RESUMEN
Lipids play crucial roles in the susceptibility and brain cellular responses to Alzheimer's disease (AD) and are increasingly considered potential soluble biomarkers in cerebrospinal fluid (CSF) and plasma. To delineate the pathological correlations of distinct lipid species, we conducted a comprehensive characterization of both spatially localized and global differences in brain lipid composition in AppNL-G-F mice with spatial and bulk mass spectrometry lipidomic profiling, using human amyloid-expressing (h-Aß) and WT mouse brains controls. We observed age-dependent increases in lysophospholipids, bis(monoacylglycerol) phosphates, and phosphatidylglycerols around Aß plaques in AppNL-G-F mice. Immunohistology-based co-localization identified associations between focal pro-inflammatory lipids, glial activation, and autophagic flux disruption. Likewise, in human donors with varying Braak stages, similar studies of cortical sections revealed co-expression of lysophospholipids and ceramides around Aß plaques in AD (Braak stage V/VI) but not in earlier Braak stage controls. Our findings in mice provide evidence of temporally and spatially heterogeneous differences in lipid composition as local and global Aß-related pathologies evolve. Observing similar lipidomic changes associated with pathological Aß plaques in human AD tissue provides a foundation for understanding differences in CSF lipids with reported clinical stage or disease severity.
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Enfermedad de Alzheimer , Encéfalo , Espectrometría de Masas , Ratones Transgénicos , Placa Amiloide , Animales , Humanos , Placa Amiloide/patología , Placa Amiloide/metabolismo , Ratones , Espectrometría de Masas/métodos , Encéfalo/metabolismo , Encéfalo/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Masculino , Femenino , Metabolismo de los Lípidos/fisiología , Lisofosfolípidos/metabolismo , Anciano , Ratones Endogámicos C57BL , Lípidos/análisis , Lipidómica/métodosRESUMEN
The paucity of currently available therapies for glioblastoma multiforme requires novel approaches to the treatment of this brain tumour. Disrupting cyclic nucleotide-signalling through phosphodiesterase (PDE) inhibition may be a promising way of suppressing glioblastoma growth. Here, we examined the effects of 28 PDE inhibitors, covering all the major PDE classes, on the proliferation of the human U87MG, A172 and T98G glioblastoma cells. The PDE10A inhibitors PF-2545920, PQ10 and papaverine, the PDE3/4 inhibitor trequinsin and the putative PDE5 inhibitor MY-5445 potently decreased glioblastoma cell proliferation. The synergistic suppression of glioblastoma cell proliferation was achieved by combining PF-2545920 and MY-5445. Furthermore, a co-incubation with drugs that block the activity of the multidrug resistance-associated protein 1 (MRP1) augmented these effects. In particular, a combination comprising the MRP1 inhibitor reversan, PF-2545920 and MY-5445, all at low micromolar concentrations, afforded nearly complete inhibition of glioblastoma cell growth. Thus, the potent suppression of glioblastoma cell viability may be achieved by combining MRP1 inhibitors with PDE inhibitors at a lower toxicity than that of the standard chemotherapeutic agents, thereby providing a new combination therapy for this challenging malignancy.
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Antineoplásicos/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Sinergismo Farmacológico , Glioblastoma , Humanos , Pirazoles/farmacología , Quinolinas/farmacologíaRESUMEN
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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Región CA1 Hipocampal/fisiología , Cognición , Homólogo 4 de la Proteína Discs Large/genética , Potenciales Postsinápticos Excitadores , Heterocigoto , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/metabolismo , Condicionamiento Clásico , Homólogo 4 de la Proteína Discs Large/metabolismo , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Plasticidad Neuronal , Neuronas/metabolismo , Neuronas/fisiologíaRESUMEN
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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Anomalías Craneofaciales/genética , Cardiopatías Congénitas/genética , N-Metiltransferasa de Histona-Lisina/genética , Discapacidad Intelectual/genética , Aprendizaje , Animales , Deleción Cromosómica , Cromosomas Humanos Par 9/genética , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/patología , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Discapacidad Intelectual/fisiopatología , Ratones , Ratones Noqueados , Células Piramidales/patología , Sinapsis/patologíaRESUMEN
Amyloid-ß pathology and neurofibrillary tangles lead to glial activation and neurodegeneration in Alzheimer's disease. In this study, we investigated the relationships between the levels of amyloid-ß oligomers, amyloid-ß plaques, glial activation and markers related to neurodegeneration in the App NL-G-F triple mutation mouse line and in a knock-in line homozygous for the common human amyloid precursor protein (App hu mouse). The relationships between neuropathological features were characterized with immunohistochemistry and imaging mass cytometry. Markers assessing human amyloid-ß proteins, microglial and astrocytic activation and neuronal and synaptic densities were used in mice between 2.5 and 12 months of age. We found that amyloid-ß oligomers were abundant in the brains of App hu mice in the absence of classical amyloid-ß plaques. These brains showed morphological changes consistent with astrocyte activation but no evidence of microglial activation or synaptic or neuronal pathology. In contrast, both high levels of amyloid-ß oligomers and numerous plaques accumulated in App NL-G-F mice in association with substantial astrocytic and microglial activation. The increase in amyloid-ß oligomers over time was more strongly correlated with astrocytic than with microglia activation. Spatial analyses suggested that activated microglia were more closely associated with amyloid-ß oligomers than with amyloid-ß plaques in App NL-G-F mice, which also showed age-dependent decreases in neuronal and synaptic density markers. A comparative study of the two models highlighted the dependence of glial and neuronal pathology on the nature and aggregation state of the amyloid-ß peptide. Astrocyte activation and neuronal pathology appeared to be more strongly associated with amyloid-ß oligomers than with amyloid-ß plaques, although amyloid-ß plaques were associated with microglia activation.
RESUMEN
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 GSK3ß 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 GSK3ß 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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Aprendizaje por Asociación/fisiología , Trastornos del Conocimiento/enzimología , Giro Dentado/enzimología , Aprendizaje Discriminativo/fisiología , Proteínas del Tejido Nervioso/fisiología , Densidad Postsináptica/enzimología , Proteínas Serina-Treonina Quinasas/fisiología , Detección de Señal Psicológica/fisiología , Percepción Espacial/fisiología , Animales , Núcleo Celular/enzimología , Trastornos del Conocimiento/fisiopatología , Giro Dentado/patología , Ácido Glutámico/fisiología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/fisiología , Glucógeno Sintasa Quinasa 3 beta , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Potenciales Postsinápticos Miniatura/fisiología , Proteínas del Tejido Nervioso/deficiencia , Neurogénesis/fisiología , Fenotipo , Fosforilación , Densidad Postsináptica/fisiología , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
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. Modelling 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 signalling pathway could rescue these cognitive impairments, Spred1-/- mice were acutely treated with the clinically relevant mitogen-activated protein 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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Manchas Café con Leche/genética , Condicionamiento Operante , Animales , Cognición , Eliminación de Gen , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
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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Aprendizaje , Mutación con Pérdida de Función , Proteínas del Tejido Nervioso/genética , Animales , Moléculas de Adhesión Celular Neuronal/genética , Femenino , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Tiempo de Reacción , Proteínas Asociadas a SAP90-PSD95/genética , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
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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Cognición , Esclerosis Múltiple/fisiopatología , Animales , Condicionamiento Operante , Cuerpo Calloso/diagnóstico por imagen , Cuprizona/toxicidad , Femenino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/etiología , Percepción VisualRESUMEN
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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Marcación de Gen/métodos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Complejos Multiproteicos/aislamiento & purificación , Proteínas del Tejido Nervioso/aislamiento & purificación , Esquizofrenia/metabolismo , Animales , Encéfalo/metabolismo , Cromatografía de Afinidad , Homólogo 4 de la Proteína Discs Large , Expresión Génica , Guanilato-Quinasas , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Ratones , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal , Mapeo de Interacción de Proteínas/métodos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Sinapsis/metabolismo , Potenciales Sinápticos , Espectrometría de Masas en TándemRESUMEN
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 30â¯min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1â¯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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Isquemia Encefálica/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Homólogo 4 de la Proteína Discs Large/antagonistas & inhibidores , Éteres/farmacología , Fármacos Neuroprotectores/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Encéfalo/patología , Isquemia Encefálica/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Éteres/efectos adversos , Éteres/uso terapéutico , Femenino , Masculino , Ratones , Neuroprotección/efectos de los fármacos , Fármacos Neuroprotectores/efectos adversos , Fármacos Neuroprotectores/uso terapéutico , Ratas , Factores de TiempoRESUMEN
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.
RESUMEN
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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Benzamidas/farmacología , Difenilamina/análogos & derivados , Hipocampo/fisiopatología , Potenciales de la Membrana/efectos de los fármacos , Proteínas Activadoras de ras GTPasa/deficiencia , Animales , Difenilamina/farmacología , Femenino , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Ratones , Mutación , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
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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Química Encefálica/fisiología , Encéfalo/diagnóstico por imagen , Encéfalo/fisiología , Biología Computacional/métodos , Sinapsis/fisiología , Animales , Conectoma/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteoma/análisis , Proteoma/fisiología , Sinapsis/químicaRESUMEN
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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Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Potenciación a Largo Plazo , Ratones Endogámicos C57BL , Mutación/genética , Neurogénesis , Fosforilación , Subunidades de Proteína/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/genética , Sinapsis/metabolismo , Ritmo Teta/fisiologíaRESUMEN
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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Aprendizaje Discriminativo , Recuerdo Mental , Reconocimiento Visual de Modelos , Aprendizaje Inverso , Recompensa , Precursor de Proteína beta-Amiloide/genética , Animales , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo II/genética , Aprendizaje Inverso/fisiología , Análisis y Desempeño de TareasRESUMEN
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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Trastornos del Conocimiento/genética , Anomalías Craneofaciales/genética , Cardiopatías Congénitas/genética , N-Metiltransferasa de Histona-Lisina/genética , Discapacidad Intelectual/genética , Aprendizaje/fisiología , Animales , Proliferación Celular/genética , Deleción Cromosómica , Cromosomas Humanos Par 9/genética , Trastornos del Conocimiento/fisiopatología , Anomalías Craneofaciales/fisiopatología , Haploinsuficiencia/genética , Haploinsuficiencia/fisiología , Cardiopatías Congénitas/fisiopatología , Hipocampo/fisiopatología , Humanos , Discapacidad Intelectual/fisiopatología , Memoria/fisiología , Ratones , Ratones Noqueados , MutaciónRESUMEN
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.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Inteligencia , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/metabolismo , Sistema Nervioso/fisiopatología , Sinapsis/metabolismo , Animales , Técnicas de Sustitución del Gen , Humanos , Ratones Noqueados , ProteómicaRESUMEN
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.
Asunto(s)
Electrofisiología/instrumentación , Electrofisiología/métodos , Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Transmisión Sináptica/fisiología , Potenciales de Acción/fisiología , Animales , Axones/fisiología , Estimulación Eléctrica/métodos , Electrodos , Potenciales Postsinápticos Excitadores/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Red Nerviosa/fisiología , Vías Nerviosas/fisiología , Técnicas de Cultivo de Órganos , Fenotipo , Receptores de N-Metil-D-Aspartato/fisiología , Especificidad de la EspecieRESUMEN
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.