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Transgenic mice overexpressing Dyrk1A (TgDyrk1A), a Down syndrome (DS) candidate gene, exhibit motor and cognitive alterations similar to those observed in DS individuals. To gain new insights into the molecular consequences of Dyrk1A overexpression underlying TgDyrk1A and possibly DS motor phenotypes, microarray studies were performed. Transcriptome analysis showed an upregulation of the NR2A subunit of the NMDA type of glutamate receptors in TgDyrk1A cerebellum. NR2A protein overexpression was also detected in TgDyrk1A cerebellar homogenates, in the synaptosome-enriched fraction and in TgDyrk1A primary cerebellar granular neuronal cultures (CGNs). In TgDyrk1A synaptosomes, calcium-imaging experiments showed a higher calcium uptake after NMDA stimulation. Similarly, NMDA administration promoted longer calcium transients in TgDyrk1A CGNs. Taken together, these results show that NMDA-induced calcium rise is altered in TgDyrk1A cerebellar neurons and indicate that calcium signaling is dysregulated in TgDyrk1A mice cerebella. These findings suggest that DYRK1A overexpression might contribute to the dysbalance in the excitatory transmission found in the cerebellum of DS individuals and DS mouse models.

Motor deficits are among the most frequent impairments in Down syndrome (DS), but their neuropathological and molecular bases remain elusive. Here we investigate the motor profile of transgenic mice overexpressing Dyrk1a, Tg(Dyrk1a)1Cff (hereafter TgDyrk1a), a candidate gene hypothesized to cause some of the neurological defects associated with DS. We have previously shown DYRK1A expression in the cerebellum and functionally related structures, most brainstem motor nuclei and spinal cord, supporting a role for Dyrk1a in controlling motor function. Here we demonstrate that TgDyrk1a mice present DYRK1A overexpression in these areas along with specific motor dysfunction. The main finding that emerged was impairment of motor learning and alteration of the organization of locomotor behavior, which agrees with reported clinical observations in subjects with DS. These results confirm and extend previous data and provide further insight to the functional domains that might be altered in TgDyrk1a mice and underlying molecular mechanisms of DS motor dysfunction.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., (© 2023 SSIEM.)

Rationale: To date, causal therapy is potentially available for GRIN2B-related neurodevelopmental disorder (NDD) due to loss-of-function (LoF) variants in GRIN2B, resulting in dysfunction of the GluN2B subunit-containing N-methyl-d-aspartate receptor (NMDAR). Recently, in vitro experiments showed that high doses of NMDAR co-agonist d-serine has the potential to boost the activity in GluN2B LoF variant-containing NMDARs. Initial reports of GRIN2B-NDD patients LoF variants, treated with l-serine using different regimens, showed varying effects on motor and cognitive performance, communication, behavior and EEG. Here, this novel treatment using a standardized protocol with an innovative developmental outcome measure is explored further in an open-label observational GRIN2B-NDD study., Methods: Initially, in vitro studies were conducted in order to functionally stratify two de novo GRIN2B variants present in two female patients (18 months and 4 years old). Functional studies showed that both variants are LoF, and thus the patients were treated experimentally according to an approved protocol with oral l-serine (500 mg/kg/day in 4 doses) for a period of 12 months. Both patients showed a heterogeneous clinical phenotype, however overlapping symptoms were present: intellectual developmental disability (IDD), behavioral abnormalities and hypotonia. Outcome measures included laboratory tests, quality of life, sleep, irritability, stool, and performance skills, measured by, among others, the Perceive-Recall-Plan-Perform System of Task Analysis (PRPP-Assessment)., Results: Both patients tolerated l-serine without adverse effects. In one patient, improvement in psychomotor development and cognitive functioning was observed after 12 months (PRPP mastery score 10% at baseline, 78% at twelve months). In the most severe clinically affected patient no significant objective improvement in validated outcomes was observed. Caregivers of both patients reported subjective increase of alertness and improved communication skills., Conclusion: Our observational study confirms that l-serine supplementation is safe in patients with GRIN2B-NDD associated with LoF variants, and may accelerate psychomotor development and ameliorate cognitive performance in some but not all patients. The PRPP-Assessment, a promising instrument to evaluate everyday activities and enhance personalized and value-based care, was not performed in the severely affected patient, meaning that possible positive results may have been missed. To generate stronger evidence for effect of l-serine in GRIN2B-NDD, we will perform placebo-controlled n-of-1 trials., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Santos-Gómez, García-Recio, Miguez-Cabello, Soto, Altafaj and Olivella.)

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.

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.

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., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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 A 2A receptors (A 2A Rs) or their degree of functional heteromerization with dopamine D 2 receptors (D 2 R). 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 A 2A R knockout mice, were used to establish correlations between behavioural and molecular studies. In addition, a new AlphaLISA-based method was implemented to detect native A 2A R-D 2 R heteromers in mouse and human brain. First, we observed a reduction of prepulse inhibition in A 2A R 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 D 2 R without changes in A 2A R expression in PCP-treated animals. In addition, PCP-treated animals showed a significant reduction of striatal A 2A R-D 2 R heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of A 2A R-D 2 R heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both D 2 R and A 2A R were upregulated. Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal A 2A R-D 2 R heteromers. The degree of A 2A R-D 2 R 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.

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.

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., (© 2020 Wiley Periodicals LLC.)

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., Competing Interests: The authors declare no conflict of interest.

Glutamate receptors of the N-methyl-D-aspartate (NMDA) family are coincident detectors of pre- and postsynaptic activity, allowing Ca 2+ 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.

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., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

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.

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., (Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

The amyloid beta-peptide (Aβ) plays a leading role in Alzheimer's disease (AD) physiopathology. Even though monomeric forms of Aβ are harmless to cells, Aβ can aggregate into β-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 Aβ aggregation. In the present study, we show that a pool of human gamma immunoglobulins (IgG) protected cortical neurons from the challenge with Aβ oligomers, as assayed by MTT reduction, caspase-3 activation and cytoskeleton integrity. In addition, we report the inhibitory effect of IgG on Aβ 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 Aβ 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 Aβ aggregation and its neuroprotective role.

The general concept of inborn error of metabolism is currently evolving into the interface between classical biochemistry and cellular biology. Basic neuroscience is providing increasing knowledge about the mechanisms of neurotransmission and novel related disorders are being described. There is a necessity of updating the classic concept of "inborn error of neurotransmitters (NT)" that considers mainly defects of synthesis and catabolism and transport of low weight NT molecules. Monogenic defects of the synaptic vesicle (SV), and especially those affecting the SV cycle are a potential new group of NT disorders since they end up in abnormal NT turnover and release. The most common clinical manifestations include epilepsy, intellectual disability, autism and movement disorders, and are in the continuum symptoms of synaptopathies. Interestingly, brain malformations and neurodegenerative conditions are also present within SV diseases. Metabolomics, proteomics, and other -omic techniques probably will provide biomarkers and contribute to therapeutic targets in the future., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The activation of N-Methyl D-Aspartate Receptor (NMDAR) by glutamate is crucial in the nervous system function, particularly in memory and learning. NMDAR is composed by two GluN1 and two GluN2 subunits. GluN2B has been reported to participate in the prevalent NMDAR subtype at synapses, the GluN1/2A/2B. Here we studied the regulation of GluN2B expression in cortical neurons finding that glutamate up-regulates GluN2B translation through the action of nitric oxide (NO), which induces the phosphorylation of the eukaryotic translation initiation factor 2 α (eIF2α). It is a process mediated by the NO-heme-regulated eIF2α kinase (HRI), as the effect was avoided when a specific HRI inhibitor or a HRI small interfering RNA (siHRI) were used. We found that the expressed GluN2B co-localizes with PSD-95 at the postsynaptic ending, which strengthen the physiological relevance of the proposed mechanism. Moreover the receptors bearing GluN2B subunits upon NO stimulation are functional as high Ca2+ entry was measured and increases the co-localization between GluN2B and GluN1 subunits. In addition, the injection of the specific HRI inhibitor in mice produces a decrease in memory retrieval as tested by the Novel Object Recognition performance. Summarizing our data suggests that glutamatergic stimulation induces HRI activation by NO to trigger GluN2B expression and this process would be relevant to maintain postsynaptic activity in cortical neurons.

N-methyl-D-aspartate glutamate receptors (NMDARs) play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs were modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser(1048) of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser(1048) hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser(1048) increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons.

Background: Zinc concentrates at excitatory synapses, both at the postsynaptic density and in a subset of glutamatergic boutons. Zinc can modulate synaptic plasticity, memory formation and nociception by regulating transmitter receptors and signal transduction pathways. Also, intracellular zinc accumulation is a hallmark of degenerating neurons in several neurological disorders. To date, no single zinc extrusion mechanism has been directly localized to synapses. Based on the presence of a canonical PDZ I motif in the Zinc Transporter-1 protein (ZnT1), we hypothesized that ZnT1 may be targeted to synaptic compartments for local control of cytosolic zinc. Using our previously developed protocol for the co-localization of reactive zinc and synaptic proteins, we further asked if ZnT1 expression correlates with presynaptic zinc content in individual synapses., Findings: Here we demonstrate that ZnT1 is a plasma membrane protein that is enriched in dendritic spines and in biochemically isolated synaptic membranes. Hippocampal CA1 synapses labelled by postembedding immunogold showed over a 5-fold increase in ZnT1 concentration at synaptic junctions compared with extrasynaptic membranes. Subsynaptic analysis revealed a peak ZnT1 density on the postsynaptic side of the synapse, < 10 nm away from the postsynaptic membrane. ZnT1 was found in the vast majority of excitatory synapses regardless of the presence of vesicular zinc in presynaptic boutons., Conclusions: Our study has identified ZnT1 as a novel postsynaptic density protein, and it may help elucidate the role of zinc homeostasis in synaptic function and disease.

Alterations in glutamatergic neurotransmission have long been associated with psychiatric and neurodevelopmental disorders (PNDD), but only recent advances in high-throughput DNA sequencing have allowed interrogation of the prevalence of mutations in glutamate receptors (GluR) among afflicted individuals. In this review we discuss recent work describing GluR mutations in the context of PNDDs. Although there are no strict relationships between receptor subunit or type and disease, some interesting preliminary conclusions have arisen. Mutations in genes coding for ionotropic glutamate receptor subunits, which are central to synaptic transmission and plasticity, are mostly associated with intellectual disability and autism spectrum disorders. In contrast, mutations of metabotropic GluRs, having a role on modulating neural transmission, are preferentially associated with psychiatric disorders. Also, the prevalence of mutations among GluRs is highly heterogeneous, suggesting a critical role of certain subunits in PNDD pathophysiology. The emerging bias between GluR subtypes and specific PNDDs may have clinical implications.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The presence of an additional copy of HSA21 chromosome in Down syndrome (DS) individuals leads to the overexpression of 30-50% of HSA21 genes. This upregulation can, in turn, trigger a deregulation on the expression of non-HSA21 genes. Moreover, the overdose of HSA21 microRNAs (miRNAs) may result in the downregulation of its target genes. Additional complexity can also arise from epigenetic changes modulating gene expression. Thus, a myriad of transcriptional and posttranscriptional alterations participate to produce abnormal phenotypes in almost all tissues and organs of DS individuals. The study of the physiological roles of genes dysregulated in DS, as well as their characterization in murine models with gene(s) dosage imbalance, pointed out several genes, and functional noncoding elements to be particularly critical in the etiology of DS. Recent findings indicate that gene therapy strategies-based on the introduction of genetic elements by means of delivery vectors-toward the correction of phenotypic abnormalities in DS are also very promising tool to identify HSA21 and non-HSA21 gene candidates, contributing to DS phenotype. In this chapter, we focus on the impact of normalizing the expression levels of up or downregulated genes to rescue particular phenotypes of DS. Attempts toward gene-based treatment approaches in mouse models will be discussed as new opportunities to ameliorate DS alterations., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Caveolin-3 is the striated muscle specific isoform of the scaffolding protein family of caveolins and has been shown to interact with a variety of proteins, including ion channels. Mutations in the human CAV3 gene have been associated with several muscle disorders called caveolinopathies and among these, the P104L mutation (Cav-3(P104L)) leads to limb girdle muscular dystrophy of type 1C characterized by the loss of sarcolemmal caveolin. There is still no clear-cut explanation as to specifically how caveolin-3 mutations lead to skeletal muscle wasting. Previous results argued in favor of a role for caveolin-3 in dihydropyridine receptor (DHPR) functional regulation and/or T-tubular membrane localization. It appeared worth closely examining such a functional link and investigating if it could result from the direct physical interaction of the two proteins. Transient expression of Cav-3(P104L) or caveolin-3 specific siRNAs in C2C12 myotubes both led to a significant decrease of the L-type Ca(2+) channel maximal conductance. Immunolabeling analysis of adult skeletal muscle fibers revealed the colocalization of a pool of caveolin-3 with the DHPR within the T-tubular membrane. Caveolin-3 was also shown to be present in DHPR-containing triadic membrane preparations from which both proteins co-immunoprecipitated. Using GST-fusion proteins, the I-II loop of Ca(v)1.1 was identified as the domain interacting with caveolin-3, with an apparent affinity of 60nM. The present study thus revealed a direct molecular interaction between caveolin-3 and the DHPR which is likely to underlie their functional link and whose loss might therefore be involved in pathophysiological mechanisms associated to muscle caveolinopathies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Individuals with trisomy 21, also known as Down syndrome (DS), develop a clinical syndrome including almost identical neuropathological characteristics of Alzheimer's disease (AD) observed in non-DS individuals. The main difference is the early age of onset of AD pathology in individuals with DS, with hish incidence of clinical symptoms in the late 40- early 50 years of age. The neuropathology of AD in persons with DS is superimposed with the developmental abnormalities causing alterations of neuronal morphology and function. Despite the ubiquitous occurrence of AD neuropathology, clinical signs of dementia do not occur in all adults with DS even at older ages. Phenotype analysis of DS mouse models has revealed a differential age-related neurodegenerative pattern that correlates with specific biochemical and molecular alterations at the cellular level. In fact, several individual genes found in trisomy in DS have been functionally related to neuronal degeneration. Thus, mouse models overexpressing HSA21 gene(s) are fundamental to understand the neurodegenerative process in DS, as described in the present review. In addition, these models might allow to define and evaluate potential drug targets and to develop therapeutic strategies that may interfere or delay the onset of AD.

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.

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.

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.

JP-45, an integral protein of the junctional face membrane of the skeletal muscle sarcoplasmic reticulum (SR), colocalizes with its Ca2+ -release channel (the ryanodine receptor), and interacts with calsequestrin and the skeletal-muscle dihydropyridine receptor Cav1. We have identified the domains of JP-45 and the Cav1.1 involved in this interaction, and investigated the functional effect of JP-45. The cytoplasmic domain of JP-45, comprising residues 1-80, interacts with Cav1.1. JP-45 interacts with two distinct and functionally relevant domains of Cav1.1, the I-II loop and the C-terminal region. Interaction between JP-45 and the I-II loop occurs through the alpha-interacting domain in the I-II loop. beta1a, a Cav1 subunit, also interacts with the cytosolic domain of JP-45, and its presence drastically reduces the interaction between JP-45 and the I-II loop. The functional effect of JP-45 on Cav1.1 activity was assessed by investigating charge movement in differentiated C2C12 myotubes after overexpression or depletion of JP-45. Overexpression of JP-45 decreased peak charge-movement and shifted VQ1/2 to a more negative potential (-10 mV). JP-45 depletion decreased both the content of Cav1.1 and peak charge-movements. Our data demonstrate that JP-45 is an important protein for functional expression of voltage-dependent Ca2+ channels.

The Xenopus oocyte is a widely used system for protein expression. Investigators have had the choice between two different techniques: injection into the cytoplasm of in vitro transcribed complementary RNA (cRNA) or injection into the nucleus of complementary DNA (cDNA). We report on a third expression technique that is based on the combined injection of cDNA and purified T7 RNA polymerase directly into the cytoplasm of oocytes.

Previous studies have shown that the skeletal dihydropyridine receptor (DHPR) pore subunit Ca(V)1.1 (alpha1S) physically interacts with ryanodine receptor type 1 (RyR1), and a molecular signal is transmitted from alpha1S to RyR1 to trigger excitation-contraction (EC) coupling. We show that the beta-subunit of the skeletal DHPR also binds RyR1 and participates in this signaling process. A novel binding site for the DHPR beta1a-subunit was mapped to the M(3201) to W(3661) region of RyR1. In vitro binding experiments showed that the strength of the interaction is controlled by K(3495)KKRR&#95; &#95;R(3502), a cluster of positively charged residues. Phenotypic expression of skeletal-type EC coupling by RyR1 with mutations in the K(3495)KKRR&#95; &#95;R(3502) cluster was evaluated in dyspedic myotubes. The results indicated that charge neutralization or deletion severely depressed the magnitude of RyR1-mediated Ca(2+) transients coupled to voltage-dependent activation of the DHPR. Meantime the Ca(2+) content of the sarcoplasmic reticulum was not affected, and the amplitude and activation kinetics of the DHPR Ca(2+) currents were slightly affected. The data show that the DHPR beta-subunit, like alpha1S, interacts directly with RyR1 and is critical for the generation of high-speed Ca(2+) signals coupled to membrane depolarization. These findings indicate that EC coupling in skeletal muscle involves the interplay of at least two subunits of the DHPR, namely alpha1S and beta1a, interacting with possibly different domains of RyR1.

Store-operated channels (SOCs) are recruited in response to the release of Ca2+ from intracellular stores. They allow a voltage-independent entry of Ca2+ into the cytoplasm also termed capacitative Ca2+ entry (CCE). In neurons, the functional significance of this Ca2+ route remains elusive. Several reports indicate that SOCs could be developmentally regulated. We verified the presence of a CCE in freshly dissociated cortical cells from E13, E14, E16, E18 fetuses and from 1-day-old mice. Intracellular Ca2+ stores were depleted by means of the SERCA pump inhibitor thapsigargin. At E13, the release of Ca2+ from thapsigargin-sensitive compartments gave rise to an entry of Ca2+ in a minority of cells. This Ca2+ route, insensitive to voltage-gated Ca2+ channel antagonists like Cd2+ and Ni2+, was blocked by the SOC inhibitor SKF-96365. After E13 and on E13 cells kept in culture, there is a marked increase in the percentage of cells with functional SOCs. The lanthanide La3+ fully inhibited the CCE from neonatal mice whereas it weakly blocked the thapsigargin-dependent Ca2+ entry at E13. This suggests that the subunit composition of the cortical SOCs is developmentally regulated with La3+-insensitive channels being expressed in the embryonic cortex whereas La3+-sensitive SOCs are found at birth. Our data argue for the presence of SOCs in embryonic cortical neurons. Their expression and pharmacological properties are developmentally regulated.

Maurocalcine (MCa) is a 33-amino-acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. External application of MCa to cultured myotubes is known to produce Ca2+ release from intracellular stores. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long lasting channel openings in a mode of smaller conductance. Here we investigated the way MCa proceeds to cross biological membranes to reach its target. A biotinylated derivative of MCa was produced (MCa(b)) and complexed with a fluorescent indicator (streptavidine-cyanine 3) to follow the cell penetration of the toxin. The toxin complex efficiently penetrated into various cell types without requiring metabolic energy (low temperature) or implicating an endocytosis mechanism. MCa appeared to share the same features as the so-called cell-penetrating peptides. Our results provide evidence that MCa has the ability to act as a molecular carrier and to cross cell membranes in a rapid manner (1-2 min), making this toxin the first demonstrated example of a scorpion toxin that translocates into cells.

Maurocalcine is a scorpion venom toxin of 33 residues that bears a striking resemblance to the domain A of the dihydropyridine voltage-dependent calcium channel type 1.1 (Cav1.1) subunit. This domain belongs to the II-III loop of Cav1.1, which is implicated in excitation-contraction coupling. Besides the structural homology, maurocalcine also modulates RyR1 channel activity in a manner akin to a synthetic peptide of domain A. Because of these similarities, we hypothesized that maurocalcine and domain A may bind onto an identical region(s) of RyR1. Using a set of RyR1 fragments, we demonstrate that peptide A and maurocalcine bind onto two discrete RyR1 regions: fragments 3 and 7 encompassing residues 1021-1631 and 3201-3661, respectively. The binding onto fragment 7 is of greater importance and was thus further investigated. We found that the amino acid region 3351-3507 of RyR1 (fragment 7.2) is sufficient for these interactions. Proof that peptide A and maurocalcine bind onto the same site is provided by competition experiments in which binding of fragment 7.2 to peptide A is inhibited by preincubation with maurocalcine. Moreover, when expressed in COS-7 cells, RyR1 carrying a deletion of fragment 7 shows a loss of interaction with both peptide A and maurocalcine. At the functional level, this deletion abolishes the maurocalcine induced stimulation of [3H]ryanodine binding onto microsomes of transfected COS-7 cells without affecting the caffeine and ATP responses.

P/Q Ca(2+) channel activity is inhibited by G protein-coupled receptor activation. Channel inhibition requires a direct Gbetagamma binding onto the pore-forming subunit, Ca(v)2.1. It is characterized by biophysical changes, including current amplitude reduction, activation kinetic slowing, and an I-V curve shift, which leads to a reluctant mode. Here, we have characterized the contribution of the auxiliary beta(3)-subunit to channel regulation by G proteins. The shift in I-V to a P/Q reluctant mode is exclusively observed in the presence of beta(3). Along with the observation that Gbetagamma has no effect on the I-V curve of Ca(v)2.1 alone, we propose that the reluctant mode promoted by Gbetagamma corresponds to a state in which the beta(3)-subunit has been displaced from its channel-binding site. We validate this hypothesis with a beta(3)-I-II(2.1) loop chimera construct. Gbetagamma binding onto the I-II(2.1) loop portion of the chimera releases the beta(3)-binding domain and makes it available for binding onto the I-II loop of Ca(v)1.2, a G protein-insensitive channel. This finding is extended to the full-length Ca(v)2.1 channel by using fluorescence resonance energy transfer. Gbetagamma injection into Xenopus oocytes displaces a Cy3-labeled beta(3)-subunit from a GFP-tagged Ca(v)2.1 channel. We conclude that beta-subunit dissociation from the channel complex constitutes a key step in P/Q calcium channel regulation by G proteins that underlies the reluctant state and is an important process for modulating neurotransmission through G protein-coupled receptors.

Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. MCa and mutated analogues were chemically synthesized, and their interaction with the skeletal muscle ryanodine receptor (RyR1) was studied on purified RyR1, sarcoplasmic reticulum (SR) vesicles, and cultured myotubes. MCa strongly potentiates [3H]ryanodine binding on SR vesicles (7-fold at pCa 5) with an apparent EC50 of 12 nm. MCa decreases the sensitivity of [3H]ryanodine binding to inhibitory high Ca2+ concentrations and increases it to the stimulatory low Ca2+ concentrations. In the presence of MCa, purified RyR1 channels show long-lasting openings characterized by a conductance equivalent to 60% of the full conductance. This effect correlates with a global increase in Ca2+ efflux as demonstrated by MCa effects on Ca2+ release from SR vesicles. In addition, we show for the first time that external application of MCa to cultured myotubes produces a cytosolic Ca2+ increase due to Ca2+ release from 4-chloro-m-cresol-sensitive intracellular stores. Using various MCa mutants, we identified a critical role of Arg24 for MCa binding onto RyR1. All of the other MCa mutants are still able to modify [3H]ryanodine binding although with a decreased EC50 and a lower stimulation efficacy. All of the active mutants produce both the appearance of a subconductance state and Ca2+ release from SR vesicles. Overall, these data identify some amino acid residues of MCa that support the effect of this toxin on ryanodine binding, RyR1 biophysical properties, and Ca2+ release from SR.

Dyrk1A and its Drosophila orthologue, the protein minibrain (mnb), belong to a family of serine/threonine kinases involved in the development of the central nervous system (CNS). However, additional roles for Dyrk1A have to be proposed, as its expression is still prominent in the adult brain. To gain insight into Dyrk1A physiological roles we have studied the distribution of this kinase in the CNS of mice in adulthood. A homogeneous diffuse immunostaining of variable intensity was detected throughout the neuropile, with the white matter displaying faint Dyrk1A immunoreactivity. Dyrk1A immunostaining was strong in the olfactory bulb, the cerebellar cortex and functionally related structures, the spinal cord and most of the motor nuclei of the midbrain and brain stem. These data agree with a possible implication of this kinase in the physiology of olfaction and motor functions. Cellular and subcellular localisation of Dyrk1A was also studied in primary cell culture of cerebellum, one of the structures showing significant Dyrk1A immunostaining in the adult. The distribution of Dyrk1A in primary cell culture showed the presence of this protein in the nucleus and the cytoplasm of both neurons and astrocytes. Moreover, studies on the subcellular distribution of Dyrk1A in whole brain homogenates of adult mice showed the presence of this protein both in nuclear and cytoplasm-enriched fractions, thus supporting selective functions of this kinase in these two subcellular compartments. The present results showing the distribution of Dyrk1A in widespread areas of the adult CNS and in different subcellular compartments, together with previous reports demonstrating its implication in developmental events concur with the idea of several spatio-temporal functional profiles.

Down's syndrome (DS) is a major cause of mental retardation, hypotonia and delayed development. Murine models of DS carrying large murine or human genomic fragments show motor alterations and memory deficits. The specific genes responsible for these phenotypic alterations have not yet been defined. DYRK1A, the human homolog of the Drosophila minibrain gene, maps to the DS critical region of human chromosome 21 and is overexpressed in DS fetal brain. DYRK1A encodes a serine-threonine kinase, probably involved in neuroblast proliferation. Mutant Drosophila minibrain flies have a reduction in both optic lobes and central brain, showing learning deficits and hypoactivity. We have generated transgenic mice (TgDyrk1A) overexpressing the full-length cDNA of Dyrk1A. TgDyrk1A mice exhibit delayed cranio-caudal maturation with functional consequences in neuromotor development. TgDyrk1A mice also show altered motor skill acquisition and hyperactivity, which is maintained to adulthood. In the Morris water maze, TgDyrk1A mice show a significant impairment in spatial learning and cognitive flexibility, indicative of hippocampal and prefrontal cortex dysfunction. In the more complex repeated reversal learning paradigm, this defect turned out to be specifically related to reference memory, whereas working memory was almost unimpaired. These alterations are comparable with those found in the partial trisomy chromosome 16 murine models of DS and suggest a causative role of DYRK1A in mental retardation and in motor anomalies of DS.

The availability of the DNA sequence of human chromosome 21 (HSA21) is a landmark contribution that will have an immediate impact on the study of the role of specific genes to Down syndrome (DS). Trisomy 21, full or partial, is a major cause of mental retardation and other phenotypic abnormalities, collectively known as Down syndrome (DS), a disorder affecting 1 in 700 births. The identification of genes on HSA21 and the elucidation of the function of the proteins encoded by these genes have been a major challenge for the human genome project and for research in DS. Over 100 of the estimated 300-500 genes of HSA21 have been identified, but the function of most remains largely unknown. It is believed that the overexpression of an unknown number of HSA21 genes is directly or indirectly responsible for the mental retardation and the other clinical features of DS. For this reason, HSA21 genes that are expressed in tissues affected in DS patients are of special interest.

The DYRK1A gene on human chromosome 21 encodes a protein kinase presumed to be involved in the pathogenesis of mental retardation in Down's syndrome. Here we describe a highly similar homolog, DYRK1B, which is, in contrast to DYRK1A, predominately expressed in muscle and testis. The human DYRK1B gene was mapped to chromosome 19 (19q12-13.11) by radiation hybrid analysis. The amino acid sequences of DYRK1A and DYRK1B are 84% identical in the N-terminus and the catalytic domain but show no extended sequence similarity in the C-terminal region. DYRK1B contains all motifs characteristic for the DYRK family of protein kinases. In addition, the sequence comprises a bipartite nuclear localization motif. A green fluorescent protein (GFP) fusion protein of DYRK1B was found mainly in the nucleus of transfected COS-7 cells. These data suggest that DYRK1B is a muscle- and testis-specific isoform of DYRK1A and is involved in the regulation of nuclear functions., (Copyright 1999 Academic Press.)