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Glutamatergic Receptors Dynamics and Regulation View all 15 Articles
Inmaculada M. Gonzalez-Gonzalez
University of Central Lancashire, United Kingdom
Antonio Sanz-Clemente
Feinberg School of Medicine, Northwestern University, United States
Mariana Vargas-Caballero
University of Southampton, United Kingdom
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ORIGINAL RESEARCH article
Front. Mol. Neurosci., 25 July 2018 | https://doi.org/10.3389/fnmol.2018.00226
Phosphoproteomic Alterations of Ionotropic Glutamate Receptors in the Hippocampus of the Ts65Dn Mouse Model of Down Syndrome
Macarena Gómez de Salazar, Cristina Grau, Francisco Ciruela and Xavier Altafaj*
Neuropharmacology Unit, Bellvitge Biomedical Research Institute (IDIBELL)—University of Barcelona, Barcelona, Spain
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.
Down syndrome (DS) is the most common genetic cause of intellectual disability (OMIM #190685). Individuals with DS present a trisomy of the human chromosome 21 (Hsa21), and pivotal genes located in Hsa21 have been proposed as candidate genes for DS pathophysiology (Antonarakis et al., 2004). To model DS pathophysiology, different animal models have been used, either overexpressing single Hsa21-homologous genes or encompassing a partial trisomy of Hsa21 syntenic region (Dierssen, 2012; Rueda et al., 2012). Among the latter, Ts65Dn mouse is the most well-established DS murine model and genetically consists of the triplication of the Hsa21 syntenic mouse chromosome Mmu16. Ts65Dn mouse recapitulates cognitive and motor alterations observed in individuals with DS (Costa et al., 1999; Galdzicki and Siarey, 2003; Altafaj et al., 2013). These alterations are neurochemically associated with an imbalance of excitatory/inhibitory neurotransmitter systems (Belichenko et al., 2009), similar to those described in individuals with DS (Risser et al., 1997; Seidl et al., 2001). This neurotransmitter imbalance resulting from the alteration of GABAergic inhibitory neurotransmission (Fernandez et al., 2009; Best et al., 2012; Contestabile et al., 2017), together with excitatory glutamatergic neurotransmission defects (Siddiqui et al., 2008; Kleschevnikov, 2012), might disturb synaptic plasticity and impair learning and memory abilities in Ts65Dn mice. Consequently, several attempts have been made toward the restoration of homeostatic excitatory/inhibitory balance. In particular, Ts65Dn treatment with memantine—a reversible N-methyl-d-aspartate glutamate receptor (NMDAR) antagonist—has been shown to partially rescue critical electrophysiological alterations (Scott-McKean and Costa, 2011) and cognitive phenotypic abnormalities of trisomic mice (Costa et al., 2007; Lockrow et al., 2011). These findings have been significantly translated to the clinical practice, with an improvement of the cognitive skills of individuals with DS (Boada et al., 2012). These data strongly support the hypothesis that Hsa21 gene overexpression (e.g., DYRK1A, APP, TIAM1, and ITSN1 gene products) might dysregulate NMDAR expression and/or function, contributing to DS-like pathogenesis in trisomic mice (Siddiqui et al., 2008). NMDARs belong to the ionotropic glutamate receptor (iGluR) family, together with α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) and kainate receptors (KARs). iGluRs importantly play a critical role in fast-mediated transmission, a process underlying synaptic plasticity and neuronal survival (Paoletti et al., 2013). The precise control of iGluR activity is tuned by numerous post-translational mechanisms (Wang et al., 2006; Traynelis et al., 2010). Among them, (de)phosphorylation events strongly influence iGluR biogenesis (assembly, trafficking, docking, surface diffusion, and internalization) and receptor channel gating. These processes modulate synaptic strength and thus modulate neuronal plasticity (Lee, 2006; Lau and Zukin, 2007; Goebel-Goody et al., 2009; Pahl et al., 2014). The phosphorylation pattern of iGluR results from the activity of several canonical protein kinases (PKA, PKC, CaMKII) (reviewed by Lussier et al., 2015) and protein phosphatases (STEP, PP2A, PP1, calcineurin) (Westphal, 1999; Chan and Sucher, 2001; Braithwaite et al., 2006; Sanderson et al., 2016) that collectively modulate the gating and subcellular location of iGluR. It has been proposed that the specific subsynaptic location of NMDARs (synaptic vs. extrasynaptic) plays a dichotomic role related with synaptic plasticity and neuronal survival (Hardingham and Bading, 2010). We have recently showed that the gene product of DS candidate gene DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1) also influences NMDAR surface expression and function similar to NMDAR regulation by Hsa21 gene products (Grau et al., 2014). To sum up, these data suggest that the overexpression of Hsa21 genes impacts glutamatergic transmission in DS. This potential functional disturbance might molecularly be associated with and/or result from a complex reorganization of the synaptic proteome composition and protein phosphopattern, with protein-specific dysregulation (subsets of proteins under-represented, over-represented, with conserved expression and with protein and site-specific phosphorylation changes). To identify these potential molecular changes, in this study, we have optimized a biochemical subfractioning method allowing the purification of subsynaptic compartments (pre-, extra-, and postsynaptic fractions) for further proteomic and phosphoproteomic analysis of Ts65Dn adult mice hippocampi. Phosphoenrichment-coupled mass spectrometry analysis revealed a subsynaptic-specific and disease-associated iGluR phosphopattern signature. This phosphopattern is in agreement with the concomitant subsynaptic-specific increase of kinase expression and phosphatase downregulation in Ts65Dn mice hippocampi. Overall, our results demonstrate an altered phosphopattern in the glutamatergic synapse, together with the identification of a protein kinase/phosphatase biochemical signature in the Ts65Dn murine model, which may represent novel therapeutic targets for DS synaptopathy.
Ts65Dn mouse colony female B6EiC3Sn a/A-Ts(1716)65Dn (Ts65Dn) and male B6C3F1/J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The mouse colony was housed and bred in the Animal Facilities of the Barcelona Biomedical Research Park (PRBB, Barcelona, Spain, EU). All animal procedures met the guidelines of the European Community Directive 86/609/EEC and were approved by the Local Ethics Committee. The mice were housed under a 12:12 h light–dark schedule (lights on at 8:00 a.m.) in controlled environmental conditions of humidity (60%) and temperature (22 ± 2°C) with food and water ad libitum. Both Ts65Dn and euploid mice were genotyped by qPCR, following the Jackson laboratories protocol (https://www.jax.org/research-and-faculty/tools/cytogenetic-and-down-syndrome-models-resource/protocols/cytogenic-qpcr-protocol).
Antibodies were obtained from commercial suppliers as it follows: anti-PSD95 (Neuromab, #P78352), anti-synaptophysin (Sigma-Aldrich, S5768), anti-β-actin (Sigma-Aldrich, A5316), anti-GluN2A (Sigma-Aldrich, M264), anti-GluN1 (Millipore, #AB9864R), anti-CaMKIIα (CST, #50049), anti-Fyn (CST, #4023), anti-pS890 GluN1 (CST, #3381), anti-pS1284 GluN2B (CST, #5355), anti-rabbit IgG-horseradish peroxidase (HRP; Dako, #P0448), and anti-mouse IgG-HRP (Dako, # P0447).
Subsynaptic Fractionation and Western Blot
Subsynaptic fractionation protocol was adapted from the protocol developed by Philips and collaborators (Phillips et al., 2001). This protocol allows the obtention of the enriched extrasynaptic (Extra), postsynaptic (Post), and presynaptic (Pre) fractions from brain regions. In brief, the frozen tissue was thawed in cold buffer A (50 mM Tris-HCl pH 7.4, 0.32 M sucrose, 5 mM EDTA, 1 mM EGTA, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1/2500 PMSF, 20 μM ZnCl2, 50 mM NaF, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, and a cocktail of protease inhibitors [Complete, Roche]) and mechanically homogenized using a potter. The homogenate was centrifuged for 10 min at 1,400 g, and the supernatant was collected. To increase the yield of protein recovery, this step was repeated thrice. The collected supernatants were pooled and centrifuged for 10 min at 700 g. The resulting supernatant was again centrifuged for 15 min at 21,000 g. The resultant pellet containing the crude membrane fraction was kept for further subfractionation, as follows. Crude membrane fraction was solubilized in buffer B (0.32 M sucrose, 50 mM Tris-HCl, pH 7.4) and loaded on a discontinuous sucrose step gradient (0.85 M/1.0 M/1.2 M). Further after centrifugation at 82,500 g for 2 h, the synaptosomes (Syns) were collected from the 1.0 M/1.2 M interface and diluted in 50 mM Tris-HCl pH 7.4 and centrifuged once more for additional 30 min at 21,000 g. Then this pellet was collected and resuspended in synaptosomal resuspension buffer, consisting of 36 mM sucrose 0.1 mM CaCl2 + 10 mM NaF + 1 mM sodium orthovanadate, 2 mM EDTA, protease, and phosphatase inhibitor cocktail (HaltTM, ThermoFisher), leading to the purified synaptosomal fraction (P2) for further subsynaptic fractionation, as follows. In brief, synaptosomes were solubilized by resuspending in 40 mM Tris-HCl pH 6.0 containing 1% triton X-100 and incubated at 4°C for 30 min under agitation. Following centrifugation at 40,000 g for 30 min, the supernatant fraction (corresponding to the extrasynaptic fraction) was acetone precipitated. The pellet, containing the synaptosomal junction, was resuspended in 20 mM Tris-HCl buffer pH 8.0 supplemented with 1% triton X-100. After 30-min incubation at 4°C, the fractions were separated by centrifugation at 40,000 g for 30 min. The resulting supernatant, corresponding to the enriched presynaptic fraction, was acetone precipitated. The pellet, containing the enriched postsynaptic fraction, was resuspended in 50 mM Tris buffer—2% SDS, as well as the resulting acetone-precipitated subsynaptic fractions.
For western blot analysis, equal protein amounts from each sample were loaded and separated using 8 and 10% SDS-PAGE. The proteins were transferred onto nitrocellulose membranes (Invitrogen) using iBlot semi-dry blotting system (Life Technologies). After transient staining of transferred proteins by Ponceau S staining, the membranes were blocked with 10% skim milk in TBST (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween-20) for 1 h. The membranes were incubated overnight at 4°C with the corresponding primary antibodies (diluted in TBST/+5% skimmed milk) and followed by incubation with HRP-conjugated anti-mouse, anti-guinea pig IgG-HRP, or anti-rabbit IgG secondary antibodies (Dako) for 1 h at room temperature. The detection was performed with chemiluminescence using Amersham ECL Prime Western blotting detection reagent (GE, Amersham) according to the manufacturers' instructions. The immunoreactive bands were visualized using a ChemiDoc MP (BioRad), and the immunoreactive signals were analyzed using the Image Lab Biorad software.
Nano-UPLC Mass Spectrometry System and Phosphopeptide Enrichment
Mass spectrometry experiments were performed from two independent biological replicates. Each replicate consisted of a pool of hippocampi (N = 4 mice/genotype) that were subsequently fractionated to obtain subsynaptic fractions, either from euploid or from trisomic mice. Following resuspension and protein quantification, subsynaptic fractions (70–200 μg) were diluted in 1% SDS and digested with the single sequence-specific protease trypsin, following the previously described FASP protocol (Wiśniewski et al., 2009). Digested peptides were subjected to phosphopeptide enrichment using the High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific). About 45% of each enriched sample was analyzed using an Orbitrap Fusion Lumos with an EASY-Spray nanosource coupled to a nano-UPLC system (EASY-nanoLC 1000 liquid chromatograph) equipped with a 50-cm C18 column (EASY-Spray; 75 μm id, PepMap RSLC C18, 2-μm particles, 45°C). Chromatographic gradients started at 5% buffer B with a flow rate of 300 nl/min and gradually increased to 22% buffer B in 79 min and to 32% in 11 min. After each analysis, the column was washed for 10 min with 95% buffer B (buffer A: 0.1% formic acid in water and buffer B: 0.1% formic acid in acetonitrile). The mass spectrometer was operated in data-dependent acquisition mode, with full MS scans over a mass range of m/z 350–1500 with detection in the Orbitrap (120-K resolution) and with auto gain control (AGC) set to 100,000. In each cycle of data-dependent acquisition analysis, following each survey scan, the most intense ions above a threshold ion count of 10,000 were selected for fragmentation with HCD at normalized collision energy of 28%. The number of selected precursor ions for fragmentation was determined by the "Top Speed" acquisition algorithm (maximum cycle time of 3 s), and a dynamic exclusion of 60 s was set. Fragment ion spectra were acquired in the ion trap with an AGC of 10,000 and a maximum injection time of 200 ms.
Raw Data Processing and Data Analysis
Acquired data were analyzed using the Proteome Discoverer software suite (v2.0, Thermo Fisher Scientific), and the Mascot search engine (v2.5.1, Matrix Science) was used for peptide identification. Data were searched against a Mus musculus protein database derived from SwissProt and included the most common contaminants. A precursor ion mass tolerance of 7 ppm at the MS1 level was used, and up to three missed cleavages for trypsin were allowed. The fragment ion mass tolerance was set to 0.5 Da oxidation of methionine; N-terminal protein acetylation and phosphorylation in serine, threonine, and tyrosine were defined as variable modification; and carbamidomethylation of cysteines was set as fixed modification. The identified peptides were filtered by 5%FDR, and only those proteins identified in both biological replicates (for each condition) were retained for further analysis (Supplementary Table 1).
Bioinformatic Analysis of Proteomic Data
The proteomic signature of the experimental groups was represented using Venn diagrams. These diagrams were created using VENNY 2.1 freeware (http://bioinfogp.cnb.csic.es/tools/venny/; Oliveros et al., 2007–2015). This graphical interface allowed the representation of proteins overlapped among groups, together with the visualization of differentially expressed proteins in the different groups/fractions.
To functionally classify the differentially expressed proteins from the different groups (subsynaptic fraction and genotype), the data were submitted to the Database for Annotation, Visualization, and Integrated Discovery (DAVID: http://david.abcc.ncifcrf.gov/). This software, based on the use of gene ontology (GO), was used for grouping the differentially expressed proteins into biological processes, molecular functions, and cellular components.
In Silico Analysis of Phosphoproteomic Data
An updated database of murine kinases and phosphatases was generated before the analysis of the phosphoproteomic data. To this end, Uniprot database was used as the reference library, proteins were sorted (e.g., "kinase" and "phosphatase" criteria), and the protein list was further manually curated. This database was used to analyze potentially differentially expressed kinases and phosphatases between genotypes (Supplementary Table 2).
The analysis of the proteome and phosphoproteome signatures was performed using, as inclusion criteria, the identification of the proteins and phosphosites in both biological replicates. Furthermore, the conserved phosphosites were classified based on their qualitative expression in the different subsynaptic compartments, as well as in their relative expression levels. Regarding the latter, proteins and phosphosites detected in both fractions were classified based on the relative TS:EU ratio, resulting from the mean MS peak area (TS) (replicates 1 and 2)/mean MS peak area (EU) (replicates 1 and 2). Ratios < 0.75 or >1.5 were considered as downregulated and upregulated protein levels, respectively (see Supplementary Table 3).
Following phosphoproteomic profile identification of trisomic subsynaptic fractions, a systems biology approach was conducted to infer the potential kinases associated with the phosphoproteomic profile. To this end, the Human protein Reference Database (Hprd.org) was used, and the putative kinases involved in the phosphoprofile were identified. Finally, an interaction network analysis was performed using the Search Tool for the Retrieval of Interacting Genes/Proteins database (STRING; http://www.string-db.org/). This bioinformatic tool allowed the generation of protein–protein interaction networks, integrating the kinases/phosphatases and iGluR phosphoprofile of Ts65Dn subsynaptic fractions.
Comparison of immunolabeling signal intensities between euploid and trisomic hippocampal fractions was evaluated using the GraphPad software. All datasets passed the Kolmogorov–Smirnov normality test and were analyzed applying a two-tailed unpaired t-test with Welch's correction. Graph bars represent the mean ± SEM for each group.
Subsynaptic Proteomic Signature of the Ts65Dn Mice Hippocampi
Neuronal communication mainly resides in the synapse, a highly specialized subcellular compartment juxtaposing crosstalking neurons. Within synapses, there is an additional morphological compartmentalization that allows the precise neurotransmitter and structural machinery positioning, underlying neuronal function. Consequently, potential specific changes on protein composition of the subsynaptic compartments can impact synaptic function and ultimately alter Ts65Dn glutamatergic neurons. To address this issue, we performed a subsynaptic fractionation protocol toward the obtention of extrasynaptic-, presynaptic-, and postsynaptic-enriched hippocampal fractions, both from euploid and from trisomic adult mice (Figure 1A).
Figure 1. Subsynaptic fractionation and proteomic analysis of Ts65Dn mouse hippocampus. (A) Experimental workflow followed for hippocampal subsynaptic fractions obtention and mass spectrometry phosphoproteomic studies (mass spectra of biological replicates 1 and 2 are figurative representations). Down left panel, Representative western blot analysis of adult hippocampal subsynaptic fractions. Antibodies directed against PSD95 and synaptophysin were used as markers of postsynaptic and extrasynaptic, respectively. Right up panel, Western blot analysis of NMDAR subunits GluN2A and GluN1 in subsynaptic fractions (Post, postsynaptic; Extra, extrasynaptic) of adult euploid (EU) and trisomic (TS) mice. (B) Venn's diagram representing total identified proteins overlap between biological replicates 1 and 2 (postsynaptic and extrasynaptic fractions from both genotypes). (C) Venn's diagram representing genotype (EU, euploid; TS, trisomic) and subsynaptic compartment (Post, postsynaptic; Extra, extrasynaptic) effect on protein composition.
Hippocampal subsynaptic fractionation protocol was validated by western blot, using specific subsynaptic compartment markers. As expected, extrasynaptic fraction showed an elevated signal of synaptophysin, whereas the postsynaptic fraction was enriched in scaffolding protein PSD95 (Figure 1A). Moreover, we observed a significant enrichment of the NMDA receptor subunits GluN1 and GluN2A in the postsynaptic fraction (Figure 1A). Overall, these biochemical studies confirmed the obtention of enriched subsynaptic compartments from murine brain tissue, and the same protocol was used to obtain enriched subsynaptic hippocampal fractions for mass spectrometry analysis.
Subsynaptic fractions (postsynaptic and extrasynaptic) were subjected to trypsin digestion, and mass spectrometry studies were performed. Data analysis was based on the comparison of the genotype effect on the subsynaptic proteomic/phosphoproteomic signature (defined as the overlapping profile of the biological replicates).
Proteomic analysis allowed the identification of 1922 proteins (M. musculus protein database, SwissProt) consistently present in both biological replicates (Figure 1B). Subsynaptic distribution analysis of these proteins showed that 938/1043 proteins are present in the postsynaptic fraction of euploid/trisomic mice, respectively, whereas 863/1059 proteins are present in the extrasynaptic fractions of euploid/trisomic mice, respectively (Figure 1C and Supplementary Table 1). Despite the proteomic profile showed the presence of conserved proteins in trisomic hippocampal synapses (60.7 and 57.8% protein coincidence for postsynaptic and extrasynaptic fractions, respectively; Figures 2A, 3A), significant protein composition changes were detected in trisomic hippocampi. Further in silico analyses were performed to analyze the potential functional outcomes of these proteomic changes.
Figure 2. Gene ontology analysis of Ts65Dn mouse hippocampal postsynaptic proteome. (A) Venn's diagram representing comparative protein composition between postsynaptic fractions of adult euploid (EU) and Ts65Dn (TS) hippocampi. The diagram represents proteins detected in euploid replicates (blue circle), in trisomic replicates (yellow circle), and commonly detected in replicates from euploid and trisomic samples (white circle). (B–E) Functional annotation analysis of proteins enriched in hippocampal postsynaptic fractions of euploid (B,D) and trisomic (C,E) hippocampi (B,C). Bar graphs representing top five significantly enriched GO terms, based on "cellular compartment" criteria (D,E). Table listing top five significantly enriched proteins related with "biological process" and "molecular function" GO terms, in the postsynaptic fraction of euploid (D) and trisomic (E) hippocampi.
Figure 3. Gene ontology analysis of Ts65Dn mouse hippocampal extrasynaptic proteome. (A) Venn's diagram representing comparative protein composition between extrasynaptic fractions of adult euploid (EU) and Ts65Dn (TS) hippocampi. The diagram represents proteins detected in euploid replicates (green circle), in trisomic replicates (red circle), and commonly detected in replicates from euploid and trisomic samples (white circle). (B–E) Functional annotation analysis of proteins enriched in hippocampal extrasynaptic fractions of euploid (B) and trisomic (C) hippocampi (B,C). Bar graphs representing top five significantly enriched GO terms, based on "cellular compartment" criteria (D,E). Table listing top five significantly enriched proteins related with "biological process" and "molecular function" GO terms, in the extrasynaptic fraction of euploid (D) and trisomic (E) hippocampi.
Gene Ontology Enrichment Analysis of Subsynaptic Ts65Dn Proteomic Signature
The genotype effect on protein composition of the postsynaptic and extrasynaptic fractions was performed following the GO-term-based protein enrichment analysis. For functional annotation studies, only those proteins showing differential subsynaptic fraction expression were considered (Figures 2A, 3A). Statistical enrichment analysis of "cellular compartment," "biological processes," and "molecular functions" revealed the presence of significant genotype-dependent differences, both in the postsynaptic and in the extrasynaptic fractions (Figures 2B–E, 3B–E).
GO-term analysis of the euploid-specific postsynaptic proteome indicated the presence of an enriched representation of dendrite-associated proteins, thereby reflecting a dendritic protein under-representation in the trisomic postsynaptic fraction (Figures 2B–D). Moreover, GO analysis of the trisomic postsynaptic fraction showed significant changes in the following cellular functions: membranes, cytoplasm, transport, and protein binding (Figures 2C–E).
Regarding the extrasynaptic fractions, GO-term analysis of the euploid-specific extrasynaptic fraction showed the presence of mitochondria and protein phosphatase-binding proteins, indicating their relative absent detection in the trisomic extrasynaptic fraction (Figures 3B–D). In contrast, GO analysis of trisomic extrasynaptic fraction indicated changes in membrane, cytoplasm, synapse transport, phosphorylation, protein kinase activity, and protein binding (Figure 3C–E). Overall, GO-term analysis suggested that the subsynaptic proteome of the trisomic hippocampus is altered, in terms of both cellular compartments and functional outcomes. In particular, the analysis strongly suggested that phosphorylation-associated processes could be affected, as described below.
Altered Expression of Kinases and Phosphatases in the Ts65Dn Mouse Hippocampal Subsynaptic Fractions
Based on the functional annotation analyses showing alterations in phosphorylation, protein kinase activity, and phosphatase binding in the Ts65Dn synapses, we aimed to elucidate potential expression changes in kinases and phosphatases. To this end, an updated mouse kinome and phosphatome database was developed, using Uniprot as the reference database and filtering the M. musculus protein entries for the extraction of mouse kinases and phosphatases (Supplementary Table 2). Afterward, the genotype effect was compared either for postynaptic or for extrasynaptic fractions. With respect to euploid mice, the postsynaptic fraction of trisomic mice showed the presence of a subset of specific/enriched kinases and phosphatases (50 and 10 proteins, respectively), together with nondetectable/reduced expression of kinases and phosphatases (21 and 7 proteins, respectively) (Figure 4 and Supplementary Figure 1A). The comparative study of the extrasynaptic fractions indicated the presence, in trisomic samples, of a subset of specific/enriched kinases and phosphatases (37 and 7 proteins, respectively), together with non-detectable/reduced expression of kinases and phosphatases that are present in euploid samples (five and eight proteins, respectively) (Figure 5 and Supplementary Figure 1B). We performed western blot analysis of MS-identified kinases with putative differential expression to validate these unbiased findings. In agreement with proteomic data, western blot showed a significant increase of CamKII expression levels in the postsynaptic fraction of trisomic mice (p = 0.033, two-tailed Student's t-test), together with a significant decrease of Fyn kinase levels in the postsynaptic and a concomitant increase in the extrasynaptic fraction of the Ts65Dn mice (p = 0.015 and 0.024, respectively; two-tailed Student's t-test; Supplementary Figure 2A).
Figure 4. Kinome and kinase-iGluR interactome analysis of Ts65Dn mouse hippocampal postsynaptic fraction. (Top) Venn's diagram representing comparative analysis of protein kinases expressed in the postsynaptic fraction of adult euploid (EU) and Ts65Dn (TS) mice. The diagram represents proteins detected in euploid replicates (blue circle), in trisomic replicates (yellow circle), and commonly detected in replicates of euploid and trisomic samples (purple circle). The diagram shows the number of kinases not detected/underrepresented in Ts65Dn mice (in red), with similar expression (TS:EU expression ratio = 0.75–1.25; black characters) and present/overrepresented in trisomic mice (in green). (Bottom) Protein–protein interaction (PPI) network of protein kinases, scaffolding proteins, and iGluRs upregulated in Ts65Dn postsynaptic fraction. Node color annotation: red, kinase activity; blue, postsynaptic density-associated kinase; green, participation in long-term potentiation; and yellow, present in the glutamatergic synapse; link color annotation: known interactions (light blue: curated databases; pink: experimentally determined), predicted interactions (green: gene neighborhood; red: gene fusions; blue: gene co-occurrence), and others (light green: text mining; black: coexpression; purple: protein homology).
Figure 5. Kinome and interactome kinase-iGluR analysis of hippocampal extrasynaptic fraction of Ts65Dn mice. (Top) Venn's diagram representing comparative analysis of protein kinases expressed in the extrasynaptic fraction of adult euploid (EU) and Ts65Dn (TS) mice. The diagram represents proteins detected in euploid replicates (green circle), in trisomic replicates (red circle), and commonly detected in replicates of euploid and trisomic samples (dark green circle). The diagram shows the number of kinases not detected/underrepresented in Ts65Dn mice (in red), with similar expression (TS:EU expression ratio = 0.75–1.25; black characters) and present/overrepresented in trisomic mice (in green). (Bottom) Protein–protein interaction (PPI) network of protein kinases and iGluRs upregulated in Ts65Dn extrasynaptic fraction. Node color annotation: red, kinase activity; blue, postsynaptic density-associated kinase; green, participation in long-term potentiation; and yellow, present in the glutamatergic synapse; link color annotation: known interactions (light blue: curated databases; pink: experimentally determined), predicted interactions (green: gene neighborhood; red: gene fusions; blue: gene co-occurrence), and others (light green: text mining; black: coexpression; purple: protein homology).
Further, we aimed to predict the potential consequences of the dysregulated trisomic synaptic kinome. We focused in silico analysis on the ionotropic family of glutamate receptors similar to the glutamatergic alterations reported in the Ts65Dn mouse model. The potential protein–protein interaction networks between iGluRs and protein kinases upregulated in the trisomic fractions (either postsynaptic or extrasynaptic) were analyzed using STRING software. Interactome analysis of the trisomic postsynaptic fraction unraveled significant kinase-iGluR interaction, as shown by the elevated number of edges (predicted number of edges: 123 vs. expected number of edges: 18) and the PPI enrichment (p-value = 0), indicating the significant interactions within the PPI network. In particular, STRING analysis identified protein kinases from the CaMKII family, Dlgap1, Agap2, MRCKβ distinctively interacting with the detected iGluR subunits (GluN1, GluN2A, GluN2B, GluN2D, GluA1, and GluA2; Figure 4). Interactome analysis of the trisomic extrasynaptic fraction unraveled significant kinase-iGluR interaction, as shown by the significant increase of the network edges (predicted number of edges: 51 vs. expected number of edges: 21) and the PPI enrichment (p-value = 2.49.10−8), indicating the significant interactions of the PPI network. Moreover, several kinases (CaMKII kinase family members, cAMP-dependent protein kinase, PKC family, and Agap2) distinctively interacting with GriA1 and GriA2 subunits of the AMPARs were also significantly detected (Figure 5).
Altered iGluR Subsynaptic Phosphopattern in the Ts65Dn Mouse Hippocampus
Hippocampal subsynaptic fractions were analyzed by phosphoenrichment-coupled mass spectrometry to evaluate the predictive kinome-iGluR interactome model. The phosphoproteome of the different fractions was analyzed, and a total of 59 iGluR phosphosites were identified in the different biological replicates (Supplementary Table 3). From these, only those iGluR phosphosites found in both replicates were retained for further genotype effect analysis (Figure 6 and Supplementary Table 3). This technique allowed the identification of several iGluR phosphosites, mostly corresponding to previously reported phosphoresidues (15 individual phosphosites); two novel phosphosites were identified. Importantly, the subsynaptic iGluR phosphopattern was characterized in the hippocampus of Ts65Dn mice. Using this unbiased method, the identified upregulated phosphosites (displaying higher MS peaks in trisomic samples) showed an elevated representation of NMDAR subunits (eight phosphosites for GluN2A, seven for GluN2B, and one for GluN1), whereas only one phosphosite was detected in GluA1 subunit (AMPAR subunit) and no phosphosites were identified in KAR subunits. We performed western blot analysis on selected MS-identified differentially expressed phosphosites to further validate our results. This analysis showed an increase of both GluN1(pS890) and GluN2B(pS1284) levels in the postsynaptic fraction of trisomic mice, although not significant, in agreement with the changes observed along unbiased phosphoproteomic analysis (Supplementary Figure 2B). Finally, the kinases putatively phosphorylating the consensus iGluR phosphoresidues were in silico predicted using "Phosphomotif Finder" software. This analysis predicted a potential enzymatic activity of CaMKII, PKC, CK1 on the identified iGluR phosphoresidues, which would be in agreement with their above mentioned upregulated expression.
Figure 6. Phosphopattern profile of NMDA and AMPA receptor subunits in the adult Ts65Dn mouse hippocampus. (A–D) Schematic representation of phosphoproteome-identified phosphosites on NMDAR subunits (GluN1 and GluN2A and GluN2B) and AMPAR subunit GluA1, in adult hippocampal subsynaptic fractions. The phosphoresidue position is indicated, together with the presence and/or relative phosphorylation of trisomic:euploid MS peak (black: phosphosite present in euploid and trisomic samples; light green: increased levels detected in trisomic fractions; dark green: phosphosites exclusively detected in trisomic samples). (E) Table listing iGluR phosphosites identified in euploid and/or trisomic hippocampal fractions, together with the predicted phosphorylating kinases. Phosphosites previously reported in (1) Munton et al. (2007), (2) Lundby et al. (2012), (3) Li et al. (2016), (4) Maki et al. (2013), (5) Trinidad et al. (2012), (6) Zhang et al. (2013), (7) Wiśniewski et al. (2010) and (8) McQueen et al. (2017).
In the present study, we have performed a phosphoenrichment-coupled mass spectrometry analysis that showed altered functional changes in the hippocampal extrasynaptic and postsynaptic fractions of the Ts65Dn mice, a trisomic mouse model recapitulating DS phenotypic alterations. The majority of kinases interacting with iGluRs remarkably show an overall increase in trisomic samples, whereas the majority of iGluR-related phosphatases show reduction, with some exceptions in both protein categories. These results are concomitant with the increased number of iGluR phosphopeptides identified by the phosphoproteomic analysis of Ts65Dn mice hippocampal postsynaptic fractions. Importantly, in silico kinome-iGluR phosphopattern analysis supported the role of specific kinases and their potential iGluR phosphosites in the neuronal pathophysiology of Ts65Dn mouse.
The initial proteome analysis showed a relative conservation of the number of proteins (roughly 1,000 proteins identified), regardless of the subsynaptic fraction and genotype. The comparative study of the genotype effect on the subsynaptic fraction composition showed a protein coincidence of 60.7 and 57.8% for the postsynaptic and extrasynaptic fractions, respectively. This quantitative analysis indicates a different molecular composition of the Ts65Dn hippocampal synapses, which might result from the overexpression of triplicated genes. Hence, several proteins are differentially expressed in the subsynaptic compartments of Ts65Dn hippocampal neurons, potentially reflecting the molecular substrates associated with Ts65Dn synaptic abnormalities. Interestingly, further GO-term-based protein enrichment analysis supported functional outcomes of the proteomic changes detected in the postsynaptic and extrasynaptic fractions of trisomic mice. In particular, the postsynaptic fraction of trisomic mice showed a relative decrease of proteins related with "dendritic function" GO term. This finding is in agreement with previous studies showing a reduction of dendritic branching and spine density in Ts65Dn mice (Belichenko et al., 2007) which, in the context of individuals with DS, might recapitulate the dendritic deterioration reported in DS fetal brains (Weitzdoerfer et al., 2001). Moreover, GO analysis revealed a differential enrichment of structural synaptic proteins within the extrasynaptic compartment. In particular, the trisomic extrasynaptic compartment showed a significant enrichment of GO-term "synapse." Further analysis showed the presence of Homer3, one of the most abundant scaffolding proteins of the postsynaptic density in euploid mice (Hayashi et al., 2009), in extrasynaptic fractions of trisomic mice. These data, together with previous electron microscopy studies showing the presence of enlarged spines in Ts65Dn neurons (Belichenko, 2004; Benavides-Piccione et al., 2004), suggest that the morphological alterations of Ts65Dn spines might be associated with a disrupted protein composition of the extrasynaptic fraction. Interestingly, GO-term enriched analysis also showed a significant reduction of mitochondrial proteins in the extrasynaptic fraction of trisomic mice. This finding is in line with previous studies reporting a reduction of mitochondrial complexes and redox activity in neuronal cultures and DS fetuses, leading to severe mitochondrial dysfunctions that can affect synaptic metabolism, energy, and function (Izzo et al., 2014).
Besides the differential enrichment of structure- and energy-associated proteins, a significant alteration of phosphorylation-associated proteins was detected in the trisomic subsynaptic compartments. Indeed, the extrasynaptic trisomic fraction showed an enrichment of "protein phosphorylation" and "protein kinase activity" GO terms, while "protein phosphatase binding" (enriched in euploid extrasynaptic fraction) was significantly reduced. Although these evidences were suggestive of an altered enzymatic activity of kinases/phosphatases in Ts65Dn hippocampi, a more detailed analysis of kinase/phosphatase subsynaptic expression levels was performed. This analysis roughly showed an increase of kinase expression, together with a reduction of phosphatase levels in the Ts65Dn mouse model. In our study, further detailed analysis of the trisomic kinome showed increased levels of CaMKII subunits (alpha, beta, delta, and gamma subunits) in hippocampal postsynaptic and extrasynaptic fractions of Ts65Dn mice, by proteomics and WB techniques. This increase could result in an enhanced CamKII activity, similar to previous studies showing increased phosphorylated CaMKII in Ts65Dn hippocampi (Siarey et al., 2006). In agreement with this kinase increase, previous work of Fernandez and collaborators showed an increased amount of phosphopeptides in crude synaptosomes from Ts65Dn mice (Fernandez et al., 2009). Overall, our proteomic data and the previously cited morphological studies support the relationship between biochemical alterations and structural changes of the trisomic hippocampal neurons. These morphological and biochemical alterations can result in a subsynaptic mislocalization of proteins, potentially affecting synaptic shape, changing the postsynaptic density molecular composition, and ultimately the function synaptic activity of trisomic neurons. In summary, these results strengthen the hypothesis that morphological and functional dendritic/synaptic abnormalities, such as those detected in our study, may directly impact synaptic plasticity (Cramer and Galdzicki, 2012) and lead to cognitive impairment.
Interactome analysis revealed significant interactions between protein kinases enriched in trisomic subsynaptic fractions and iGluR subunits. In the postsynaptic fraction, these kinases interact with both NMDAR and AMPAR subunits. In contrast, the interactome analysis only revealed potential protein–protein interactions of these kinases with GluA1 and GluA2 subunits of the AMPAR, which are proportionally more largely represented than NMDAR subunits in the extrasynaptic fraction. Following this predictive analysis, we performed an accurate characterization of the iGluR phosphopattern in hippocampal subsynaptic fractions of Ts65Dn mice. Further bioinformatic analyses were focused on the phosphorylation of these receptors to unveil potential changes on iGluR phosphopattern. In sample preparation, the fractions followed tryptic digestion with a single protease (trypsin) for further unbiased MS method. Technically, the use of a single protease limited the extent of detectable phosphopeptides (Dephoure et al., 2013). Moreover, the reported low abundance of phosphopeptides (Parker et al., 2010) might also contribute to underestimate the complete phosphoproteome of trisomic hippocampal synapses. Despite these intrinsic technical limitations, a total amount of 17 individual iGluR phosphosites were identified in our study, 15 of them corresponding to previously reported phosphosites (PhosphositePlus, Cell Signaling Technology Inc.) (Lussier et al., 2015) (Figure 6E). In the past, Siarey and collaborators showed increased phosphorylation levels of iGluR phosphosites in Ts65Dn mice, such as GluA1(pS831) (Siarey et al., 2006). In line with these, our results showed an over-representation of iGluR phosphoresidues in specific Ts65Dn synaptic compartments identified, supporting the hypothesis that iGluR phosphopattern is associated with the synaptic transmission and glutamatergic dysfunctions reported in the Ts65Dn mouse model of DS. Furthermore, iGluR phosphoprofile analysis provided potential functional links between increased levels of iGluR sububnits in Ts65Dn synaptic fractions and in silico predicted kinases phosphorylating those phosphoresidues. This group of candidate kinases contained CaMKII, PKC, and CK1, previously described as iGluR regulatory kinases (Lan et al., 2001; Chergui, 2005; Sanz-Clemente et al., 2013; Lussier et al., 2015). Our proteomic study significantly showed an upregulation of these kinases in trisomic hippocampi, supporting the association between their overexpression and the changes on iGluR phosphopattern. Regarding the latter, a significant elevated representation of GluN2A and GluN2B subunit phosphopeptide was detected in trisomic postsynaptic fractions. This biochemical signature suggests a relevant contribution of abnormally phosphorylated GluN2A- and GluN2B-containing NMDARs in Ts65Dn phenotype. Indeed, our group and others have previously provided biochemical and pharmacological evidences supporting the role of NMDAR alterations in the DS murine models (Costa et al., 2007; Altafaj et al., 2008; Hanson et al., 2013; Grau et al., 2014). It has been reported that, specifically, CA3-CA1 hippocampal LTP is functionally reduced in Ts65Dn mice (Siarey et al., 2006). Moreover, trisomic mice exhibit an increased NMDAR-dependent LTD that can be pharmacologically rescued with memantine, an NMDAR antagonist (Scott-McKean and Costa, 2011). The alterations on NMDAR activity observed in Ts65Dn mice might probably be influenced by changes in NMDAR expression levels and/or the NMDAR (and other iGluR) phosphopattern signature described in the present study. Likewise, our previous studies showed that in the TgDyrk1A transgenic mouse model, DYRK1A overexpression is associated with GluN2A subunit upregulation (Altafaj et al., 2008). This increased density mechanistically results from a Dyrk1A-mediated phosphorylation of GluN2A subunit (GluN2A(pS1048)) that reduces NMDAR internalization and functionally increases NMDAR-mediated currents and modifies NMDAR gating properties (Grau et al., 2014). Unfortunately, the methodology followed in this study (trypsin digestion) was not compatible with GluN2A(pS1048) detection. Although our study revealed Dyrk1A presence in the hippocampal postsynaptic fraction and previous studies showed DYRK1A overexpression in DS, our data suggest that Dyrk1a-mediated phosphorylation GluN2A(pS1048) might contribute to DS synaptic alterations, a hypothesis that might be explored in the future. Interestingly, several iGluR phosphosites identified in this study have been associated with synaptic plasticity mechanisms. Among these, GluN1(pS890) has been showed to disturb GluN1 surface clusters (Tingley et al., 1997), and GluN2A(pS929) phosphorylation has been described as modulating GluN2A subunit containing NMDAR desensitization (Maki et al., 2013). Regarding GluN2B subunit phosphorylation, GluN2B(pS1303) phosphorylation has been shown to increase GluN1/GluN2B currents (Liao et al., 2001), and GluN2B(pS1116) phosphorylation potentiates NMDAR currents and Ca2+ permeability (Murphy et al., 2014). Overall, the presence of discrete NMDAR phosphosites with relevant function in synaptic function and the identification of their relative overrepresentation in trisomic mice support the contribution of glutamatergic neurotransmission alterations in Ts65Dn mice phenotype.
Furthermore, some of the phosphosites found to be over-represented in our study had previously been described to be involved in synaptic plasticity. For example, GluN1 S890 phosphorylation disperses the surface clusters of GluN1 (Tingley et al., 1997). GluN2A S929 phosphorylation modulates desensitization of GluN2A/NMDA receptors (Maki et al., 2013). GluN2B S1303 phosphorylation increases GluN1/GluN2B currents (Liao et al., 2001). GluN2B S1116 phosphorylation potentiates the receptor currents and its permeability to Ca2+ (Murphy et al., 2014). Consequently, their upregulation could lead to alterations in synaptic plasticity. Overall these findings suggest that the altered kinome and iGluR phosphopattern (mainly affecting GluN2A and GluN2B subunits) can contribute to the glutamatergic neurotransmission dysfunctions present in the Ts65Dn mouse model of DS.
In summary, our study revealed an altered subsynaptic composition of the adult Ts65Dn mouse hippocampus. These molecular subsynaptic alterations might functionally be underlying the Ts65Dn-associated dendritic deterioration, altered synapse organization, and mitochondrial dysfunction. Furthermore, this phosphoproteomic study provides an extensive repertoire of the iGluR phosphosites, together with the respective predicted kinases. Importantly, although the mechanistic studies remain elusive, the characterization of the iGluR phosphopattern and the predicted associated kinases in the Ts65Dn hippocampi may help to identify novel therapeutic targets for DS synaptopathy.
XA and MG contributed to the conception and design of the study. MG performed the experimental and bioinformatic analyses, with the technical help from CG. MG, FC, and XA wrote the manuscript. All authors contributed to the revision of the manuscript revision and also read and approved the submitted version.
This work was supported by the Grants La Marató (Project N. 20140210), PI16/00851 (ISCIII), PCIN-2014-105 (MINECO) and Miguel Servet Program (CPII16/00021, ISCIII) to XA. MG and CG contract were funded by Fundació La Marató (Project N. 20140210).
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.
The authors would like to thank G. Gou (Hospital de la Santa Creu i de Sant Pau-UAB, Barcelona, Spain) and X. Morató and M. López (Unitat Neurofarmacologia, Universitat Barcelona, Spain) for their technical support in the synaptosomes preparation. We also acknowledge Dr. M. López Heredia (Unitat Genètica, IDIBELL), G. Gou, and Dr. À. Bayés (Hospital de la Santa Creu i de Sant Pau-UAB, Barcelona, Spain) for guidance in bioinformatic analysis. We thank Dr. Eduard Sabidó and Cristina Chiva (Proteomics Unit, Center for Genomic Regulation, Barcelona, Spain) for technical help in MS studies and data managing.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol.2018.00226/full#supplementary-material
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Wang, J. Q., Liu, X., Zhang, G., Parelkar, N. K., Arora, A., Haines, M., et al. (2006). Phosphorylation of glutamate receptors: a potential mechanism for the regulation of receptor function and psychostimulant action. J. Neurosci. Res. 84, 1621–1629. doi: 10.1002/jnr.21050
Weitzdoerfer, R., Dierssen, M., Fountoulakis, M., and Lubec, G. (2001). Fetal life in Down syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure. J. Neural Transm. 61, 59–70. doi: 10.1007/978-3-7091-6262-0_5
Westphal, R. S. (1999). Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex. Science 285, 93–96. doi: 10.1126/science.285.5424.93
Wiśniewski, J. R., Nagaraj, N., Zougman, A., Gnad, F., and Mann, M. (2010). Brain phosphoproteome obtained by a FASP-based method reveals plasma membrane protein topology. J. Proteome Res. 9, 3280–3289. doi: 10.1021/pr1002214
Wiśniewski, J. R., Zougman, A., Nagaraj, N., and Mann, M. (2009). Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362. doi: 10.1038/nmeth.1322
Zhang, F., Guo, A., Liu, C., Comb, M., and Hu, B. (2013). Phosphorylation and assembly of glutamate receptors after brain ischemia. Stroke 44, 170–176. doi: 10.1161/STROKEAHA.112.667253
Keywords: Down syndrome, ionotropic glutamate receptors, NMDA receptor, kinases, proteomics, phosphoproteomics
Citation: Gómez de Salazar M, Grau C, Ciruela F and Altafaj X (2018) Phosphoproteomic Alterations of Ionotropic Glutamate Receptors in the Hippocampus of the Ts65Dn Mouse Model of Down Syndrome. Front. Mol. Neurosci. 11:226. doi: 10.3389/fnmol.2018.00226
Received: 01 March 2018; Accepted: 11 June 2018;
Published: 25 July 2018.
Inmaculada Maria Gonzalez-Gonzalez, University of Central Lancashire, United Kingdom
Antonio Sanz-Clemente, Feinberg School of Medicine, Northwestern University, United States
Mariana Vargas-Caballero, University of Southampton, United Kingdom
Copyright © 2018 Gómez de Salazar, Grau, Ciruela and Altafaj. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Xavier Altafaj, xaltafaj@idibell.cat
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The European Defence Fund continues to deliver with another set of EU-funded defence research projects. The Fund, launched by President Juncker in June 2017, is a catalyst for the creation of a strong EU defence industry. It boosts defence capabilities and builds new partnerships across borders.
Elżbieta Bieńkowska, Commissioner for Internal Market, Industry, Entrepreneurship and SMEs said: "The European Defence Fund is essential to bring more cooperation between defence companies and the military in the European Union and to ensure Europe's strategic autonomy. The pan-European collaborative defence research projects supported by the Fund are designed to ensure Europe's technological leadership, lay the foundations for future defence capabilities and support a more innovative and competitive European defence industry."
The Ocean2020 project supports maritime surveillance and interdiction missions at sea and to that end will integrate drones and unmanned submarines into fleet operations. The information acquired will be combined with conventional systems to build up a comprehensive picture of developing situations for military commanders.
The project will be run by a consortium led by Leonardo S.p.A, bringing together 42 partners from 15 EU countries. It will receive a grant of around €35 million. In 2019, the project will stage two real-life demonstrations – one in the Baltics led by the Swedish navy and one in the Mediterranean led by the Italian navy.
ACAMSII will develop adaptive camouflage that will protect soldiers against sensors operating in several wavelength ranges.
Gossra will improve the compatibility of complex system elements (e.g. sensors or digital goggles) carried by soldiers.
Vestlife seeks to develop ultralight body armour for dismounted soldiers.
The grant agreements for these projects will be signed in the coming weeks.
Today's announcement on support for these defence research projects comes on the opening day of the annual Munich Security Conference, where President Juncker will present further steps to promote a stronger Europe on security and defence. The President will be accompanied by Vice-President Frans Timmermans, Commissioners Johannes Hahn, Elżbieta Bieńkowska, and Julian King.
Under the research strand of the European Defence Fund, €90 million will be allocated to defence research grants fully and directly funded from the EU budget (2017-2019). Projects to be funded have been selected following the first call for defence research proposals in 2017. In December 2017, the first grant agreement was signed with the PYTHIA consortium. This project aims to identify key trends in the fast evolving world of innovative defence technologies.
The Commission will also soon adopt the defence research work-programme for 2018 and ask the European Defence Agency to open the new calls for defence research proposals for this year. These calls will focus on electronic design technologies for defence applications and a European high-power laser effector. This next batch of research grants for defence will be signed by the end of 2018.
As announced in June 2017, the Commission will come forward with a proposal to establish, post 2020, a €1.5 billion per year European Defence Fund to support defence capabilities, out of which an estimated annual budget of €500 million will be dedicated to defence research, making the EU one of the biggest defence research investors in Europe.
Ocean 2020: the project aims to enhance situational awareness in a maritime environment by using manned and unmanned systems and building a complete picture on the basis of many different inputs. Defence Ministries in Estonia, France, Greece, Italy, Lithuania, the Netherlands, Portugal, Spain, Sweden and the United Kingdom are involved. The industrial partners are: Indra, Safran, Saab, MBDA, PGZ/CTM Hensoldt, Intracom-IDE, Fincantieri and Qinetiq. Research centres include Fraunhofer, the Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek (TNO), the Centre for Maritime Research and Experimentation (CMRE NATO) and the Italian Istituto Affari Internazionali (IAI).
ACAMSII: this project will develop adaptive camouflage for soldiers that will protect them against sensors operating in several wavelength ranges. Partners from France, Germany, Lithuania, the Netherlands, Portugal and Sweden are involved. Participating companies are from the textile, aerospace and defence system integrators sectors: CITEVE, Damel and Safran. Research centres are the Swedish Totalförsvarets forskningsinstitut (FOI), Fraunhofer, and the Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek (TNO).
GOSSRA: this project focuses onensuring thatcomplex system elements worn by soldiers work together. Soldiers are equipped with a range of devices which are required to work together. The project aims to create a methodology for specifying how components connect, making it much easier to develop new devices that can work with existing equipment. Partners from Germany, Italy, the Netherlands, Poland, Portugal, Spain and Sweden will run the project. The companies involved are Rheinmetall, Indra, GMV aerospace and defence, Leonardo, Larimart and Saab; SMEs Tekever and iTTi and the research institute Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek (TNO).
Vestlife: the goal of this project is to create protective clothing for soldiers. It will work on developing clothes that are effective for defence purposes, but lighter, more flexible and comfortable at the same time. Partners from Finland, Italy, the Netherlands, Portugal and Spain, together with companies CITEVE and FY-composites, research institutes AITEX and TECNALIA are involved. Two small and medium enterprises (SMEs) BRAPA and Petroceramics are also taking part in the project.


In February 2008, HE President Teodoro Obiang Nguema Mbasogo unveiled a historic national economic and social development plan called Horizon 2020, which outlined its main objectives over the next 12 years. The country was dedicated to laying the foundation for economic and social development; transforming infrastructure to strengthen economic competitiveness; improving quality of life; and building good governance.
The main priority for public investment policy has been infrastructure and the country has built nearly 2,500 kilometers of roads and several schools and health centers and public buildings throughout the country. As a result, human capital has been a noteworthy beneficiary. Student per classroom rates are now in line with international standards and efforts to eradicate poverty are on track to meet UN Millennium Development Goals.
Equatorial Guinea has placed considerable emphasis on good governance as part of Horizon 2020. In 2011, the government introduced constitutional reforms to improve transparency and accountability. This resulted in the creation of a bicameral parliament, a court of auditors, a national ombudsman, the Economic and Social Council and the Council of the Republic. The government also established an anti-corruption judiciary and prosecutor and created the National Agency of Financial Research to honor transparency commitments.


text:
/**
*============================================================================
*  The Ohio State University Research Foundation, The University of Chicago -
*  Argonne National Laboratory, Emory University, SemanticBits LLC,
*  and Ekagra Software Technologies Ltd.
*
*  Distributed under the OSI-approved BSD 3-Clause License.
*  See http://ncip.github.com/cagrid-portal/LICENSE.txt for details.
*============================================================================
**/
package gov.nih.nci.cagrid.portal.dao;

import gov.nih.nci.cagrid.portal.DBTestBase;
import gov.nih.nci.cagrid.portal.domain.NotificationLogMessage;
import static org.junit.Assert.*;
import org.junit.Test;

/**
 * User: kherm
 *
 * @author kherm manav.kher@semanticbits.com
 */
public class NotificationLogMessageDaoTest extends DBTestBase<NotificationLogMessageDao> {

    @Test
    public void get() {

        assertNotNull(getDao().getAll());
        NotificationLogMessage loaded = getDao().getById(-1);
        assertFalse(loaded.isSendSuccessful());
        loaded.setSendSuccessful(true);
        getDao().save(loaded);
        NotificationLogMessage reloaded = getDao().getById(-1);
        assertTrue(reloaded.isSendSuccessful());

    }
}

redpajama_set_name: RedPajamaGithub


An Ocala woman shot a naked intruder who forced his way into her home, the Marion County Sheriff's Office stated in a media release on Monday.
Sometime Sunday evening, a 54-year-old Ocala woman was in her kitchen when 27-year-old Victor Alex Etherington allegedly knocked on her window and demanded that he wanted to come inside, deputies stated.
Since the woman did not recognize the man, she told him to leave. The suspect allegedly kicked her door open and the victim ran upstairs to her bedroom, called 911 and armed herself, according to deputies.
"The suspect then followed her upstairs to the bedroom at which time she locked herself in the bedroom closet. The suspect forced entry into the bedroom, took off his shorts, (which he had defecated in) and forced entry into the closet where the victim was hiding," deputies stated.
The woman then fired her .22 caliber handgun and hit the suspect in the abdomen, which stopped him, deputies explained.
When deputies arrive at the home, they allegedly found Etherington naked and hiding behind the bedroom door.
Etherington was transported to Ocala Regional Medical Center where he underwent surgery and is listed in stable condition.
Deputies stated that they believe Etherington was under the influence of alcohol at the time of the incident and may have lived in the home 12 years earlier.


Researchers from ZeroFox build a tool that employs neural networks to trick unsuspecting Twitter users.
Phishing is big business for attackers, and being able to craft the right message to a target is a valuable skill. Social networks are great resources for potential attackers, both to learn about a target victim and to deploy a phishing message that will be clicked.
At the recent Black Hat USA conference, researchers from security firm ZeroFox detailed their research using neural networking skills to build an automated spear phishing capability that goes after Twitter users. ZeroFox Senior Data Scientist Phil Tully earned a Ph.D. in neural networks by doing research to understand how patterns and spikes could be simulated in software to model and describe how networks and neurons store memories.
In an interview with eSecurityPlanet, Tully noted that he's often asked by former colleagues why he's working at ZeroFox on IT security. He explained that fundamentally understanding the neural network of the human body is somewhat similar to understanding an artificial environment like IT.
"In the same way that our eyes, ears and nose receive different stimuli on a millisecond basis and then make sense of it for conscious thought and memory, the way the ZeroFox platform also works on a second-by-second basis," Tully said. "With the scale of Twitter for example, our platform is able to monitor social networks and is able to distill the large amount of posts into actionable information."
That actionable information could include data on potential phishing links and fraud, which leads to the research with a new tool dubbed SNAP_R (Social Network Automated Phishing with Reconnaissance). Most attacks to date on social media including Twitter have largely been low-effort phishing attempts that have low-success rates. Rather than using this scattershot approach, the SNAP_R tool starts with an automated target discovery capability, said John Seymour, a data scientist at ZeroFox.
The output of the discovery is that a number of different Twitter users can be targeted with tweets that contain the target's username, so it's more likely targets will click on them. The targeted tweet is also appended with a shortened URL. The SNAP_R tool can be used to grab usernames that follow or post to a certain hashtag, for example #infosec. The algorithm will then be able extract information from the user's Twitter account, including the user's biography, posting frequency and how many followers they have.
"Our tool will help select users that we then deem to be high-value targets," Tully explained. "SNAP_R then relies on a neural network to generate a tweet out to the high-value user."
The neural network uses what is known as an LSTM, which is an acronym of Long, Short-Term Memory. The ZeroFox LSTM neural network was pre-trained with several million tweets, so it could produce Twitter messages that seem authentic.
SNAP_R can go to a target victim's timeline, extract information and then select a topic that is important to the victim. The system is also able to understand what time of day the user is most active. The LSTM neural network is loaded with information on the given topic, and SNAP_R is able to tweet a relevant message to the target at a time when the target is typically active.
The SNAP_R system is able to target thousands of users at a time. As part of the research, the appended links were benign and not malicious. In one experiment, ZeroFox researchers sent out 90 phishing posts to people that had posted on Twitter using the hashtag #cat. After two hours, the SNAP_R-generated link had a 17 percent clickthrough rate, which grew to 66 percent after two days.
"People make a split second judgment whether to click a link," Seymour said. "If I see a post, with a profile picture, posting on a topic that I engage with frequently and it seems to be legitimate, my initial reaction is to trust it."
Seymour added that the goal of the research is help bring awareness to the issue of social engineering on social networks.


text:
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We apologize for any inconvenience this may cause. Please bear with us and you will receive your order as soon as possible.
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Wall Street Journal Review: Return of the Strong Gods is "a thoughtful contribution to American political debate"
December 30, 2019 | ballison | News
Excerpt from Barton Swaim's Wall Street Journal review, Politics: Liberalism Is as Liberals Do (Dec. 27, 2019).
After reading R.R. Reno's "Return of the Strong Gods: Nationalism, Populism, and the Future of the West" , I'm inclined to think Mr. Traub's most revealing lines come at the end of a chapter on liberal antitotalitarianism: "Liberals defended a commitment to truth and reason in the face of the big lie, and to incrementalism in the face of visionary madness," Mr. Traub writes. "It is true that the threat they faced has long since faded; ours is of a different nature. But we have learned, as they knew, that the liberal virtues of reason, pragmatism, and tolerance are always in jeopardy—not from outside forces, but from ourselves." Mr. Reno has written his book precisely because this is how today's liberals and, he believes, many conservatives still think: Totalitarianism is always upon us, and what we must do is embrace "pragmatism" instead.
Midcentury intellectuals such as Karl Popper and Friedrich Hayek countered the totalitarian ideologies of communism and fascism by insisting that the Western democratic polity concern itself only with questions of individual and economic liberty and not with cosmic questions of national destiny—with "incrementalism" and not with "visionary madness," as Mr. Traub has it. The problem, for Mr. Reno, is that American intellectuals still take this attitude, even to the point of absurdly suggesting, as Mr. Traub does obliquely, that the present administration in Washington poses some form of a totalitarian threat.
The struggle against totalitarian ideologies was real—half a century ago. Now, "defeating them has become a destructive preoccupation," Mr. Reno says. "Today, the greatest threat to the political health of the West is not fascism or a resurgent Ku Klux Klan but a decline in solidarity and the breakdown of the trust between leaders and the led."
According to what Mr. Reno calls the "postwar consensus," the only way to combat the "metaphysical" visions that brought Europe to destruction between 1914 and 1945 was to "go small": to embrace critique rather than transcendence, meaning rather than truth, peace rather than unity. This has led to a worldview of "pure negation": antifascism, antiracism, antihomophobia, resistance. These "anti imperatives" are the "weak gods." But human societies will not be satisfied with negation forever; the "sacralizing impulse in public life is fundamental." Eventually the strong gods return. The task is to ensure they are life-affirming and not degrading, ennobling and not violent.
"Return of the Strong Gods," an expansion of a 2017 essay that appeared in the journal First Things, which Mr. Reno edits, is a thoughtful contribution to American political debate. It is incisively written and full of mordant observations. Mr. Reno explains, better than any book I can remember, the present-day progressive's paranoid fear of fascism and neurotic determination to ferret out racism where none exists.
I would register one criticism. Mr. Reno contends that the post-1945 struggle between right and left in America has often amounted to little more than a "sibling rivalry" because both adopted the negating assumptions of the postwar consensus. The right wants deregulation of the economy, the left deregulation of the culture. That's fair, and he may legitimately complain that segments of the American right fixate on economic gains at the expense of metaphysical concerns, but he is wrong to imply that arguments for the free market have only materialist ends in view. The prosperity produced by free markets may debase and coarsen in the absence of higher values; but it's also true that free markets encourage work and industry, whereas command economies, though they gesture at patriotism, promote sloth, resentment and dependency. Mr. Reno writes vaguely of "economic solidarity," but as a practical matter I'm not sure what members of today's federal apparatus can be trusted to put his noble aims into practice. The wiser course, spiritually and materially, is still to deregulate. If the strong gods are back, they will need hard workers.
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redpajama_set_name: RedPajamaCommonCrawl


Cutie patooties! And great job on the memory verses Mom. You are arming them young which is great!!!
I have dial up so I can't watch the video, but I saw the last ones you posted. They are so cute, and great job on the verses! My daughter is in Awanas and it is great to hear he say her verses every week too!


VRBO is Vacation Rental By Owner. We have done many VRBO rentals and they have been outstanding. What makes it good, as opposed to AirBnB and others, is there is no middleman; you can be in direct contact with the owner of the property so you can get information easily. You can pay the owner directly. You are able to rate the owner/property and they are able to rate you, so it keeps everyone honest.
VRBO has the largest number of vacation rental properties and has listings all over the world. We may continue to use VRBO but there are others out there as well: homeescape.com and hometogo.com work in a similar way to VRBO, but do not seem to have the depth of listings.
In all cases, listings come with numerous photos of the property so you can get a pretty good idea in advance of what you will be getting. There are reviews from former renters and they are important to read to get a sense of what the place is like. The listing includes the available amenities, eg. internet, cable, washer/dryer (very important for long term stays, and allows you to pack much lighter as well). We prefer staying where there is a well-equipped kitchen where we can prepare our own meals. This cuts down on the expense of eating out all the time and allows you to explore foods that are available locally. Our typical pattern is to eat a light breakfast at home, go out for lunch, then a light dinner at home again. We also like having a "home base" where we can unpack and not have to live out of a suitcase. We take a lot of day trips and occasionally a short overnight at a hotel if the distance is too far, but all in all, it is more economical and much less stressful than traveling from place to place.
I recommend the idea of vacation rental sites to anyone thinking about traveling and staying somewhere for a period of time. I think less than a week would not be worth it, but a week or more, makes sense. There is a lot to consider. Do your homework, research as much as you can, but we have had good luck using vacation rental sites.


text:
Journal cut positive comments, author claims
Essex scholar raises fears over peer review integrity
By Paul Jump
Twitter: @PaulJump
Source: Getty
Any good news? All comments should be reported to author, some argue
An academic has called for authors to have the right to see everything written about them in referees' reports after discovering that positive comments made about a rejected manuscript had been removed.
Mark Harvey, director of the Centre for Research in Economic Sociology and Innovation at the University of Essex, submitted a paper to the Routledge journal Economy and Society last year. It was rejected after being reviewed by five referees, some of whom had been suggested by Professor Harvey.
But by comparing the version of the reviewers' comments he received with those submitted by two of his recommended referees, he found that several positive comments about his paper had been removed.
Professor Harvey told Times Higher Education he feared that the comments may have been removed to justify the rejection. He added that the form for referees' comments used by Economy and Society did not suggest that reports could be edited before being shown to authors.
According to Professor Harvey, the journal's managing editor, Fran Tonkiss, reader in sociology at the London School of Economics, told him that only direct recommendations for publication had been excised from the reports. However, Professor Harvey disputed this and noted that a recommendation against publication in one report remained uncut.
He said Dr Tonkiss had told him that the journal would alter the wording of its referees' reporting form to indicate that they could be edited. But Professor Harvey argued that this would merely give editors licence to continue unfairly editing reports. He said that "clear rules" should be laid down stating explicitly that authors should be shown the full version of the reports.
All the colleagues with whom he and one of the referees of his paper - Norman Geras, professor emeritus in politics at the University of Manchester - discussed the case had expressed "surprise and shock" at Economy and Society's behaviour, Professor Harvey said.
Dr Tonkiss could not be contacted for comment. A Routledge spokeswoman said that information on editorial decisions provided to authors by Economy and Society "combines the feedback from referee reports, board members and the editor".
Liz Wager, a publication consultant and former chair of the Committee on Publication Ethics, said it was not uncommon for referees' reports to be edited, and most editors agreed that "offensive, ad hominem attacks" should be cut.
But in her view, "if the editor disagrees with the reviewer - especially over recommendations about whether to publish or not - then it is up to the editor to explain this to the author".
Irene Hames, an editorial consultant and a Cope council member, agreed. She said reports could also be edited to tidy up ambiguous language or to remove explicitly confidential material. However, "journals shouldn't use selective editing of reviewer reports to help them justify or be a better fit for a decision".
"Decision-making should be transparent," she added.
paul.jump@tsleducation.com
Reader's comments (2)
#1 Submitted by LalehK on May 7, 2013 - 9:34am
Two comments: "But by comparing the version of the reviewers' comments he received with those submitted by two of his recommended referees"... 1) Does that mean that the article author was mates with the referees so he could receive and compare the unedited version? 2) Shouldn't we want journals to be as selective as possible? Doesn't the integrity of a journal lie in its selectivity? 3) Ultimately, the editors of a journal decide whether or not an article is suitable for publishing, weighing various referee reports against each other (and taking into account whether a referee is "recommended" by the author or chosen by the editorial board).
#2 Submitted by Paul Jump on May 7, 2013 - 2:01pm
Re. the comment above, my understanding is that the journal invited authors to nominate three referees. One of those nominated refereees, Professor Geras, told Professor Harvey he had written a review. But Professor Harvey was unable to identify which report he had written, so sent him all the reports. It was at this point he noticed the cuts t his review. Professor Harvey added: "As to the procedure of an author being requested to nominate referees, I personally have not encountered it before, and do not in general support the practice. Both referee and author assumed that the editors would take into account which reports were written by nominated referees when making their decision."
University of California defiant as Elsevier cuts journal access
US' top producer of research papers hopes stance will force major gains in open access
By Paul Basken
Research publications: does piling them high sell them short?
All modern academics know that it's publish or perish, but is regular publication a gateway or a barrier to groundbreaking scholarship? Simon Baker assesses the data on the relationship between research volume and quality and asks which, if either, should be prioritised
By Simon Baker
Code to tackle 'ethics dumping' gaining momentum
Uclan joins University of Cape Town in adopting rules to stamp out export of unethical research practices to poorer countries
By Nick Mayo
More clarity is needed over hybrid journals' Plan S compliance
Recent confusion over The Lancet's stance on green open access highlights the difficulty for support staff in preparing researchers for the new rules, says Alice Gibson
By Alice Gibson
Postdoctoral Researcher Level 1 on DCU Research Career Framework
Dublin City University
Associate Professor, Department of Chemical and Petroleum Engineering
Enid Linder Foundation / Royal College of Surgeons Consultant Senior Lecturer in Clinical Trials


Has anyone tried Chatham boots at all? What did you think of them? What is the sizing like on them?
Not had those. Are they Shires?
I'm looking at some of their country/riding boots. I read a review that says they came out big, although not the style of boot I'm looking at, I had hoped someone else had tried them.
Well just bit the bullet and ordered myself a pair of the paddock boots, really hope they are nice and they fit now!


text:
Mobilink Becomes 1st Pakistani Telco to Implement Facebook at Work
Posted 3 years ago by Press Release
In a move to increase efficiency and collaboration amongst employees spread across the country and ensure communication can become more efficient, Mobilink has launched Facebook at Work, the business version of Facebook that allows companies to build more productive, efficient and collaborative workplaces.
The enterprise service shares a similar look and feel to the personal Facebook network but is completely separate from the consumer Facebook service. The service also has built in enterprise-grade security and administration tools.
As we move towards digitalization, Facebook at Work will serve as a platform which will not only connect our employees better but will also serve as an informal platform for information sharing. – Jeffrey Hedberg, President and CEO – Mobilink
"Facebook at Work has not only increased employee engagement, but has removed typical line management and hierarchical barriers to communication. It has also improved two-way communications and reduced outbound email traffic," added Ehtisham Rao, Head of Digital Services – Mobilink.
With many of Mobilink's employees already using the consumer version of Facebook, adoption of Facebook at Work throughout the organization was a simple process for Mobilink's HR and Digital teams. The Telco initiated various creative engagement activities i.e. a Dubsmash Competition, Knowledge Share, and other similar activities to promote the use of the enterprise service amongst employees.
With Facebook at Work, we have a tool which allows us to further embed our work culture across the organization. We want to derive an open, collaborative and non-hierarchical culture through the utilization where teams can communicate, share and solve problems in ways that traditional tools cannot. – Asghar Jameel, Chief Human Resource Officer – Mobilink
Facebook at Work looks and feels the same as the Facebook that we use every day. The key difference is that it allows workers to create an additional work account that is separate from their own personal Facebook account. It uses familiar Facebook features like the news feed, groups, messages, and events, but has been designed purely for sharing content and collaborating with co-workers to build a more connected and productive workplace.
Umer says:
What is your source? I request you to not write on topics, which you do not have enough information and knowledge. Facebook has launched [email protected] as Pilot and has been in use by many other companies including Telenor :)
Muhammad Faheem says:
brother its press release
Well done Mobilink
Its Me says:
uffff very angry young man Umer… itna Ghusa