RESUMO
Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. Here, we show that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. We identify the regulation of GABA availability by mitochondrial activity as a biologically relevant mechanism and demonstrate its contribution to social behavior. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. We identify Aralar as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases our understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , Ácido gama-Aminobutírico/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Ácido Aspártico/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Glucose/metabolismo , Homeostase , Humanos , Masculino , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Comportamento Social , Transmissão Sináptica , Ácido gama-Aminobutírico/genéticaRESUMO
Studies using induced pluripotent stem cells (iPSCs) are gaining momentum in brain disorder modelling, but optimal study designs are poorly defined. Here, we compare commonly used designs and statistical analysis for different research aims. Furthermore, we generated immunocytochemical, electrophysiological, and proteomic data from iPSC-derived neurons of five healthy subjects, analysed data variation and conducted power simulations. These analyses show that published case-control iPSC studies are generally underpowered. Designs using isogenic iPSC lines typically have higher power than case-control designs, but generalization of conclusions is limited. We show that, for the realistic settings used in this study, a multiple isogenic pair design increases absolute power up to 60% or requires up to 5-fold fewer lines. A free web tool is presented to explore the power of different study designs, using any (pilot) data.
Assuntos
Encefalopatias , Células-Tronco Pluripotentes Induzidas , Humanos , Proteômica , Estudos de Casos e Controles , Voluntários SaudáveisRESUMO
In the rapidly moving proteomics field, a diverse patchwork of data analysis pipelines and algorithms for data normalization and differential expression analysis is used by the community. We generated a mass spectrometry downstream analysis pipeline (MS-DAP) that integrates both popular and recently developed algorithms for normalization and statistical analyses. Additional algorithms can be easily added in the future as plugins. MS-DAP is open-source and facilitates transparent and reproducible proteome science by generating extensive data visualizations and quality reporting, provided as standardized PDF reports. Second, we performed a systematic evaluation of methods for normalization and statistical analysis on a large variety of data sets, including additional data generated in this study, which revealed key differences. Commonly used approaches for differential testing based on moderated t-statistics were consistently outperformed by more recent statistical models, all integrated in MS-DAP. Third, we introduced a novel normalization algorithm that rescues deficiencies observed in commonly used normalization methods. Finally, we used the MS-DAP platform to reanalyze a recently published large-scale proteomics data set of CSF from AD patients. This revealed increased sensitivity, resulting in additional significant target proteins which improved overlap with results reported in related studies and includes a large set of new potential AD biomarkers in addition to previously reported.
Assuntos
Doença de Alzheimer , Software , Humanos , Proteômica/métodos , Benchmarking , Fluxo de Trabalho , Doença de Alzheimer/diagnóstico , Proteoma/análise , Espectrometria de Massas/métodos , BiomarcadoresRESUMO
Synapse development requires spatiotemporally regulated recruitment of synaptic proteins. In this study, we describe a novel presynaptic mechanism of cis-regulated oligomerization of adhesion molecules that controls synaptogenesis. We identified synaptic adhesion-like molecule 1 (SALM1) as a constituent of the proposed presynaptic Munc18/CASK/Mint1/Lin7b organizer complex. SALM1 preferentially localized to presynaptic compartments of excitatory hippocampal neurons. SALM1 depletion in excitatory hippocampal primary neurons impaired Neurexin1ß- and Neuroligin1-mediated excitatory synaptogenesis and reduced synaptic vesicle clustering, synaptic transmission, and synaptic vesicle release. SALM1 promoted Neurexin1ß clustering in an F-actin- and PIP2-dependent manner. Two basic residues in SALM1's juxtamembrane polybasic domain are essential for this clustering. Together, these data show that SALM1 is a presynaptic organizer of synapse development by promoting F-actin/PIP2-dependent clustering of Neurexin.
Assuntos
Actinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sinapses/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , NeurogêneseRESUMO
INTRODUCTION: Cerebrospinal fluid (CSF) biomarkers for specific cellular disease processes are lacking for tauopathies. In this translational study we aimed to identify CSF biomarkers reflecting early tau pathology-associated unfolded protein response (UPR) activation. METHODS: We employed mass spectrometry proteomics and targeted immunoanalysis in a combination of biomarker discovery in primary mouse neurons in vitro and validation in patient CSF from two independent large multicentre cohorts (EMIF-AD MBD, n = 310; PRIDE, n = 771). RESULTS: First, we identify members of the protein disulfide isomerase (PDI) family in the neuronal UPR-activated secretome and validate secretion upon tau aggregation in vitro. Next, we demonstrate that PDIA1 and PDIA3 levels correlate with total- and phosphorylated-tau levels in CSF. PDIA1 levels are increased in CSF from AD patients compared to controls and patients with tau-unrelated frontotemporal and Lewy body dementia (LBD). HIGHLIGHTS: Neuronal unfolded protein response (UPR) activation induces the secretion of protein disulfide isomerases (PDIs) in vitro. PDIA1 is secreted upon tau aggregation in neurons in vitro. PDIA1 and PDIA3 levels correlate with total and phosphorylated tau levels in CSF. PDIA1 levels are increased in CSF from Alzheimer's disease (AD) patients compared to controls. PDIA1 levels are not increased in CSF from tau-unrelated frontotemporal dementia (FTD) and Lewy body dementia (LBD) patients.
Assuntos
Doença de Alzheimer , Doença por Corpos de Lewy , Animais , Camundongos , Doença por Corpos de Lewy/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Isomerases de Dissulfetos de Proteínas , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Fosforilação , Doença de Alzheimer/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidianoRESUMO
The pentameric γ-Aminobutyric acid type A receptors (GABAARs) are ligand-gated ion channels that mediate the majority of inhibitory neurotransmission in the brain. In the cerebellum, the two main receptor subtypes are the 2α1/2ß/γ and 2α6/2ß/δ subunits. In the present study, an interaction proteomics workflow was used to reveal additional subtypes that contain both α1 and α6 subunits. Immunoprecipitation of the α6 subunit from mouse brain cerebellar extract co-purified the α1 subunit. In line with this, pre-incubation of the cerebellar extract with anti-α6 antibodies and analysis by blue native gel electrophoresis mass-shifted part of the α1 complexes, indicative of the existence of an α1α6-containing receptor. Subsequent mass spectrometry of the blue native gel showed the α1α6-containing receptor subtype to exist in two main forms, i.e., with or without Neuroligin-2. Immunocytochemistry on a cerebellar granule cell culture revealed co-localization of α6 and α1 in post-synaptic puncta that apposed the presynaptic marker protein Vesicular GABA transporter, indicative of the presence of this synaptic GABAAR subtype.
Assuntos
Receptores de GABA-A , Receptores de GABA , Camundongos , Animais , Receptores de GABA/metabolismo , Receptores de GABA-A/metabolismo , Eletroforese em Gel de Poliacrilamida Nativa , Cerebelo/metabolismo , Anticorpos/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Protein phosphatase 2B (PP2B) is critical for synaptic plasticity and learning, but the molecular mechanisms involved remain unclear. Here we identified different types of proteins that interact with PP2B, including various structural proteins of the postsynaptic densities (PSDs) of Purkinje cells (PCs) in mice. Deleting PP2B reduced expression of PSD proteins and the relative thickness of PSD at the parallel fiber to PC synapses, whereas reexpression of inactive PP2B partly restored the impaired distribution of nanoclusters of PSD proteins, together indicating a structural role of PP2B. In contrast, lateral mobility of surface glutamate receptors solely depended on PP2B phosphatase activity. Finally, the level of motor learning covaried with both the enzymatic and nonenzymatic functions of PP2B. Thus, PP2B controls synaptic function and learning both through its action as a phosphatase and as a structural protein that facilitates synapse integrity.SIGNIFICANCE STATEMENT Phosphatases are generally considered to serve their critical role in learning and memory through their enzymatic operations. Here, we show that protein phosphatase 2B (PP2B) interacts with structural proteins at the synapses of cerebellar Purkinje cells. Differentially manipulating the enzymatic and structural domains of PP2B leads to different phenotypes in cerebellar learning. We propose that PP2B is crucial for cerebellar learning via two complementary actions, an enzymatic and a structural operation.
Assuntos
Calcineurina/metabolismo , Aprendizagem/fisiologia , Plasticidade Neuronal/fisiologia , Células de Purkinje/fisiologia , Animais , Movimentos Oculares/fisiologia , Camundongos , Densidade Pós-Sináptica/metabolismoRESUMO
An enigma in studies of neuropsychiatric disorders is how to translate polygenic risk into disease biology. For schizophrenia, where > 145 significant GWAS loci have been identified and only a few genes directly implicated, addressing this issue is a particular challenge. We used a combined cellomics and proteomics approach to show that polygenic risk can be disentangled by searching for shared neuronal morphology and cellular pathway phenotypes of candidate schizophrenia risk genes. We first performed an automated high-content cellular screen to characterize neuronal morphology phenotypes of 41 candidate schizophrenia risk genes. The transcription factors Tcf4 and Tbr1 and the RNA topoisomerase Top3b shared a neuronal phenotype marked by an early and progressive reduction in synapse numbers upon knockdown in mouse primary neuronal cultures. Proteomics analysis subsequently showed that these three genes converge onto the syntaxin-mediated neurotransmitter release pathway, which was previously implicated in schizophrenia, but for which genetic evidence was weak. We show that dysregulation of multiple proteins in this pathway may be due to the combined effects of schizophrenia risk genes Tcf4, Tbr1, and Top3b. Together, our data provide new biological functions for schizophrenia risk genes and support the idea that polygenic risk is the result of multiple small impacts on common neuronal signaling pathways.
Assuntos
Esquizofrenia , Animais , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Camundongos , Herança Multifatorial/genética , Neurônios , Fenótipo , Polimorfismo de Nucleotídeo Único , Proteômica , Esquizofrenia/genéticaRESUMO
Granulovacuolar degeneration (GVD) is a common feature in Alzheimer's disease (AD). The occurrence of GVD is closely associated with that of neurofibrillary tangles (NFTs) and GVD is even considered to be a pre-NFT stage in the disease process of AD. Currently, the composition of GVD bodies, the mechanisms associated with GVD and how GVD exactly relates to NFTs is not well understood. By combining immunohistochemistry (IHC) and laser microdissection (LMD) we isolated neurons with GVD and those bearing tangles separately from human post-mortem AD hippocampus (n = 12) using their typical markers casein kinase (CK)1δ and phosphorylated tau (AT8). Control neurons were isolated from cognitively healthy cases (n = 12). 3000 neurons per sample were used for proteome analysis by label free LC-MS/MS. In total 2596 proteins were quantified across samples and a significant change in abundance of 115 proteins in GVD and 197 in tangle bearing neurons was observed compared to control neurons. With IHC the presence of PPIA, TOMM34, HSP70, CHMP1A, TPPP and VXN was confirmed in GVD containing neurons. We found multiple proteins localizing specifically to the GVD bodies, with VXN and TOMM34 being the most prominent new protein markers for GVD bodies. In general, protein groups related to protein folding, proteasomal function, the endolysosomal pathway, microtubule and cytoskeletal related function, RNA processing and glycolysis were found to be changed in GVD neurons. In addition to these protein groups, tangle bearing neurons show a decrease in ribosomal proteins, as well as in various proteins related to protein folding. This study, for the first time, provides a comprehensive human based quantitative assessment of protein abundances in GVD and tangle bearing neurons. In line with previous functional data showing that tau pathology induces GVD, our data support the model that GVD is part of a pre-NFT stage representing a phase in which proteostasis and cellular homeostasis is disrupted. Elucidating the molecular mechanisms and cellular processes affected in GVD and its relation to the presence of tau pathology is highly relevant for the identification of new drug targets for therapy.
Assuntos
Doença de Alzheimer/metabolismo , Degeneração Neural/metabolismo , Emaranhados Neurofibrilares/metabolismo , Neurônios/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Feminino , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Masculino , Pessoa de Meia-Idade , Degeneração Neural/patologia , Emaranhados Neurofibrilares/patologia , Neurônios/patologia , Proteoma , Vacúolos/metabolismo , Vacúolos/patologiaRESUMO
The pentameric glycine receptor (GlyR), comprising the α1 and ß subunits, is a major inhibitory ionotropic receptor in brainstem and spinal cord. GlyRs interact with gephyrin (GPHN), a scaffold protein that anchors the GlyR in the plasma membrane and enables it to form clusters in glycinergic postsynapses. Using an interaction proteomics approach, evidence of the ArfGEFs IQ motif and Sec7 domain 3 (IQSEC3) and IQ motif and Sec7 domain 2 (IQSEC2) as two novel synaptic proteins interacting with GlyR complexes is provided. When the affinity-isolated GlyR complexes are fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1ß-GPHN appears as the most abundant complex with a molecular weight of ≈1 MDa, and GlyR α1ß-GPHN-IQSEC3 as a minor protein complex of ≈1.2 MDa. A third GlyR α1ß-GPHN-IQSEC2 complex exists at the lowest amount with a mass similar to the IQSEC3 containing complex. Using yeast two-hybrid it is demonstrated that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N-terminal of the IQSEC3 IQ-like domain. The data provide direct evidence of the interaction of IQSEC3 with GlyR-GPHN complexes, underscoring a potential role of these ArfGEFs in the function of glycinergic synapses.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Eletroforese/métodos , Proteoma/análise , Proteômica/métodos , Receptores de Glicina/metabolismo , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de Glicina/genética , Sinapses/metabolismoRESUMO
The MIR137 locus is a replicated genetic risk factor for schizophrenia. The risk-associated allele is reported to increase miR-137 expression and miR-137 overexpression alters synaptic transmission in mouse hippocampus. We investigated the cellular mechanisms underlying these observed effects in mouse hippocampal neurons in culture. First, we correlated the risk allele to expression of the genes in the MIR137 locus in human postmortem brain. Some evidence for increased MIR137HG expression was observed, especially in hippocampus of the disease-associated genotype. Second, in mouse hippocampal neurons, we confirmed previously observed changes in synaptic transmission upon miR-137 overexpression. Evoked synaptic transmission and spontaneous release were 50% reduced. We identified defects in release probability as the underlying cause. In contrast to previous observations, no evidence was obtained for selective synaptic vesicle docking defects. Instead, ultrastructural morphometry revealed multiple effects of miR-137 overexpression on docking, active zone length and total vesicle number. Moreover, proteomic analyses of neuronal protein showed that expression of Syt1 and Cplx1, previously reported as downregulated upon miR-137 overexpression, was unaltered. Immunocytochemistry of synapses overexpressing miR-137 showed normal Synaptotagmin1 and Complexin1 protein levels. Instead, our proteomic analyses revealed altered expression of genes involved in synaptogenesis. Concomitantly, synaptogenesis assays revealed 31% reduction in synapse formation. Taken together, these data show that miR-137 regulates synaptic function by regulating synaptogenesis, synaptic ultrastructure and synapse function. These effects are plausible contributors to the increased schizophrenia risk associated with miR-137 overexpression.
Assuntos
MicroRNAs/genética , Proteômica , Esquizofrenia/genética , Animais , Autopsia , Exocitose/genética , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Humanos , Camundongos , Neurônios/patologia , Esquizofrenia/fisiopatologia , Sinapses/genética , Transmissão Sináptica/genética , Vesículas Sinápticas/genéticaRESUMO
Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal-regulated kinase (ERK) that mediates CB1R- and mGluR2/3-induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock-induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18-1. Mimicking constitutive phosphorylation of Munc18-1 results in a drastic decrease in synaptic transmission. ERK-mediated phosphorylation of Munc18-1 ultimately leads to degradation by the ubiquitin-proteasome system. Conversely, preventing ERK-dependent Munc18-1 phosphorylation increases synaptic strength. CB1R- and mGluR2/3-induced synaptic inhibition and depolarization-induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK-dependent Munc18-1 phosphorylation is blocked. Thus, ERK-dependent Munc18-1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Munc18/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transmissão Sináptica , Animais , Estimulação Elétrica , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores , Feminino , Células HEK293 , Hipocampo/fisiologia , Humanos , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Neurônios/fisiologia , Neurônios/ultraestrutura , Fosforilação , Gravidez , Ratos Wistar , Estresse Psicológico/metabolismoRESUMO
A simple and fast immunoprecipitation (IP) protocol is designed with the sample preparation incorporated, applicable to both low and high throughput. This new protocol combines two procedures based on magnetic beads in 96-well plate format. Protein complexes are captured by antibodies and magnetic beads conjugated with protein A. Proteins are washed and on-bead digested by using Single-Pot solid-phase sample preparation (SP3). The whole IP-SP3 approach can be completed in one day, which is considerably faster compared to the classical approach. No major quantitative differences are found between SP3 and FASP (filter-aided sample preparation) or a longer incubation protocol. Taken together, the IP-SP3 protocol is a fast and economical approach easily applicable for large-scale protein interactome analysis.
Assuntos
Imunoprecipitação/métodos , Complexos Multiproteicos/genética , Proteoma/genética , Proteômica/métodos , Anticorpos/genética , Anticorpos/imunologia , Imunoprecipitação/economia , Imãs , Complexos Multiproteicos/química , Proteômica/economia , Manejo de Espécimes/economia , Proteína Estafilocócica A/química , Proteína Estafilocócica A/genéticaRESUMO
Data-independent acquisition (DIA) is an emerging technology for quantitative proteomics. Current DIA focusses on the identification and quantitation of fragment ions that are generated from multiple peptides contained in the same selection window of several to tens of m/z. An alternative approach is WiSIM-DIA, which combines conventional DIA with wide-SIM (wide selected-ion monitoring) windows to partition the precursor m/z space to produce high-quality precursor ion chromatograms. However, WiSIM-DIA has been underexplored; it remains unclear if it is a viable alternative to DIA. We demonstrate that WiSIM-DIA quantified more than 24 000 unique peptides over five orders of magnitude in a single 2 h analysis of a neuronal synapse-enriched fraction, compared to 31 000 in DIA. There is a strong correlation between abundance values of peptides quantified in both the DIA and WiSIM-DIA datasets. Interestingly, the S/N ratio of these peptides is not correlated. We further show that peptide identification directly from DIA spectra identified >2000 proteins, which included unique peptides not found in spectral libraries generated by DDA.
Assuntos
Interpretação Estatística de Dados , Hipocampo/metabolismo , Espectrometria de Massas/métodos , Proteoma/metabolismo , Software , Sinaptossomos/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , ProteômicaRESUMO
Synapse disruption takes place in many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the mechanistic understanding of this process is still limited. We set out to study a possible role for dynein in synapse integrity. Cytoplasmic dynein is a multisubunit intracellular molecule responsible for diverse cellular functions, including long-distance transport of vesicles, organelles, and signaling factors toward the cell center. A less well-characterized role dynein may play is the spatial clustering and anchoring of various factors including mRNAs in distinct cellular domains such as the neuronal synapse. Here, in order to gain insight into dynein functions in synapse integrity and disruption, we performed a screen for novel dynein interactors at the synapse. Dynein immunoprecipitation from synaptic fractions of the ALS model mSOD1(G93A) and wild-type controls, followed by mass spectrometry analysis on synaptic fractions of the ALS model mSOD1(G93A) and wild-type controls, was performed. Using advanced network analysis, we identified Staufen1, an RNA-binding protein required for the transport and localization of neuronal RNAs, as a major mediator of dynein interactions via its interaction with protein phosphatase 1-beta (PP1B). Both in vitro and in vivo validation assays demonstrate the interactions of Staufen1 and PP1B with dynein, and their colocalization with synaptic markers was altered as a result of two separate ALS-linked mutations: mSOD1(G93A) and TDP43(A315T). Taken together, we suggest a model in which dynein's interaction with Staufen1 regulates mRNA localization along the axon and the synapses, and alterations in this process may correlate with synapse disruption and ALS toxicity.
Assuntos
Esclerose Lateral Amiotrófica/genética , Dineínas do Citoplasma/genética , Proteômica , Proteínas de Ligação a RNA/biossíntese , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Axônios/metabolismo , Axônios/patologia , Dineínas do Citoplasma/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação , Proteínas de Ligação a RNA/genética , Sinapses/genética , Sinapses/metabolismo , Sinapses/patologia , Sinaptossomos/metabolismo , Sinaptossomos/patologiaRESUMO
The relative abundance of synaptic proteins shapes protein complex formation and is essential for synapse function and behavioral fitness. Here, we have used a panel of highly diverse inbred strains of mice-NOD/LtJ, A/J, 129S1/SvImJ, FVB/NJ, C57BL/6J, WSB/EiJ, PWK/PhJ, and CAST/EiJ-to quantify the effects of genetic variation on the synaptic proteome between strains. Using iTRAQ-based quantitative proteome analyses, we detected significant differences in â¼20% of 400 core synaptic proteins. Surprisingly, the differentially abundant proteins showed a modest range of variation across strains, averaging about 1.3-fold. Analysis of protein abundance covariation across the eight strains identified known protein-protein relations (proteins of Arp2/3 complex), as well as novel relations (e.g. Dlg family, Fscn1). Moreover, covariation of synaptic proteins was substantially tighter (â¼fourfold more dense than chance level) than corresponding networks of synaptic transcripts (â¼twofold more dense than chance). The tight stoichiometry and coherent synaptic protein covariation networks suggest more intense evolutionary selection at this level of molecular organization. In conclusion, genetic diversity in the mouse genome differentially affects the transcriptome and proteome, and only partially penetrates the synaptic proteome. Protein abundance correlation analyses in genetically divergent strains can complement protein-protein interaction network analyses, to provide insight into protein interactomes.
Assuntos
Variação Genética/genética , Camundongos Endogâmicos/genética , Proteoma/análise , Animais , Camundongos , Mapas de Interação de Proteínas , Proteoma/genética , Proteoma/metabolismo , ProteômicaRESUMO
The group 1 metabotropic glutamate receptors 1 and 5 (mGluR1/5) have been implicated in mechanisms of synaptic plasticity and may serve as potential therapeutic targets in autism spectrum disorders. The interactome of group 1 mGluRs has remained largely unresolved. Using a knockout-controlled interaction proteomics strategy we examined the mGluR5 protein complex in two brain regions, hippocampus and cortex, and identified mGluR1 as its major interactor in addition to the well described Homer proteins. We confirmed the presence of mGluR1/5 complex by (i) reverse immunoprecipitation using an mGluR1 antibody to pulldown mGluR5 from hippocampal tissue, (ii) coexpression in HEK293 cells followed by coimmunoprecipitation to reveal the direct interaction of mGluR1 and 5, and (iii) superresolution microscopy imaging of hippocampal primary neurons to show colocalization of the mGluR1/5 in the synapse.
Assuntos
Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Receptor de Glutamato Metabotrópico 5/análise , Receptores de Glutamato Metabotrópico/análiseRESUMO
In brain, specific RNA-binding proteins (RBPs) associate with localized mRNAs and function as regulators of protein synthesis at synapses exerting an indirect control on neuronal activity. Thus, the Fragile X Mental Retardation protein (FMRP) regulates expression of the scaffolding postsynaptic density protein PSD95, but the mode of control appears to be different from other FMRP target mRNAs. Here, we show that the fragile X mental retardation-related protein 2 (FXR2P) cooperates with FMRP in binding to the 3'-UTR of mouse PSD95/Dlg4 mRNA. Absence of FXR2P leads to decreased translation of PSD95/Dlg4 mRNA in the hippocampus, implying a role for FXR2P as translation activator. Remarkably, mGluR-dependent increase of PSD95 synthesis is abolished in neurons lacking Fxr2. Together, these findings show a coordinated regulation of PSD95/Dlg4 mRNA by FMRP and FXR2P that ultimately affects its fine-tuning during synaptic activity.
Assuntos
Regulação da Expressão Gênica/fisiologia , Guanilato Quinases/biossíntese , Proteínas de Membrana/biossíntese , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Western Blotting , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases/genética , Imuno-Histoquímica , Imunoprecipitação , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Biossíntese de Proteínas/fisiologia , Proteínas de Ligação a RNA/genéticaRESUMO
NMDA receptors play a central role in shaping the strength of synaptic connections throughout development and in mediating synaptic plasticity mechanisms that underlie some forms of learning and memory formation in the CNS. In the hippocampus and the neocortex, GluN1 is combined primarily with GluN2A and GluN2B, which are differentially expressed during development and confer distinct molecular and physiological properties to NMDA receptors. The contribution of each subunit to the synaptic traffic of NMDA receptors and therefore to their role during development and in synaptic plasticity is still controversial. We report a critical role for the GluN2B subunit in regulating NMDA receptor synaptic targeting. In the absence of GluN2B, the synaptic levels of AMPA receptors are increased and accompanied by decreased constitutive endocytosis of GluA1-AMPA receptor. We used quantitative proteomic analysis to identify changes in the composition of postsynaptic densities from GluN2B(-/-) mouse primary neuronal cultures and found altered levels of several ubiquitin proteasome system components, in particular decreased levels of proteasome subunits. Enhancing the proteasome activity with a novel proteasome activator restored the synaptic levels of AMPA receptors in GluN2B(-/-) neurons and their endocytosis, revealing that GluN2B-mediated anchoring of the synaptic proteasome is responsible for fine tuning AMPA receptor synaptic levels under basal conditions.
Assuntos
Encéfalo/citologia , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Animais , Células Cultivadas , Endocitose/fisiologia , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hidrazonas/farmacologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/fisiologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico/genética , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Sinapses/efeitos dos fármacos , Tetrodotoxina/farmacologia , Fatores de Tempo , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
The inbred strains C57BL/6J and DBA/2J (DBA) display striking differences in a number of behavioral tasks depending on hippocampal function, such as contextual memory. Historically, this has been explained through differences in postsynaptic protein expression underlying synaptic transmission and plasticity. We measured the synaptic hippocampal protein content (iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) and mass spectrometry), CA1 synapse ultrastructural morphology, and synaptic functioning in adult C57BL/6J and DBA mice. DBA mice showed a prominent decrease in the Ras-GAP calcium-sensing protein RASAL1. Furthermore, expression of several presynaptic markers involved in exocytosis, such as syntaxin (Stx1b), Ras-related proteins (Rab3a/c), and rabphilin (Rph3a), was reduced. Ultrastructural analysis of CA1 hippocampal synapses showed a significantly lower number of synaptic vesicles and presynaptic cluster size in DBA mice, without changes in postsynaptic density or active zone. In line with this compromised presynaptic morphological and molecular phenotype in DBA mice, we found significantly lower paired-pulse facilitation and enhanced short term depression of glutamatergic synapses, indicating a difference in transmitter release and/or refilling mechanisms. Taken together, our data suggest that in addition to strain-specific postsynaptic differences, the change in dynamic properties of presynaptic transmitter release may underlie compromised synaptic processing related to cognitive functioning in DBA mice.