expression in the glutamatergic synapse and 5 controls. Samples from patients diagnosed with MCP2, CDKL5-, GRINpathies and STXBP1-related encephalopathies were included. We have performed univariate (UVA) and multivariate statistical analysis (MVA), using Wilcoxon rank-sum test, principal component analysis (PCA), and OPLS-DA. By using the results of both analyses, we have identified the metabolites that were significantly altered and that were important in clustering the respective groups. On these, we performed pathway- and network-based analyses to define which metabolic pathways were possibly altered in each pathology. We have observed alterations in the tryptophan and branched-chain amino acid metabolism pathways, which interestingly converge on LAT1 transporter-dependency to cross the blood-brain barrier (BBB). Analysis of the expression of LAT1 transporter in brain samples from a mouse model of Rett syndrome (MECP2) revealed a decrease in the transporter expression, that was already noticeable at pre-symptomatic stages. The study of the glutamatergic synapse from this perspective advances the understanding of their pathophysiology, shining light on an understudied feature as is their metabolic signature.

High Resolution Ambient MS Imaging of Biological Samples by Desorption Electro-Flow Focussing Ionization.

In this study, we examine the suitability of desorption electro-flow focusing ionization (DEFFI) for mass spectrometry imaging (MSI) of biological tissue. We also compare the performance of desorption electrospray ionization (DESI) with and without the flow focusing setup. The main potential advantages of applying the flow focusing mechanism in DESI is its rotationally symmetric electrospray jet, higher intensity, more controllable parameters, and better portability due to the robustness of the sprayer. The parameters for DEFFI have therefore been thoroughly optimized, primarily for spatial resolution but also for intensity. Once the parameters have been optimized, DEFFI produces similar images to the existing DESI. MS images for mouse brain samples, acquired at a nominal pixel size of 50 µm, are comparable for both DESI setups, albeit the new sprayer design yields better sensitivity. Furthermore, the two methods are compared with regard to spectral intensity as well as the area of the desorbed crater on rhodamine-coated slides. Overall, the implementation of a flow focusing mechanism in DESI is shown to be highly suitable for imaging biological tissue and has potential to overcome some of the shortcomings experienced with the current geometrical design of DESI.

GRIN database: A unified and manually curated repertoire of GRIN variants.

Glutamatergic neurotransmission is crucial for brain development, wiring neuronal function, and synaptic plasticity mechanisms. Recent genetic studies showed the existence of autosomal dominant de novo GRIN gene variants associated with GRIN-related disorders (GRDs), a rare pediatric neurological disorder caused by N-methyl- d-aspartate receptor (NMDAR) dysfunction. Notwithstanding, GRIN variants identification is exponentially growing and their clinical, genetic, and functional annotations remain highly fragmented, representing a bottleneck in GRD patient's stratification. To shorten the gap between GRIN variant identification and patient stratification, we present the GRIN database (GRINdb), a publicly available, nonredundant, updated, and curated database gathering all available genetic, functional, and clinical data from more than 4000 GRIN variants. The manually curated GRINdb outputs on a web server, allowing query and retrieval of reported GRIN variants, and thus representing a fast and reliable bioinformatics resource for molecular clinical advice. Furthermore, the comprehensive mapping of GRIN variants' genetic and clinical information along NMDAR structure revealed important differences in GRIN variants' pathogenicity and clinical phenotypes, shedding light on GRIN-specific fingerprints. Overall, the GRINdb and web server is a resource for molecular stratification of GRIN variants, delivering clinical and investigational insights into GRDs. GRINdb is accessible at http://lmc.uab.es/grindb.

Paradigmatic De Novo GRIN1 Variants Recapitulate Pathophysiological Mechanisms Underlying GRIN1-Related Disorder Clinical Spectrum.

BACKGROUND: GRIN-related disorders (GRD), the so-called grinpathies, is a group of rare encephalopathies caused by mutations affecting GRIN genes (mostly GRIN1, GRIN2A and GRIN2B genes), which encode for the GluN subunit of the N-methyl D-aspartate (NMDA) type ionotropic glutamate receptors. A growing number of functional studies indicate that GRIN-encoded GluN1 subunit disturbances can be dichotomically classified into gain- and loss-of-function, although intermediate complex scenarios are often present. METHODS: In this study, we aimed to delineate the structural and functional alterations of GRIN1 disease-associated variants, and their correlations with clinical symptoms in a Spanish cohort of 15 paediatric encephalopathy patients harbouring these variants. RESULTS: Patients harbouring GRIN1 disease-associated variants have been clinically deeply-phenotyped. Further, using computational and in vitro approaches, we identified different critical checkpoints affecting GluN1 biogenesis (protein stability, subunit assembly and surface trafficking) and/or NMDAR biophysical properties, and their association with GRD clinical symptoms. CONCLUSIONS: Our findings show a strong correlation between GRIN1 variants-associated structural and functional outcomes. This structural-functional stratification provides relevant insights of genotype-phenotype association, contributing to future precision medicine of GRIN1-related encephalopathies.

Metabotropic glutamate type 5 receptor requires contactin-associated protein 1 to control memory formation.

The hippocampus is a key brain region for memory formation. Metabotropic glutamate type 5 receptors (mGlu5R) are strongly expressed in CA1 pyramidal neurons and fine-tune synaptic plasticity. Accordingly, mGlu5R pharmacological manipulation may represent an attractive therapeutic strategy to manage hippocampal-related neurological disorders. Here, by means of a membrane yeast two-hybrid screening, we identified contactin-associated protein 1 (Caspr1), a type I transmembrane protein member of the neurexin family, as a new mGlu5R partner. We report that mGlu5R and Caspr1 co-distribute and co-assemble both in heterologous expression systems and in rat brain. Furthermore, downregulation of Caspr1 in rat hippocampal primary cultures decreased mGlu5R-mediated signaling. Finally, silencing Caspr1 expression in the hippocampus impaired the impact of mGlu5R on spatial memory. Our results indicate that Caspr1 plays a pivotal role controlling mGlu5R function in hippocampus-dependent memory formation. Hence, this new protein-protein interaction may represent novel target for neurological disorders affecting hippocampal glutamatergic neurotransmission.

Comprehensive Analysis of GABAA-A1R Developmental Alterations in Rett Syndrome: Setting the Focus for Therapeutic Targets in the Time Frame of the Disease.

Rett syndrome, a serious neurodevelopmental disorder, has been associated with an altered expression of different synaptic-related proteins and aberrant glutamatergic and γ-aminobutyric acid (GABA)ergic neurotransmission. Despite its severity, it lacks a therapeutic option. Through this work we aimed to define the relationship between MeCP2 and GABAA.-A1 receptor expression, emphasizing the time dependence of such relationship. For this, we analyzed the expression of the ionotropic receptor subunit in different MeCP2 gene-dosage and developmental conditions, in cells lines, and in primary cultured neurons, as well as in different developmental stages of a Rett mouse model. Further, RNAseq and systems biology analysis was performed from post-mortem brain biopsies of Rett patients. We observed that the modulation of the MeCP2 expression in cellular models (both Neuro2a (N2A) cells and primary neuronal cultures) revealed a MeCP2 positive effect on the GABAA.-A1 receptor subunit expression, which did not occur in other proteins such as KCC2 (Potassium-chloride channel, member 5). In the Mecp2+/- mouse brain, both the KCC2 and GABA subunits expression were developmentally regulated, with a decreased expression during the pre-symptomatic stage, while the expression was variable in the adult symptomatic mice. Finally, the expression of the gamma-aminobutyric acid (GABA) receptor-related synaptic proteins from the postmortem brain biopsies of two Rett patients was evaluated, specifically revealing the GABA A1R subunit overexpression. The identification of the molecular changes along with the Rett syndrome prodromic stages strongly endorses the importance of time frame when addressing this disease, supporting the need for a neurotransmission-targeted early therapeutic intervention.

Normalization of Dyrk1A expression by AAV2/1-shDyrk1A attenuates hippocampal-dependent defects in the Ts65Dn mouse model of Down syndrome.

The cognitive dysfunctions of Down Syndrome (DS) individuals are the most disabling alterations caused by the trisomy of human chromosome 21 (HSA21). In trisomic Ts65Dn mice, a genetic model for DS, the overexpression of HSA21 homologous genes has been associated with strong visuo-spatial cognitive alterations, ascribed to hippocampal dysfunction. In the present study, we evaluated whether the normalization of the expression levels of Dyrk1A (Dual specificity tyrosine-phosphorylation-regulated kinase 1A), a candidate gene for DS, might correct hippocampal defects in Ts65Dn mice. In the hippocampus of 2 month-old Ts65Dn mice, such normalization was achieved through the stereotaxical injection of adeno-associated viruses containing a short hairpin RNA against Dyrk1A (AAV2/1-shDyrk1A) and a luciferase reporter gene. The injected hippocampi were efficiently transduced, as shown by bioluminescence in vivo imaging, luciferase activity quantification and immunohistochemical analysis. At the molecular level, viral infusion allowed the normalization of the targeted Dyrk1A expression, as well as of the key players of the MAPK/CREB pathway. The electrophysiological recordings of hippocampal slices from Ts65Dn mice injected with AAV2/1-shDyrk1A displayed attenuation of the synaptic plasticity defects of trisomic mice. In contrast, contralateral hippocampal injection with an AAV2/1 control virus containing a scrambled sequence, showed neither the normalization of Dyrk1A levels nor changes of synaptic plasticity. In the Morris water maze task, although long-term consolidation of the task was not achieved, treated Ts65Dn mice displayed initially a normalized thigmotactic behavior, similar to euploid littermates, indicating the partial improvement in their hippocampal-dependent search strategy. Taken together, these results show Dyrk1A as a critical player in the pathophysiology of DS and define Dyrk1A as a therapeutic target in adult trisomic mice.

Identification of homologous GluN subunits variants accelerates GRIN variants stratification.

The clinical spectrum of GRIN-related neurodevelopmental disorders (GRD) results from gene- and variant-dependent primary alterations of the NMDA receptor, disturbing glutamatergic neurotransmission. Despite GRIN gene variants' functional annotations being dually critical for stratification and precision medicine design, genetically diagnosed pathogenic GRIN variants currently outnumber their relative functional annotations. Based on high-resolution crystal 3D models and topological domains conservation between GluN1, GluN2A, and GluN2B subunits of the NMDAR, we have generated GluN1-GluN2A-GluN2B subunits structural superimposition model to find equivalent positions between GluN subunits. We have developed a GRIN structural algorithm that predicts functional changes in the equivalent structural positions in other GluN subunits. GRIN structural algorithm was computationally evaluated to the full GRIN missense variants repertoire, consisting of 4,525 variants. The analysis of this structure-based model revealed an absolute predictive power for GluN1, GluN2A, and GluN2B subunits, both in terms of pathogenicity-association (benign vs. pathogenic variants) and functional impact (loss-of-function, benign, gain-of-function). Further, we validated this computational algorithm experimentally, using an in silico library of GluN2B-equivalent GluN2A artificial variants, designed from pathogenic GluN2B variants. Thus, the implementation of the GRIN structural algorithm allows to computationally predict the pathogenicity and functional annotations of GRIN variants, resulting in the duplication of pathogenic GRIN variants assignment, reduction by 30% of GRIN variants with uncertain significance, and increase by 70% of functionally annotated GRIN variants. Finally, GRIN structural algorithm has been implemented into GRIN variants Database (http://lmc.uab.es/grindb), providing a computational tool that accelerates GRIN missense variants stratification, contributing to clinical therapeutic decisions for this neurodevelopmental disorder.

Targeting Dyrk1A with AAVshRNA attenuates motor alterations in TgDyrk1A, a mouse model of Down syndrome.

Genetic-dissection studies carried out with Down syndrome (DS) murine models point to the critical contribution of Dyrk1A overexpression to the motor abnormalities and cognitive deficits displayed in DS individuals. In the present study we have used a murine model overexpressing Dyrk1A (TgDyrk1A mice) to evaluate whether functional CNS defects could be corrected with an inhibitory RNA against Dyrk1A, delivered by bilateral intrastriatal injections of adeno-associated virus type 2 (AAVshDyrk1A). We report that AAVshDyrk1A efficiently transduced HEK293 cells and primary neuronal cultures, triggering the specific inhibition of Dyrk1A expression. Injecting the vector into the striata of TgDyrk1A mice resulted in a restricted, long-term transduction of the striatum. This gene therapy was found to be devoid of toxicity and succeeded in normalizing Dyrk1A protein levels in TgDyrk1A mice. Importantly, the behavioral studies of the adult TgDyrk1A mice treated showed a reversal of corticostriatal-dependent phenotypes, as revealed by the attenuation of their hyperactive behavior, the restoration of motor-coordination defects, and an improvement in sensorimotor gating. Taken together, the data demonstrate that normalizing Dyrk1A gene expression in the striatum of adult TgDyrk1A mice, by means of AAVshRNA, clearly reverses motor impairment. Furthermore, these results identify Dyrk1A as a potential target for therapy in DS.

An integrated workflow for enhanced taxonomic and functional coverage of the mouse fecal metaproteome.

Intestinal microbiota plays a key role in shaping host homeostasis by regulating metabolism, immune responses and behavior. Its dysregulation has been associated with metabolic, immune and neuropsychiatric disorders and is accompanied by changes in bacterial metabolic regulation. Although proteomics is well suited for analysis of individual microbes, metaproteomics of fecal samples is challenging due to the physical structure of the sample, presence of contaminating host proteins and coexistence of hundreds of taxa. Furthermore, there is a lack of consensus regarding preparation of fecal samples, as well as downstream bioinformatic analyses following metaproteomics data acquisition. Here we assess sample preparation and data analysis strategies applied to mouse feces in a typical mass spectrometry-based metaproteomic experiment. We show that subtle changes in sample preparation protocols may influence interpretation of biological findings. Two-step database search strategies led to significant underestimation of false positive protein identifications. Unipept software provided the highest sensitivity and specificity in taxonomic annotation of the identified peptides of unknown origin. Comparison of matching metaproteome and metagenome data revealed a positive correlation between protein and gene abundances. Notably, nearly all functional categories of detected protein groups were differentially abundant in the metaproteome compared to what would be expected from the metagenome, highlighting the need to perform metaproteomics when studying complex microbiome samples.

Decreased striatal adenosine A2A-dopamine D2 receptor heteromerization in schizophrenia.

According to the adenosine hypothesis of schizophrenia, the classically associated hyperdopaminergic state may be secondary to a loss of function of the adenosinergic system. Such a hypoadenosinergic state might either be due to a reduction of the extracellular levels of adenosine or alterations in the density of adenosine A2A receptors (A2ARs) or their degree of functional heteromerization with dopamine D2 receptors (D2R). In the present study, we provide preclinical and clinical evidences for this latter mechanism. Two animal models for the study of schizophrenia endophenotypes, namely the phencyclidine (PCP) mouse model and the A2AR knockout mice, were used to establish correlations between behavioural and molecular studies. In addition, a new AlphaLISA-based method was implemented to detect native A2AR-D2R heteromers in mouse and human brain. First, we observed a reduction of prepulse inhibition in A2AR knockout mice, similar to that observed in the PCP animal model of sensory gating impairment of schizophrenia, as well as a significant upregulation of striatal D2R without changes in A2AR expression in PCP-treated animals. In addition, PCP-treated animals showed a significant reduction of striatal A2AR-D2R heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of A2AR-D2R heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both D2R and A2AR were upregulated. Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal A2AR-D2R heteromers. The degree of A2AR-D2R heteromer formation in schizophrenia might constitute a hallmark of the illness, which indeed should be further studied to establish possible correlations with chronic antipsychotic treatments.

DYRK1A autophosphorylation on serine residue 520 modulates its kinase activity via 14-3-3 binding.

Dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) proteins are an evolutionarily conserved family of protein kinases, with members identified from yeast to humans, that participate in a variety of cellular processes. DYRKs are serine/threonine protein kinases that are activated by autophosphorylation on a tyrosine residue in the activation loop. The family member DYRK1A has been shown to phosphorylate several cytosolic proteins and a number of splicing and transcription factors, including members of the nuclear factor of activated T cells family. In the present study, we show that DYRK1A autophosphorylates, via an intramolecular mechanism, on Ser-520, in the PEST domain of the protein. We also show that phosphorylation of this residue, which we show is subjected to dynamic changes in vivo, mediates the interaction of DYRK1A with 14-3-3beta. A second 14-3-3 binding site is present within the N-terminal of the protein. In the context of the DYRK1A molecule, neither site can act independently of the other. Bacterially produced DYRK1A and the mutant DYRK1A/S520A have similar kinase activities, suggesting that Ser-520 phosphorylation does not affect the intrinsic kinase activity on its own. Instead, we demonstrate that this phosphorylation allows the binding of 14-3-3beta, which in turn stimulates the catalytic activity of DYRK1A. These findings provide evidence for a novel mechanism for the regulation of DYRK1A kinase activity.

A primate-specific short GluN2A-NMDA receptor isoform is expressed in the human brain.

Glutamate receptors of the N-methyl-D-aspartate (NMDA) family are coincident detectors of pre- and postsynaptic activity, allowing Ca2+ influx into neurons. These properties are central to neurological disease mechanisms and are proposed to be the basis of associative learning and memory. In addition to the well-characterised canonical GluN2A NMDAR isoform, large-scale open reading frames in human tissues had suggested the expression of a primate-specific short GluN2A isoform referred to as GluN2A-S. Here, we confirm the expression of both GluN2A transcripts in human and primate but not rodent brain tissue, and show that they are translated to two corresponding GluN2A proteins present in human brain. Furthermore, we demonstrate that recombinant GluN2A-S co-assembles with the obligatory NMDAR subunit GluN1 to form functional NMDA receptors. These findings suggest a more complex NMDAR repertoire in human brain than previously thought.

L-Serine dietary supplementation is associated with clinical improvement of loss-of-function GRIN2B-related pediatric encephalopathy.

Autosomal dominant mutations in GRIN2B are associated with severe encephalopathy, but little is known about the pathophysiological outcomes and any potential therapeutic interventions. Genetic studies have described the association between de novo mutations of genes encoding the subunits of the N-methyl-d-aspartate receptor (NMDAR) and severe neurological conditions. Here, we evaluated a missense mutation in GRIN2B, causing a proline-to-threonine switch (P553T) in the GluN2B subunit of NMDAR, which was found in a 5-year-old patient with Rett-like syndrome with severe encephalopathy. Structural molecular modeling predicted a reduced pore size of the mutant GluN2B-containing NMDARs. Electrophysiological recordings in a HEK-293T cell line expressing the mutated subunit confirmed this prediction and showed an associated reduced glutamate affinity. Moreover, GluN2B(P553T)-expressing primary murine hippocampal neurons showed decreased spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of the AMPA receptor subunit GluA1 at stimulated synapses. Furthermore, the naturally occurring coagonist d-serine restored function to GluN2B(P553T)-containing NMDARs. l-Serine dietary supplementation of the patient was hence initiated, resulting in the increased abundance of d-serine in the plasma and brain. The patient has shown notable improvements in motor and cognitive performance and communication after 11 and 17 months of l-serine dietary supplementation. Our data suggest that l-serine supplementation might ameliorate GRIN2B-related severe encephalopathy and other neurological conditions caused by glutamatergic signaling deficiency.

Maurocalcine interacts with the cardiac ryanodine receptor without inducing channel modification.

We have previously shown that MCa (maurocalcine), a toxin from the venom of the scorpion Maurus palmatus, binds to RyR1 (type 1 ryanodine receptor) and induces strong modifications of its gating behaviour. In the present study, we investigated the ability of MCa to bind to and modify the gating process of cardiac RyR2. By performing pull-down experiments we show that MCa interacts directly with RyR2 with an apparent affinity of 150 nM. By expressing different domains of RyR2 in vitro, we show that MCa binds to two domains of RyR2, which are homologous with those previously identified on RyR1. The effect of MCa binding to RyR2 was then evaluated by three different approaches: (i) [(3)H]ryanodine binding experiments, showing a very weak effect of MCa (up to 1 muM), (ii) Ca(2+) release measurements from cardiac sarcoplasmic reticulum vesicles, showing that MCa up to 1 muM is unable to induce Ca(2+) release, and (iii) single-channel recordings, showing that MCa has no effect on the open probability or on the RyR2 channel conductance level. Long-lasting opening events of RyR2 were observed in the presence of MCa only when the ionic current direction was opposite to the physiological direction, i.e. from the cytoplasmic face of RyR2 to its luminal face. Therefore, despite the conserved MCa binding ability of RyR1 and RyR2, functional studies show that, in contrast with what is observed with RyR1, MCa does not affect the gating properties of RyR2. These results highlight a different role of the MCa-binding domains in the gating process of RyR1 and RyR2.

Phosphoproteomic Alterations of Ionotropic Glutamate Receptors in the Hippocampus of the Ts65Dn Mouse Model of Down Syndrome.

Down syndrome (DS), the main genetic cause of intellectual disability, is associated with an imbalance of excitatory/inhibitory neurotransmitter systems. The phenotypic assessment and pharmacotherapy interventions in DS murine models strongly pointed out glutamatergic neurotransmission alterations (specially affecting ionotropic glutamate receptors [iGluRs]) that might contribute to DS pathophysiology, which is in agreement with DS condition. iGluRs play a critical role in fast-mediated excitatory transmission, a process underlying synaptic plasticity. Neuronal plasticity is biochemically modulated by post-translational modifications, allowing rapid and reversible adaptation of synaptic strength. Among these modifications, phosphorylation/dephosphorylation processes strongly dictate iGluR protein-protein interactions, cell surface trafficking, and subsynaptic mobility. Hence, we hypothesized that dysregulation of phosphorylation/dephosphorylation balance might affect neuronal function, which in turn could contribute to the glutamatergic neurotransmitter alterations observed in DS. To address this point, we biochemically purified subsynaptic hippocampal fractions from adult Ts65Dn mice, a trisomic mouse model recapitulating DS phenotypic alterations. Proteomic analysis showed significant alterations of the molecular composition of subsynaptic compartments of hippocampal trisomic neurons. Further, we characterized iGluR phosphopattern in the hippocampal glutamatergic synapse of trisomic mice. Phosphoenrichment-coupled mass spectrometry analysis revealed specific subsynaptic- and trisomy-associated iGluR phosphorylation signature, concomitant with differential subsynaptic kinase and phosphatase composition of Ts65Dn hippocampal subsynaptic compartments. Furthermore, biochemical data were used to build up a genotype-kinome-iGluR phosphopattern matrix in the different subsynaptic compartments. Overall, our results provide a precise profile of iGluR phosphopattern alterations in the glutamatergic synapse of the Ts65Dn mouse model and support their contribution to DS-associated synaptopathy. The alteration of iGluR phosphoresidues in Ts65Dn hippocampi, together with the kinase/phosphatase signature, identifies potential novel therapeutic targets for the treatment of glutamatergic dysfunctions in DS.

Rett-like Severe Encephalopathy Caused by a De Novo GRIN2B Mutation Is Attenuated by D-serine Dietary Supplement.

BACKGROUND: N-Methyl-D-aspartate receptors (NMDARs) play pivotal roles in synaptic development, plasticity, neural survival, and cognition. Despite recent reports describing the genetic association between de novo mutations of NMDAR subunits and severe psychiatric diseases, little is known about their pathogenic mechanisms and potential therapeutic interventions. Here we report a case study of a 4-year-old Rett-like patient with severe encephalopathy carrying a missense de novo mutation in GRIN2B(p.P553T) coding for the GluN2B subunit of NMDAR. METHODS: We generated a dynamic molecular model of mutant GluN2B-containing NMDARs. We expressed the mutation in cell lines and primary cultures, and we evaluated the putative morphological, electrophysiological, and synaptic plasticity alterations. Finally, we evaluated D-serine administration as a therapeutic strategy and translated it to the clinical practice. RESULTS: Structural molecular modeling predicted a reduced pore size of mutant NMDARs. Electrophysiological recordings confirmed this prediction and also showed gating alterations, a reduced glutamate affinity associated with a strong decrease of NMDA-evoked currents. Moreover, GluN2B(P553T)-expressing neurons showed decreased spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of GluA1 at stimulated synapses. Notably, the naturally occurring coagonist D-serine was able to attenuate hypofunction of GluN2B(p.P553T)-containing NMDARs. Hence, D-serine dietary supplementation was initiated. Importantly, the patient has shown remarkable motor, cognitive, and communication improvements after 17 months of D-serine dietary supplementation. CONCLUSIONS: Our data suggest that hypofunctional NMDARs containing GluN2B(p.P553T) can contribute to Rett-like encephalopathy and that their potentiation by D-serine treatment may underlie the associated clinical improvement.

The antigen-binding fragment of human gamma immunoglobulin prevents amyloid ß-peptide folding into ß-sheet to form oligomers.

The amyloid beta-peptide (Aß) plays a leading role in Alzheimer's disease (AD) physiopathology. Even though monomeric forms of Aß are harmless to cells, Aß can aggregate into ß-sheet oligomers and fibrils, which are both neurotoxic. Therefore, one of the main therapeutic approaches to cure or delay AD onset and progression is targeting Aß aggregation. In the present study, we show that a pool of human gamma immunoglobulins (IgG) protected cortical neurons from the challenge with Aß oligomers, as assayed by MTT reduction, caspase-3 activation and cytoskeleton integrity. In addition, we report the inhibitory effect of IgG on Aß aggregation, as shown by Thioflavin T assay, size exclusion chromatography and atomic force microscopy. Similar results were obtained with Palivizumab, a human anti-sincitial virus antibody. In order to dissect the important domains, we cleaved the pool of human IgG with papain to obtain Fab and Fc fragments. Using these cleaved fragments, we functionally identified Fab as the immunoglobulin fragment inhibiting Aß aggregation, a result that was further confirmed by an in silico structural model. Interestingly, bioinformatic tools show a highly conserved structure able to bind amyloid in the Fab region. Overall, our data strongly support the inhibitory effect of human IgG on Aß aggregation and its neuroprotective role.