Publications by authors named "Maksym V Kopanitsa"

20 Publications

  • Page 1 of 1

Impaired instrumental learning in Spred1 mice, a model for a rare RASopathy.

Genes Brain Behav 2021 Feb 23:e12727. Epub 2021 Feb 23.

Department of Human Genetics, KU Leuven, Leuven, Belgium.

RASopathies are neuro-cardio-facio-cutaneous disorders stemming from mutations in genes regulating the RAS-MAPK pathway. Legius syndrome is a rare RASopathy disorder caused by mutations in the SPRED1 gene. SPRED1 protein negatively regulates activation of Ras by inhibiting RAS/RAF and by its interaction with neurofibromin, a Ras GTPase-activating protein (RAS-GAP). Cognitive impairments have been reported in Legius syndrome as well as in other RASopathy disorders. Modeling these cognitive deficits in a Spred1 mouse model for Legius syndrome has demonstrated spatial learning and memory deficits, but other cognitive domains remained unexplored. Here, we attempted to utilize a cognitive touchscreen battery to investigate if Spred1 mice exhibit deficits in other cognitive domains. We show that Spred1 mice had heterogeneous performance in instrumental operant learning, with a large subgroup (n = 9/20) failing to reach the standard criterion on touchscreen operant pretraining, precluding further cognitive testing. To examine whether targeting the RAS-MAPK signaling pathway could rescue these cognitive impairments, Spred1 mice were acutely treated with the clinically relevant mitogen-activated protein kinase kinase (MEK) inhibitor PD325901. However, MEK inhibition did not improve their instrumental learning. We conclude that Spred1 mice can model severe cognitive impairments that cannot be reversed in adulthood.
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http://dx.doi.org/10.1111/gbb.12727DOI Listing
February 2021

Learning and reaction times in mouse touchscreen tests are differentially impacted by mutations in genes encoding postsynaptic interacting proteins SYNGAP1, NLGN3, DLGAP1, DLGAP2 and SHANK2.

Genes Brain Behav 2021 Jan 29;20(1):e12723. Epub 2020 Dec 29.

Synome Ltd, Babraham Research Campus, Cambridge, UK.

The postsynaptic terminal of vertebrate excitatory synapses contains a highly conserved multiprotein complex that comprises neurotransmitter receptors, cell-adhesion molecules, scaffold proteins and enzymes, which are essential for brain signalling and plasticity underlying behaviour. Increasingly, mutations in genes that encode postsynaptic proteins belonging to the PSD-95 protein complex, continue to be identified in neurodevelopmental disorders (NDDs) such as autism spectrum disorder, intellectual disability and epilepsy. These disorders are highly heterogeneous, sharing genetic aetiology and comorbid cognitive and behavioural symptoms. Here, by using genetically engineered mice and innovative touchscreen-based cognitive testing, we sought to investigate whether loss-of-function mutations in genes encoding key interactors of the PSD-95 protein complex display shared phenotypes in associative learning, updating of learned associations and reaction times. Our genetic dissection of mice with loss-of-function mutations in Syngap1, Nlgn3, Dlgap1, Dlgap2 and Shank2 showed that distinct components of the PSD-95 protein complex differentially regulate learning, cognitive flexibility and reaction times in cognitive processing. These data provide insights for understanding how human mutations in these genes lead to the manifestation of diverse and complex phenotypes in NDDs.
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http://dx.doi.org/10.1111/gbb.12723DOI Listing
January 2021

Cognitive disturbances in the cuprizone model of multiple sclerosis.

Genes Brain Behav 2021 Jan 25;20(1):e12663. Epub 2020 May 25.

Charles River Discovery Services, Kuopio, Finland.

Cognitive problems frequently accompany neurological manifestations of multiple sclerosis (MS). However, during screening of preclinical candidates, assessments of behaviour in mouse models of MS typically focus on locomotor activity. In the present study, we analysed cognitive behaviour of 9 to 10-week-old female C57Bl/6J mice orally administered with the toxin cuprizone that induces demyelination, a characteristic feature of MS. Animals received 400 mg/kg cuprizone daily for 2 or 4 weeks, and their performance was compared with that of vehicle-treated mice. Cuprizone-treated animals showed multiple deficits in short touchscreen-based operant tasks: they responded more slowly to visual stimuli, rewards and made more errors in a simple rule-learning task. In contextual/cued fear conditioning experiments, cuprizone-treated mice showed significantly lower levels of contextual freezing than vehicle-treated mice. Diffusion tensor imaging showed treatment-dependent changes in fractional anisotropy as well as in axial and mean diffusivities in different white matter areas. Lower values of fractional anisotropy and axial diffusivity in cuprizone-treated mice indicated developing demyelination and/or axonal damage. Several diffusion tensor imaging measurements correlated with learning parameters. Our results show that translational touchscreen operant tests and fear conditioning paradigms can reliably detect cognitive consequences of cuprizone treatment. The suggested experimental approach enables screening novel MS drug candidates in longitudinal experiments for their ability to improve pathological changes in brain structure and reverse cognitive deficits.
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http://dx.doi.org/10.1111/gbb.12663DOI Listing
January 2021

Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor.

Neuropharmacology 2019 05 2;150:100-111. Epub 2019 Mar 2.

Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials.
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http://dx.doi.org/10.1016/j.neuropharm.2019.02.035DOI Listing
May 2019

The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences.

Cell Rep 2018 10;25(4):841-851.e4

Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK; The Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK; Centre for Clinical Brain Sciences, University of Edinburgh Chancellor's Building, Edinburgh, UK. Electronic address:

The GluN2 subtype (2A versus 2B) determines biophysical properties and signaling of forebrain NMDA receptors (NMDARs). During development, GluN2A becomes incorporated into previously GluN2B-dominated NMDARs. This "switch" is proposed to be driven by distinct features of GluN2 cytoplasmic C-terminal domains (CTDs), including a unique CaMKII interaction site in GluN2B that drives removal from the synapse. However, these models remain untested in the context of endogenous NMDARs. We show that, although mutating the endogenous GluN2B CaMKII site has secondary effects on GluN2B CTD phosphorylation, the developmental changes in NMDAR composition occur normally and measures of plasticity and synaptogenesis are unaffected. Moreover, the switch proceeds normally in mice that have the GluN2A CTD replaced by that of GluN2B and commences without an observable decline in GluN2B levels but is impaired by GluN2A haploinsufficiency. Thus, GluN2A expression levels, and not GluN2 subtype-specific CTD-driven events, are the overriding factor in the developmental switch in NMDAR composition.
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http://dx.doi.org/10.1016/j.celrep.2018.09.089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218242PMC
October 2018

Impaired Performance of the Q175 Mouse Model of Huntington's Disease in the Touch Screen Paired Associates Learning Task.

Front Behav Neurosci 2018 2;12:226. Epub 2018 Oct 2.

Charles River Discovery Services, Kuopio, Finland.

Cognitive disturbances often predate characteristic motor dysfunction in individuals with Huntington's disease (HD) and place an increasing burden on the HD patients and caregivers with the progression of the disorder. Therefore, application of maximally translational cognitive tests to animal models of HD is imperative for the development of treatments that could alleviate cognitive decline in human patients. Here, we examined the performance of the Q175 mouse knock-in model of HD in the touch screen version of the paired associates learning (PAL) task. We found that 10-11-month-old heterozygous Q175 mice had severely attenuated learning curve in the PAL task, which was conceptually similar to previously documented impaired performance of individuals with HD in the PAL task of the Cambridge Neuropsychological Test Automated Battery (CANTAB). Besides high rate of errors in PAL task, Q175 mice exhibited considerably lower responding rate than age-matched wild-type (WT) animals. Our examination of effortful operant responding during fixed ratio (FR) and progressive ratio (PR) reinforcement schedules in a separate cohort of similar age confirmed slower and unselective performance of mutant animals, as observed during PAL task, but suggested that motivation to work for nutritional reward in the touch screen setting was similar in Q175 and WT mice. We also demonstrated that pronounced sensorimotor disturbances in Q175 mice can be detected at early touch screen testing stages, (e.g., during "Punish Incorrect" phase of operant pretraining), so we propose that shorter test routines may be utilised for more expedient studies of treatments aimed at the rescue of HD-related phenotype.
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http://dx.doi.org/10.3389/fnbeh.2018.00226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176131PMC
October 2018

Architecture of the Mouse Brain Synaptome.

Neuron 2018 08 2;99(4):781-799.e10. Epub 2018 Aug 2.

Genes to Cognition Program, Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh EH16 4SB, UK. Electronic address:

Synapses are found in vast numbers in the brain and contain complex proteomes. We developed genetic labeling and imaging methods to examine synaptic proteins in individual excitatory synapses across all regions of the mouse brain. Synapse catalogs were generated from the molecular and morphological features of a billion synapses. Each synapse subtype showed a unique anatomical distribution, and each brain region showed a distinct signature of synapse subtypes. Whole-brain synaptome cartography revealed spatial architecture from dendritic to global systems levels and previously unknown anatomical features. Synaptome mapping of circuits showed correspondence between synapse diversity and structural and functional connectomes. Behaviorally relevant patterns of neuronal activity trigger spatiotemporal postsynaptic responses sensitive to the structure of synaptome maps. Areas controlling higher cognitive function contain the greatest synapse diversity, and mutations causing cognitive disorders reorganized synaptome maps. Synaptome technology and resources have wide-ranging application in studies of the normal and diseased brain.
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http://dx.doi.org/10.1016/j.neuron.2018.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117470PMC
August 2018

Chronic treatment with a MEK inhibitor reverses enhanced excitatory field potentials in Syngap1 mice.

Pharmacol Rep 2018 Aug 23;70(4):777-783. Epub 2018 Jun 23.

Centre for Clinical Brain Science, University of Edinburgh, Edinburgh, UK.

Background: Synaptic Ras-GTPase-activating protein 1 (SYNGAP1) is an abundant brain-specific protein localized at the postsynaptic density of mammalian excitatory synapses. SYNGAP1 functions as a crucial regulator of downstream intracellular signaling triggered by N-methyl-d-aspartate receptor activation. One of the most important signaling pathways regulated by SYNGAP1 is the Ras-Raf-MEK-ERK pathway. SYNGAP1 deficiency is associated with hyperphosphorylation of MEK and ERK kinases and with altered synaptic function in Syngap1 mice. Loss-of-function mutations in the SYNGAP1 gene have been documented in many human cognitive and neurological disorders. However, there are currently no approaches that reverse the phenotypes of SYNGAP1 deficiency.

Methods: Using electrophysiological recordings of field responses in hippocampal slices, we examined if disturbances of synaptic physiology in the hippocampus of 7-8-month old Syngap1 mice were sensitive to the effect of the MEK inhibitor PD-0325901 given orally for 6days.

Results: We found that in hippocampal slices from vehicle-treated Syngap1 mice, basal synaptic responses were higher and their long-term potentiation (LTP) was lower than in slices from wild-type littermates. Chronic administration of PD-0325901 normalized basal synaptic responses, but did not reverse LTP deficit.

Conclusions: The differential sensitivity of basal synaptic transmission and LTP to MEK inhibition indicates that the effects of SYNGAP1 deficiency on these synaptic parameters are mediated by distinct pathways. Our findings also suggest that at least some physiological phenotypes of the germline Syngap1 mutation can be ameliorated by pharmacological treatment of adult animals.
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http://dx.doi.org/10.1016/j.pharep.2018.02.021DOI Listing
August 2018

Enhanced cognition and dysregulated hippocampal synaptic physiology in mice with a heterozygous deletion of PSD-95.

Eur J Neurosci 2018 01 10;47(2):164-176. Epub 2018 Jan 10.

Synome Ltd, Cambridge, UK.

PSD-95 is one of the most abundant proteins of the postsynaptic density of excitatory synapses. It functions as the backbone of protein supercomplexes that mediate signalling between membrane glutamate receptors and intracellular pathways. Homozygous deletion of the Dlg4 gene encoding PSD-95 was previously found to cause a profound impairment in operant and Pavlovian conditioning in Dlg4 mice studied in touch screen chambers that precluded evaluation of PSD-95's role in shaping more subtle forms of learning and memory. In this study, using a battery of touch screen tests, we investigated cognitive behaviour of mice with a heterozygous Dlg4 mutation. We found that in contrast to learning deficits of Dlg4 mice, Dlg4 animals demonstrated enhanced performance in the Visual Discrimination, Visual Discrimination Reversal and Paired-Associates Learning touch screen tasks. The divergent directions of learning phenotypes observed in Dlg4 and Dlg4 mice also contrasted with qualitatively similar changes in the amplitude and plasticity of field excitatory postsynaptic potentials recorded in the CA1 area of hippocampal slices from both mutants. Our results have important repercussions for the studies of genetic models of human diseases, because they demonstrate that reliance on phenotypes observed solely in homozygous mice may obscure qualitatively different changes in heterozygous animals and potentially weaken the validity of translational comparisons with symptoms seen in heterozygous human carriers.
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http://dx.doi.org/10.1111/ejn.13792DOI Listing
January 2018

Arc Requires PSD95 for Assembly into Postsynaptic Complexes Involved with Neural Dysfunction and Intelligence.

Cell Rep 2017 Oct;21(3):679-691

Genes to Cognition Programme, The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK; Genes to Cognition Programme, Centre for Clinical Brain Science, University of Edinburgh, Edinburgh, UK. Electronic address:

Arc is an activity-regulated neuronal protein, but little is known about its interactions, assembly into multiprotein complexes, and role in human disease and cognition. We applied an integrated proteomic and genetic strategy by targeting a tandem affinity purification (TAP) tag and Venus fluorescent protein into the endogenous Arc gene in mice. This allowed biochemical and proteomic characterization of native complexes in wild-type and knockout mice. We identified many Arc-interacting proteins, of which PSD95 was the most abundant. PSD95 was essential for Arc assembly into 1.5-MDa complexes and activity-dependent recruitment to excitatory synapses. Integrating human genetic data with proteomic data showed that Arc-PSD95 complexes are enriched in schizophrenia, intellectual disability, autism, and epilepsy mutations and normal variants in intelligence. We propose that Arc-PSD95 postsynaptic complexes potentially affect human cognitive function.
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http://dx.doi.org/10.1016/j.celrep.2017.09.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656750PMC
October 2017

Acquisition and reversal of visual discrimination learning in APPSwDI/Nos2 (CVN) mice.

Neurosci Lett 2017 05 26;650:126-133. Epub 2017 Apr 26.

Charles River Discovery Research Services, Microkatu 1, 70210 Kuopio, Finland. Electronic address:

Studies of cognitive behavior in rodent models of Alzheimer's disease (AD) are the mainstay of academic and industrial efforts to find effective treatments for this disorder. However, in the majority of such studies, the nature of rodent behavioral tests is considerably different from the setting associated with cognitive assessments of individuals with AD. The recently developed touchscreen technique provides a more translational way of rodent cognitive testing because the stimulus (images in different locations on the screen) and reaction (touch) are similar to those employed in human test routines, such as the Cambridge Neuropsychological Test Automated Battery. Here, we used Visual Discrimination and Reversal of Visual Discrimination touchscreen tasks to assess cognitive performance of APPSwDI/Nos2 (CVN) mice, which express mutated human APP and have a homozygous deletion of the Nos2 gene. We revealed that CVN mice made more first-time errors and received more correction trials than WT mice across both discrimination and reversal phases, although mutation effect size was larger during the latter phase. These results indicate sensitivity of touchscreen-based measurements to AD-relevant mutations in CVN mice and warrant future touchscreen experiments aimed at evaluating other cognitive and motivational phenotypes in this AD mouse model.
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http://dx.doi.org/10.1016/j.neulet.2017.04.049DOI Listing
May 2017

Haploinsufficiency of EHMT1 improves pattern separation and increases hippocampal cell proliferation.

Sci Rep 2017 01 10;7:40284. Epub 2017 Jan 10.

Department of Cognitive Neuroscience, Radboudumc, Donders Institute for Brain, Cognition and Behaviour, Nijmegen, the Netherlands.

Heterozygous mutations or deletions of the human Euchromatin Histone Methyltransferase 1 (EHMT1) gene are the main causes of Kleefstra syndrome, a neurodevelopmental disorder that is characterized by impaired memory, autistic features and mostly severe intellectual disability. Previously, Ehmt1 heterozygous knockout mice were found to exhibit cranial abnormalities and decreased sociability, phenotypes similar to those observed in Kleefstra syndrome patients. In addition, Ehmt1 knockout mice were impaired at fear extinction and novel- and spatial object recognition. In this study, Ehmt1 and wild-type mice were tested on several cognitive tests in a touchscreen-equipped operant chamber to further investigate the nature of learning and memory changes. Performance of Ehmt1 mice in the Visual Discrimination &Reversal learning, object-location Paired-Associates learning- and Extinction learning tasks was found to be unimpaired. Remarkably, Ehmt1 mice showed enhanced performance on the Location Discrimination test of pattern separation. In line with improved Location Discrimination ability, an increase in BrdU-labelled cells in the subgranular zone of the dentate gyrus was observed. In conclusion, reduced levels of EHMT1 protein in Ehmt1 mice does not result in general learning deficits in a touchscreen-based battery, but leads to increased adult cell proliferation in the hippocampus and enhanced pattern separation ability.
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http://dx.doi.org/10.1038/srep40284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223204PMC
January 2017

PSD95 nanoclusters are postsynaptic building blocks in hippocampus circuits.

Sci Rep 2016 04 25;6:24626. Epub 2016 Apr 25.

Centre for Clinical Brain Sciences (CCBS), University of Edinburgh, Edinburgh, United Kingdom.

The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity.
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http://dx.doi.org/10.1038/srep24626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842999PMC
April 2016

Evolution of GluN2A/B cytoplasmic domains diversified vertebrate synaptic plasticity and behavior.

Nat Neurosci 2013 Jan 2;16(1):25-32. Epub 2012 Dec 2.

Genes to Cognition Programme, Wellcome Trust Sanger Institute, Cambridge, UK.

Two genome duplications early in the vertebrate lineage expanded gene families, including GluN2 subunits of the NMDA receptor. Diversification between the four mammalian GluN2 proteins occurred primarily at their intracellular C-terminal domains (CTDs). To identify shared ancestral functions and diversified subunit-specific functions, we exchanged the exons encoding the GluN2A (also known as Grin2a) and GluN2B (also known as Grin2b) CTDs in two knock-in mice and analyzed the mice's biochemistry, synaptic physiology, and multiple learned and innate behaviors. The eight behaviors were genetically separated into four groups, including one group comprising three types of learning linked to conserved GluN2A/B regions. In contrast, the remaining five behaviors exhibited subunit-specific regulation. GluN2A/B CTD diversification conferred differential binding to cytoplasmic MAGUK proteins and differential forms of long-term potentiation. These data indicate that vertebrate behavior and synaptic signaling acquired increased complexity from the duplication and diversification of ancestral GluN2 genes.
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http://dx.doi.org/10.1038/nn.3277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979286PMC
January 2013

Hippocampal dysfunction in the Euchromatin histone methyltransferase 1 heterozygous knockout mouse model for Kleefstra syndrome.

Hum Mol Genet 2013 Mar 21;22(5):852-66. Epub 2012 Nov 21.

Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Nijmegen, the Netherlands.

Euchromatin histone methyltransferase 1 (EHMT1) is a highly conserved protein that catalyzes mono- and dimethylation of histone H3 lysine 9, thereby epigenetically regulating transcription. Kleefstra syndrome (KS), is caused by haploinsufficiency of the EHMT1 gene, and is an example of an emerging group of intellectual disability (ID) disorders caused by genes encoding epigenetic regulators of neuronal gene activity. Little is known about the mechanisms underlying this disorder, prompting us to study the Euchromatin histone methyltransferase 1 heterozygous knockout (Ehmt1(+/-)) mice as a model for KS. In agreement with the cognitive disturbances observed in patients with KS, we detected deficits in fear extinction learning and both novel and spatial object recognition in Ehmt1(+/-) mice. These learning and memory deficits were associated with a significant reduction in dendritic arborization and the number of mature spines in hippocampal CA1 pyramidal neurons of Ehmt1(+/-) mice. In-depth analysis of the electrophysiological properties of CA3-CA1 synapses revealed no differences in basal synaptic transmission or theta-burst induced long-term potentiation (LTP). However, paired-pulse facilitation (PPF) was significantly increased in Ehmt1(+/-) neurons, pointing to a potential deficiency in presynaptic neurotransmitter release. Accordingly, a reduction in the frequency of miniature excitatory post-synaptic currents (mEPSCs) was observed in Ehmt1(+/-) neurons. These data demonstrate that Ehmt1 haploinsufficiency in mice leads to learning deficits and synaptic dysfunction, providing a possible mechanism for the ID phenotype in patients with KS.
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http://dx.doi.org/10.1093/hmg/dds490DOI Listing
March 2013

TNiK is required for postsynaptic and nuclear signaling pathways and cognitive function.

J Neurosci 2012 Oct;32(40):13987-99

Genes to Cognition Programme, The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom.

Traf2 and NcK interacting kinase (TNiK) contains serine-threonine kinase and scaffold domains and has been implicated in cell proliferation and glutamate receptor regulation in vitro. Here we report its role in vivo using mice carrying a knock-out mutation. TNiK binds protein complexes in the synapse linking it to the NMDA receptor (NMDAR) via AKAP9. NMDAR and metabotropic receptors bidirectionally regulate TNiK phosphorylation and TNiK is required for AMPA expression and synaptic function. TNiK also organizes nuclear complexes and in the absence of TNiK, there was a marked elevation in GSK3β and phosphorylation levels of its cognate phosphorylation sites on NeuroD1 with alterations in Wnt pathway signaling. We observed impairments in dentate gyrus neurogenesis in TNiK knock-out mice and cognitive testing using the touchscreen apparatus revealed impairments in pattern separation on a test of spatial discrimination. Object-location paired associate learning, which is dependent on glutamatergic signaling, was also impaired. Additionally, TNiK knock-out mice displayed hyperlocomotor behavior that could be rapidly reversed by GSK3β inhibitors, indicating the potential for pharmacological rescue of a behavioral phenotype. These data establish TNiK as a critical regulator of cognitive functions and suggest it may play a regulatory role in diseases impacting on its interacting proteins and complexes.
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http://dx.doi.org/10.1523/JNEUROSCI.2433-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3978779PMC
October 2012

Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins.

Mol Syst Biol 2009 19;5:269. Epub 2009 May 19.

Genes to Cognition Programme, The Wellcome Trust Sanger Institute, Cambridge, UK.

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD-95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD-95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K+ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage-dependent K+ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.
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http://dx.doi.org/10.1038/msb.2009.27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2694677PMC
July 2009

Neurotransmitters drive combinatorial multistate postsynaptic density networks.

Sci Signal 2009 Apr 28;2(68):ra19. Epub 2009 Apr 28.

Genes to Cognition, Wellcome Trust Sanger Institute, Cambridgeshire, UK.

The mammalian postsynaptic density (PSD) comprises a complex collection of approximately 1100 proteins. Despite extensive knowledge of individual proteins, the overall organization of the PSD is poorly understood. Here, we define maps of molecular circuitry within the PSD based on phosphorylation of postsynaptic proteins. Activation of a single neurotransmitter receptor, the N-methyl-D-aspartate receptor (NMDAR), changed the phosphorylation status of 127 proteins. Stimulation of ionotropic and metabotropic glutamate receptors and dopamine receptors activated overlapping networks with distinct combinatorial phosphorylation signatures. Using peptide array technology, we identified specific phosphorylation motifs and switching mechanisms responsible for the integration of neurotransmitter receptor pathways and their coordination of multiple substrates in these networks. These combinatorial networks confer high information-processing capacity and functional diversity on synapses, and their elucidation may provide new insights into disease mechanisms and new opportunities for drug discovery.
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http://dx.doi.org/10.1126/scisignal.2000102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3280897PMC
April 2009

Kinase networks integrate profiles of N-methyl-D-aspartate receptor-mediated gene expression in hippocampus.

J Biol Chem 2008 Dec 23;283(49):34101-7. Epub 2008 Sep 23.

Genes to Cognition, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom.

The postsynaptic N-methyl-d-aspartate (NMDA) receptor activates multiple kinases and changes the phosphorylation of many postsynaptic proteins organized in signaling networks. Because the NMDA receptor is known to regulate gene expression, it is important to examine whether networks of kinases control signaling to gene expression. We examined the requirement of multiple kinases and NMDA receptor-interacting proteins for gene expression in mouse hippocampal slices. Protocols that induce long-term depression (LTD) and long-term potentiation (LTP) activated common kinases and overlapping gene expression profiles. Combinations of kinases were required for induction of each gene. Distinct combinations of kinases were required to up-regulate Arc, Npas4, Egr2, and Egr4 following either LTP or LTD protocols. Consistent with the combinatorial data, a mouse mutant model of the human cognition disease gene SAP102, which couples ERK kinase to the NMDA receptor, showed deregulated expression of specific genes. These data support a network model of postsynaptic integration where kinase signaling networks are recruited by differential synaptic activity and control both local synaptic events and activity-dependent gene expression.
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http://dx.doi.org/10.1074/jbc.M804951200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2662229PMC
December 2008

Recording long-term potentiation of synaptic transmission by three-dimensional multi-electrode arrays.

BMC Neurosci 2006 Aug 30;7:61. Epub 2006 Aug 30.

Genes to Cognition Programme, The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Background: Multi-electrode arrays (MEAs) have become popular tools for recording spontaneous and evoked electrical activity of excitable tissues. The majority of previous studies of synaptic transmission in brain slices employed MEAs with planar electrodes that had limited ability to detect signals coming from deeper, healthier layers of the slice. To overcome this limitation, we used three-dimensional (3D) MEAs with tip-shaped electrodes to probe plasticity of field excitatory synaptic potentials (fEPSPs) in the CA1 area of hippocampal slices of 129S5/SvEvBrd and C57BL/6J-TyrC-Brd mice.

Results: Using 3D MEAs, we were able to record larger fEPSPs compared to signals measured by planar MEAs. Several stimulation protocols were used to induce long-term potentiation (LTP) of synaptic responses in the CA1 area recorded following excitation of Schäffer collateral/commissural fibres. Either two trains of high frequency tetanic stimulation or three trains of theta-burst stimulation caused a persistent, pathway specific enhancement of fEPSPs that remained significantly elevated for at least 60 min. A third LTP induction protocol that comprised 150 pulses delivered at 5 Hz, evoked moderate LTP if excitation strength was increased to 1.5x of the baseline stimulus. In all cases, we observed a clear spatial plasticity gradient with maximum LTP levels detected in proximal apical dendrites of pyramidal neurones. No significant differences in the manifestation of LTP were observed between 129S5/SvEvBrd and C57BL/6J-TyrC-Brd mice with the three protocols used. All forms of plasticity were sensitive to inhibition of N-methyl-D-aspartate (NMDA) receptors.

Conclusion: Principal features of LTP (magnitude, pathway specificity, NMDA receptor dependence) recorded in the hippocampal slices using MEAs were very similar to those seen in conventional glass electrode experiments. Advantages of using MEAs are the ability to record from different regions of the slice and the ease of conducting several experiments on a multiplexed platform which could be useful for efficient screening of novel transgenic mice.
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http://dx.doi.org/10.1186/1471-2202-7-61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1574331PMC
August 2006