RESUMO
MAPK interacting protein kinases 1 and 2 (Mnk1/2) regulate a plethora of functions, presumably via phosphorylation of their best characterized substrate, eukaryotic translation initiation factor 4E (eIF4E) on Ser209. Here, we show that, whereas deletion of Mnk1/2 (Mnk double knockout) impairs synaptic plasticity and memory in mice, ablation of phospho-eIF4E (Ser209) does not affect these processes, suggesting that Mnk1/2 possess additional downstream effectors in the brain. Translational profiling revealed only a small overlap between the Mnk1/2- and phospho-eIF4E(Ser209)-regulated translatome. We identified the synaptic Ras GTPase activating protein 1 (Syngap1), encoded by a syndromic autism gene, as a downstream target of Mnk1 because Syngap1 immunoprecipitated with Mnk1 and showed reduced phosphorylation (S788) in Mnk double knockout mice. Knockdown of Syngap1 reversed memory deficits in Mnk double knockout mice and pharmacological inhibition of Mnks rescued autism-related phenotypes in Syngap1+/- mice. Thus, Syngap1 is a downstream effector of Mnk1, and the Mnks-Syngap1 axis regulates memory formation and autism-related behaviours.
Assuntos
Transtorno Autístico , Fator de Iniciação 4E em Eucariotos , Animais , Camundongos , Fator de Iniciação 4E em Eucariotos/genética , Camundongos Knockout , Fosforilação , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder. Separate lines of evidence suggest that synapses and astrocytes play a role in the pathological mechanisms underlying ALS. Given that astrocytes make specialised contacts with some synapses, called tripartite synapses, we hypothesise that tripartite synapses could act as the fulcrum of disease in ALS. To test this hypothesis, we have performed an extensive microscopy-based investigation of synapses and tripartite synapses in the spinal cord of ALS model mice and post-mortem human tissue from ALS cases. We reveal widescale synaptic changes at the early symptomatic stages of the SOD1G93a mouse model. Super-resolution microscopy reveals that large complex postsynaptic structures are lost in ALS mice. Most surprisingly, tripartite synapses are selectively lost, while non-tripartite synapses remain in equal number to healthy controls. Finally, we also observe a similar selective loss of tripartite synapses in human post-mortem ALS spinal cords. From these data we conclude that tripartite synaptopathy is a key hallmark of ALS.
Assuntos
Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Medula Espinal/patologia , Superóxido Dismutase , Superóxido Dismutase-1/genética , Sinapses/patologiaRESUMO
In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1,000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons, the extent of synapse diversity across the brain is largely unknown. This is mainly due to the limitations of current techniques. Here, we report an efficient method for the purification of synaptic protein complexes, fusing a high-affinity tag to endogenous PSD95 in specific cell types. We also developed a strategy, which enables the visualisation of endogenous PSD95 with fluorescent-protein tag in Cre-recombinase-expressing cells. We demonstrate the feasibility of proteomic analysis of synaptic protein complexes and visualisation of these in specific cell types. We find that the composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. The results suggest that there might be differential interactions in the PSD95 complexes in different brain regions. We have detected differentially interacting proteins by comparing data sets from the whole hippocampus and the CA3 subfield of the hippocampus. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific brain regions and subsets of neuronal cells, but also provide an opportunity to better understand brain region- and cell-type-specific alterations associated with various psychiatric/neurological diseases. These newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will facilitate research on the molecular complexity of synapses.
Assuntos
Proteômica , Sinapses , Animais , Proteína 4 Homóloga a Disks-Large/metabolismo , Hipocampo/metabolismo , Camundongos , Neurônios/metabolismo , Proteoma/metabolismo , Sinapses/metabolismoRESUMO
Schizophrenia is a common disease with a complex aetiology, probably involving multiple and heterogeneous genetic factors. Here, by analysing the exome sequences of 2,536 schizophrenia cases and 2,543 controls, we demonstrate a polygenic burden primarily arising from rare (less than 1 in 10,000), disruptive mutations distributed across many genes. Particularly enriched gene sets include the voltage-gated calcium ion channel and the signalling complex formed by the activity-regulated cytoskeleton-associated scaffold protein (ARC) of the postsynaptic density, sets previously implicated by genome-wide association and copy-number variation studies. Similar to reports in autism, targets of the fragile X mental retardation protein (FMRP, product of FMR1) are enriched for case mutations. No individual gene-based test achieves significance after correction for multiple testing and we do not detect any alleles of moderately low frequency (approximately 0.5 to 1 per cent) and moderately large effect. Taken together, these data suggest that population-based exome sequencing can discover risk alleles and complements established gene-mapping paradigms in neuropsychiatric disease.
Assuntos
Herança Multifatorial/genética , Mutação/genética , Esquizofrenia/genética , Transtorno Autístico/genética , Canais de Cálcio/genética , Proteínas do Citoesqueleto/genética , Variações do Número de Cópias de DNA/genética , Proteína 4 Homóloga a Disks-Large , Feminino , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Deficiência Intelectual/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
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.
Assuntos
Região CA1 Hipocampal/fisiologia , Cognição , Proteína 4 Homóloga a Disks-Large/genética , Potenciais Pós-Sinápticos Excitadores , Heterozigoto , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Condicionamento Clássico , Proteína 4 Homóloga a Disks-Large/metabolismo , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
PSD95 is an abundant postsynaptic scaffold protein in glutamatergic synapses that assembles into supercomplexes composed of over 80 proteins including neurotransmitter receptors, ion channels and adhesion proteins. How these diverse constituents are organized into PSD95 supercomplexes in vivo is poorly understood. Here, we dissected the supercomplexes in mice combining endogenous gene-tagging, targeted mutations and quantitative biochemical assays. Generating compound heterozygous mice with two different gene-tags, one on each Psd95 allele, showed that each ~1.5 MDa PSD95-containing supercomplex contains on average two PSD95 molecules. Gene-tagging the endogenous GluN1 and PSD95 with identical Flag tags revealed N-methyl D-aspartic acid receptors (NMDARs) containing supercomplexes that represent only 3% of the total population of PSD95 supercomplexes, suggesting there are many other subtypes. To determine whether this extended population of different PSD95 supercomplexes use genetically defined mechanisms to specify their assembly, we tested the effect of five targeted mouse mutations on the assembly of known PSD95 interactors, Kir2.3, Arc, IQsec2/BRAG1 and Adam22. Unexpectedly, some mutations were highly selective, whereas others caused widespread disruption, indicating that PSD95 interacting proteins are organized hierarchically into distinct subfamilies of ~1.5 MDa supercomplexes, including a subpopulation of Kir2.3-NMDAR ion channel-channel supercomplexes. Kir2.3-NMDAR ion channel-channel supercomplexes were found to be anatomically restricted to particular brain regions. These data provide new insight into the mechanisms that govern the constituents of postsynaptic supercomplexes and the diversity of synapse types. Read the Editorial Highlight for this article on page 500. Cover Image for this issue: doi. 10.1111/jnc.13811.
Assuntos
Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Sinapses/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteína 4 Homóloga a Disks-Large , Masculino , Camundongos , Camundongos Transgênicos , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/genéticaRESUMO
Previous studies have hypothesized that diverse genetic causes of intellectual disability (ID) and autism spectrum disorders (ASDs) converge on common cellular pathways. Testing this hypothesis requires detailed phenotypic analyses of animal models with genetic mutations that accurately reflect those seen in the human condition (i.e., have structural validity) and which produce phenotypes that mirror ID/ASDs (i.e., have face validity). We show that SynGAP haploinsufficiency, which causes ID with co-occurring ASD in humans, mimics and occludes the synaptic pathophysiology associated with deletion of the Fmr1 gene. Syngap(+/-) and Fmr1(-/y) mice show increases in basal protein synthesis and metabotropic glutamate receptor (mGluR)-dependent long-term depression that, unlike in their wild-type controls, is independent of new protein synthesis. Basal levels of phosphorylated ERK1/2 are also elevated in Syngap(+/-) hippocampal slices. Super-resolution microscopy reveals that Syngap(+/-) and Fmr1(-/y) mice show nanoscale alterations in dendritic spine morphology that predict an increase in biochemical compartmentalization. Finally, increased basal protein synthesis is rescued by negative regulators of the mGlu subtype 5 receptor and the Ras-ERK1/2 pathway, indicating that therapeutic interventions for fragile X syndrome may benefit patients with SYNGAP1 haploinsufficiency. SIGNIFICANCE STATEMENT: As the genetics of intellectual disability (ID) and autism spectrum disorders (ASDs) are unraveled, a key issue is whether genetically divergent forms of these disorders converge on common biochemical/cellular pathways and hence may be amenable to common therapeutic interventions. This study compares the pathophysiology associated with the loss of fragile X mental retardation protein (FMRP) and haploinsufficiency of synaptic GTPase-activating protein (SynGAP), two prevalent monogenic forms of ID. We show that Syngap(+/-) mice phenocopy Fmr1(-/y) mice in the alterations in mGluR-dependent long-term depression, basal protein synthesis, and dendritic spine morphology. Deficits in basal protein synthesis can be rescued by pharmacological interventions that reduce the mGlu5 receptor-ERK1/2 signaling pathway, which also rescues the same deficit in Fmr1(-/y) mice. Our findings support the hypothesis that phenotypes associated with genetically diverse forms of ID/ASDs result from alterations in common cellular/biochemical pathways.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/biossíntese , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Proteínas Ativadoras de ras GTPase/biossíntese , Animais , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Proteína do X Frágil da Deficiência Intelectual/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Association studies implicate multiple PDZ domain protein (MPDZ/MUPP1) sequence and/or expression in risk for alcoholism in humans and ethanol withdrawal (EW) in mice, but confirmation has been hindered by the dearth of targeted genetic models. We report the creation of transgenic (MPDZ-TG) and knockout heterozygote (Mpdz(+/-) ) mice, with increased (2.9-fold) and decreased (53%) target expression, respectively. Both models differ in EW compared with wild-type littermates (P ≤ 0.03), providing compelling evidence for an inverse relationship between Mpdz expression and EW severity. Additionally, ethanol consumption is reduced up to 18% (P = 0.006) in Mpdz(+/-) , providing the first evidence implicating Mpdz in ethanol self-administration.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Convulsões por Abstinência de Álcool/genética , Proteínas de Transporte/genética , Depressores do Sistema Nervoso Central/efeitos adversos , Etanol/efeitos adversos , Convulsões por Abstinência de Álcool/etiologia , Animais , Técnicas de Silenciamento de Genes , Proteínas de Membrana , Camundongos , Camundongos Transgênicos , Síndrome de Abstinência a Substâncias/etiologia , Síndrome de Abstinência a Substâncias/genéticaRESUMO
Insufficient sleep is a global problem with serious consequences for cognition and mental health.1 Synapses play a central role in many aspects of cognition, including the crucial function of memory consolidation during sleep.2 Interference with the normal expression or function of synapse proteins is a cause of cognitive, mood, and other behavioral problems in over 130 brain disorders.3 Sleep deprivation (SD) has also been reported to alter synapse protein composition and synapse number, although with conflicting results.4,5,6,7 In our study, we conducted synaptome mapping of excitatory synapses in 125 regions of the mouse brain and found that sleep deprivation selectively reduces synapse diversity in the cortex and in the CA1 region of the hippocampus. Sleep deprivation targeted specific types and subtypes of excitatory synapses while maintaining total synapse density (synapse number/area). Synapse subtypes with longer protein lifetimes exhibited resilience to sleep deprivation, similar to observations in aging and genetic perturbations. Moreover, the altered synaptome architecture affected the responses to neural oscillations, suggesting that sleep plays a vital role in preserving cognitive function by maintaining the brain's synaptome architecture.
RESUMO
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.
Assuntos
Aprendizagem por Associação/fisiologia , Transtornos Cognitivos/enzimologia , Giro Denteado/enzimologia , Aprendizagem por Discriminação/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Densidade Pós-Sináptica/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Detecção de Sinal Psicológico/fisiologia , Percepção Espacial/fisiologia , Animais , Núcleo Celular/enzimologia , Transtornos Cognitivos/fisiopatologia , Giro Denteado/patologia , Ácido Glutâmico/fisiologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/fisiologia , Glicogênio Sintase Quinase 3 beta , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Proteínas do Tecido Nervoso/deficiência , Neurogênese/fisiologia , Fenótipo , Fosforilação , Densidade Pós-Sináptica/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/fisiologiaRESUMO
Signaling by laminins and axonal neuregulin has been implicated in regulating axon sorting by myelin-forming Schwann cells. However, the signal transduction mechanisms are unknown. Focal adhesion kinase (FAK) has been linked to alpha6beta1 integrin and ErbB receptor signaling, and we show that myelination by Schwann cells lacking FAK is severely impaired. Mutant Schwann cells could interdigitate between axon bundles, indicating that FAK signaling was not required for process extension. However, Schwann cell FAK was required to stimulate cell proliferation, suggesting that amyelination was caused by insufficient Schwann cells. ErbB2 receptor and AKT were robustly phosphorylated in mutant Schwann cells, indicating that neuregulin signaling from axons was unimpaired. These findings demonstrate the vital relationship between axon defasciculation and Schwann cell number and show the importance of FAK in regulating cell proliferation in the developing nervous system.
Assuntos
Axônios/enzimologia , Comunicação Celular/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Sistema Nervoso/enzimologia , Células de Schwann/metabolismo , Animais , Axônios/patologia , Axônios/ultraestrutura , Contagem de Células , Feminino , Quinase 1 de Adesão Focal/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Bainha de Mielina/enzimologia , Bainha de Mielina/patologia , Sistema Nervoso/embriologia , Sistema Nervoso/patologia , Gravidez , Células de Schwann/patologia , Células de Schwann/ultraestrutura , Transdução de Sinais/fisiologiaRESUMO
Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino-acid substitution in the polyproline-binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol-3-kinase-C2alpha to the SH3-binding site of PSD95. In wild-type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.
Assuntos
Inflamação/complicações , Inflamação/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dor/complicações , Dor/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Domínios de Homologia de src , Animais , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases , Hipocampo/enzimologia , Hipocampo/patologia , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Plasticidade Neuronal , Mutação Puntual/genética , Ligação Proteica , Relação Estrutura-Atividade , Sinapses/enzimologiaRESUMO
Through protein interactions mediated by their cytoplasmic C termini the GluN2A and GluN2B subunits of NMDA receptors (NMDARs) have a key role in the formation of NMDAR signaling complexes at excitatory synapses. Although these signaling complexes are thought to have a crucial role in NMDAR-dependent forms of synaptic plasticity such as long-term potentiation (LTP), the role of the C terminus of GluN2A in coupling NMDARs to LTP enhancing and/or suppressing signaling pathways is unclear. To address this issue we examined the induction of LTP in the hippocampal CA1 region in mice lacking the C terminus of endogenous GluN2A subunits (GluN2AΔC/ΔC). Our results show that truncation of GluN2A subunits produces robust, but highly frequency-dependent, deficits in LTP and a reduction in basal levels of extracellular signal regulated kinase 2 (ERK2) activation and phosphorylation of AMPA receptor GluA1 subunits at a protein kinase A site (serine 845). Consistent with the notion that these signaling deficits contribute to the deficits in LTP in GluN2AΔC/ΔC mice, activating ERK2 and increasing GluA1 S845 phosphorylation through activation of ß-adrenergic receptors rescued the induction of LTP in these mutants. Together, our results indicate that the capacity of excitatory synapses to undergo plasticity in response to different patterns of activity is dependent on the coupling of specific signaling pathways to the intracellular domains of the NMDARs and that abnormal plasticity resulting from mutations in NMDARs can be reduced by activation of key neuromodulatory transmitter receptors that engage converging signaling pathways.
Assuntos
Hipocampo/fisiologia , Potenciação de Longa Duração/genética , Mutação/genética , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Fenômenos Biofísicos/genética , Estimulação Elétrica/métodos , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Isoproterenol/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , N-Metilaspartato/farmacologia , Técnicas de Patch-Clamp/métodos , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Receptores de N-Metil-D-Aspartato/deficiência , Receptores de N-Metil-D-Aspartato/genética , Serina/metabolismoRESUMO
Neurodevelopmental disorders of genetic origin delay the acquisition of normal abilities and cause disabling phenotypes. Nevertheless, spontaneous attenuation and even complete amelioration of symptoms in early childhood and adolescence can occur in many disorders, suggesting that brain circuits possess an intrinsic capacity to overcome the deficits arising from some germline mutations. We examined the molecular composition of almost a trillion excitatory synapses on a brain-wide scale between birth and adulthood in mice carrying a mutation in the homeobox transcription factor Pax6, a neurodevelopmental disorder model. Pax6 haploinsufficiency had no impact on total synapse number at any age. By contrast, the molecular composition of excitatory synapses, the postnatal expansion of synapse diversity and the acquisition of normal synaptome architecture were delayed in all brain regions, interfering with networks and electrophysiological simulations of cognitive functions. Specific excitatory synapse types and subtypes were affected in two key developmental age-windows. These phenotypes were reversed within 2-3 weeks of onset, restoring synapse diversity and synaptome architecture to the normal developmental trajectory. Synapse subtypes with rapid protein turnover mediated the synaptome remodeling. This brain-wide capacity for remodeling of synapse molecular composition to recover and maintain the developmental trajectory of synaptome architecture may help confer resilience to neurodevelopmental genetic disorders.
Assuntos
Transtornos do Neurodesenvolvimento , Sinapses , Animais , Humanos , Camundongos , Encéfalo/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Fenótipo , Sinapses/metabolismoRESUMO
The lifetime of proteins in synapses is important for their signaling, maintenance, and remodeling, and for memory duration. We quantified the lifetime of endogenous PSD95, an abundant postsynaptic protein in excitatory synapses, at single-synapse resolution across the mouse brain and lifespan, generating the Protein Lifetime Synaptome Atlas. Excitatory synapses have a wide range of PSD95 lifetimes extending from hours to several months, with distinct spatial distributions in dendrites, neurons, and brain regions. Synapses with short protein lifetimes are enriched in young animals and in brain regions controlling innate behaviors, whereas synapses with long protein lifetimes accumulate during development, are enriched in the cortex and CA1 where memories are stored, and are preferentially preserved in old age. Synapse protein lifetime increases throughout the brain in a mouse model of autism and schizophrenia. Protein lifetime adds a further layer to synapse diversity and enriches prevailing concepts in brain development, aging, and disease.
Assuntos
Longevidade , Sinapses , Camundongos , Animais , Sinapses/fisiologia , Neurônios/fisiologia , Encéfalo/fisiologia , Proteína 4 Homóloga a Disks-Large/metabolismoRESUMO
Lipid rafts are dynamic membrane microdomains enriched in cholesterol and sphingolipids involved in the compartmentalization of signaling pathways, trafficking and sorting of proteins. At synapses, the glutamatergic NMDA receptor and its cytoplasmic scaffold protein PSD-95 move between postsynaptic density (PSD) and rafts following learning or ischemia. However it is not known whether the signaling complexes formed by these proteins are different in rafts nor the molecular mechanisms that govern their localization. To examine these issues in vivo we used mice carrying genetically encoded tags for purification of protein complexes and specific mutations in NMDA receptors, PSD-95 and other postsynaptic scaffold proteins. Isolation of PSD-95 complexes from mice carrying tandem affinity purification tags showed differential composition in lipid rafts, postsynaptic density and detergent-soluble fractions. Raft PSD-95 complexes showed less CaMKIIalpha and SynGAP and enrichment in Src and Arc/Arg3.1 compared with PSD complexes. Mice carrying knock-outs of PSD-95 or PSD-93 show a key role for PSD-95 in localizing NR2A-containing NMDA receptor complexes to rafts. Deletion of the NR2A C terminus or the C-terminal valine residue of NR2B, which prevents all PDZ interactions, reduced the NR1 association with rafts. Interestingly, the deletion of the NR2B valine residue increased the total amount of lipid rafts. These data show critical roles for scaffold proteins and their interactions with NMDA receptor subunits in organizing the differential expression in rafts and postsynaptic densities of synaptic signaling complexes.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/fisiologia , Animais , Toxina da Cólera , Proteína 4 Homóloga a Disks-Large , Gangliosidose GM1/metabolismo , Guanilato Quinases , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Proteínas de Membrana/deficiência , Camundongos , Camundongos Transgênicos , Mutação/genética , Domínios PDZ/genética , Domínios PDZ/fisiologia , Estrutura Terciária de Proteína/fisiologia , Receptores de N-Metil-D-Aspartato/genética , Valina/genéticaRESUMO
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.
Assuntos
Aprendizagem , Mutação com Perda de Função , Proteínas do Tecido Nervoso/genética , Animais , Moléculas de Adesão Celular Neuronais/genética , Feminino , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Tempo de Reação , Proteínas Associadas SAP90-PSD95/genética , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
BACKGROUND: Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments.The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample.Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. RESULTS: We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time.We went on to experimentally validate a number of these automated designs by Southern blotting. The majority of probes we tested performed well confirming our in silico prediction methodology and the general usefulness of the software for automated genomic Southern probe design. CONCLUSIONS: Software and supplementary information are freely available at: http://www.genes2cognition.org/software/southern_blot.
Assuntos
Southern Blotting/métodos , Biologia Computacional/métodos , Sondas de DNA/síntese química , Animais , Automação , Camundongos , SoftwareRESUMO
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.
Assuntos
Marcação de Genes/métodos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Complexos Multiproteicos/isolamento & purificação , Proteínas do Tecido Nervoso/isolamento & purificação , Esquizofrenia/metabolismo , Animais , Encéfalo/metabolismo , Cromatografia de Afinidade , Proteína 4 Homóloga a Disks-Large , Expressão Gênica , Guanilato Quinases , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Camundongos , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Sinapses/metabolismo , Potenciais Sinápticos , Espectrometria de Massas em TandemRESUMO
Synapses connect neurons together to form the circuits of the brain, and their molecular composition controls innate and learned behavior. We analyzed the molecular and morphological diversity of 5 billion excitatory synapses at single-synapse resolution across the mouse brain from birth to old age. A continuum of changes alters synapse composition in all brain regions across the life span. Expansion in synapse diversity produces differentiation of brain regions until early adulthood, and compositional changes cause dedifferentiation in old age. The spatiotemporal synaptome architecture of the brain potentially accounts for life-span transitions in intellectual ability, memory, and susceptibility to behavioral disorders.