RESUMO
Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity of synaptic proteomes remains largely unexplored. We prepared synaptosomes from 7 different transgenic mouse lines with fluorescently labeled presynaptic terminals. Combining microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting (FASS), we isolated and analyzed the proteomes of 18 different synapse types. We discovered â¼1,800 unique synapse-type-enriched proteins and allocated thousands of proteins to different types of synapses (https://syndive.org/). We identify shared synaptic protein modules and highlight the proteomic hotspots for synapse specialization. We reveal unique and common features of the striatal dopaminergic proteome and discover the proteome signatures that relate to the functional properties of different interneuron classes. This study provides a molecular systems-biology analysis of synapses and a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.
Assuntos
Encéfalo , Proteoma , Sinapses , Animais , Camundongos , Encéfalo/metabolismo , Camundongos Transgênicos , Proteoma/metabolismo , Proteômica , Sinapses/metabolismo , Sinaptossomos/metabolismoRESUMO
Local translation meets protein turnover and plasticity demands at synapses, however, the location of its energy supply is unknown. We found that local translation in neurons is powered by mitochondria and not by glycolysis. Super-resolution microscopy revealed that dendritic mitochondria exist as stable compartments of single or multiple filaments. To test if these mitochondrial compartments can serve as local energy supply for synaptic translation, we stimulated individual synapses to induce morphological plasticity and visualized newly synthesized proteins. Depletion of local mitochondrial compartments abolished both the plasticity and the stimulus-induced synaptic translation. These mitochondrial compartments serve as spatially confined energy reserves, as local depletion of a mitochondrial compartment did not affect synaptic translation at remote spines. The length and stability of dendritic mitochondrial compartments and the spatial functional domain were altered by cytoskeletal disruption. These results indicate that cytoskeletally tethered local energy compartments exist in dendrites to fuel local translation during synaptic plasticity.
Assuntos
Mitocôndrias/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas/fisiologia , Animais , Citoesqueleto/metabolismo , Dendritos/metabolismo , Espinhas Dendríticas/metabolismo , Feminino , Masculino , Mitocôndrias/fisiologia , Plasticidade Neuronal/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismoRESUMO
As our understanding of the cell interior has grown, we have come to appreciate that most cellular operations are localized, that is, they occur at discrete and identifiable locations or domains. These cellular domains contain enzymes, machines, and other components necessary to carry out and regulate these localized operations. Here, we review these features of one such operation: the localization and translation of mRNAs within subcellular compartments observed across cell types and organisms. We describe the conceptual advantages and the "ingredients" and mechanisms of local translation. We focus on the nature and features of localized mRNAs, how they travel and get localized, and how this process is regulated. We also evaluate our current understanding of protein synthesis machines (ribosomes) and their cadre of regulatory elements, that is, the translation factors.
Assuntos
Biossíntese de Proteínas , Ribossomos , Ribossomos/genética , Ribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying molecular mechanisms are poorly understood and important players are lacking. Here, we discovered a Rab5 effector, the five-subunit endosomal Rab5 and RNA/ribosome intermediary (FERRY) complex, that recruits mRNAs and ribosomes to early endosomes through direct mRNA-interaction. FERRY displays preferential binding to certain groups of transcripts, including mRNAs encoding mitochondrial proteins. Deletion of FERRY subunits reduces the endosomal localization of transcripts in cells and has a significant impact on mRNA levels. Clinical studies show that genetic disruption of FERRY causes severe brain damage. We found that, in neurons, FERRY co-localizes with mRNA on early endosomes, and mRNA loaded FERRY-positive endosomes are in close proximity of mitochondria. FERRY thus transforms endosomes into mRNA carriers and plays a key role in regulating mRNA distribution and transport.
Assuntos
Endossomos , Proteínas rab5 de Ligação ao GTP , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Endossomos/metabolismo , Transporte Biológico , Endocitose/fisiologiaRESUMO
Neurons are characterized by a complex morphology that enables the generation of subcellular compartments with unique biochemical and biophysical properties, such as dendrites, axons, and synapses. To sustain these different compartments and carry a wide array of elaborate operations, neurons express a diverse repertoire of gene products. Extensive regulation at both the messenger RNA (mRNA) and protein levels allows for the differentiation of subcellular compartments as well as numerous forms of plasticity in response to variable stimuli. Among the multiple mechanisms that control cellular functions, mRNA translation is manipulated by neurons to regulate where and when a protein emerges. Interestingly, transcriptomic and translatomic profiles of both dendrites and axons have revealed that the mRNA population only partially predicts the local protein population and that this relation significantly varies between different gene groups. Here, we describe the space that local translation occupies within the large molecular and regulatory complexity of neurons, in contrast to other modes of regulation. We then discuss the specialized organization of mRNAs within different neuronal compartments, as revealed by profiles of the local transcriptome. Finally, we discuss the features and functional implications of both locally correlated-and anticorrelated-mRNA-protein relations both under baseline conditions and during synaptic plasticity.
Assuntos
Axônios , Dendritos , Axônios/metabolismo , Dendritos/genética , Dendritos/metabolismo , Plasticidade Neuronal/genética , Neurônios/metabolismo , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Species that depend on membership in social groups for survival exhibit changes in neuronal gene expression and behaviour when they face restricted social interactions or isolation1-3. Here we show that, across the lifespan of zebrafish (Danio rerio), social isolation specifically decreased the level of transcription of pth2, the gene that encodes the vertebrate-specific neuropeptide Pth2. However, 30 minutes of exposure to conspecifics was sufficient to initiate a significant rescue of pth2 transcript levels in previously isolated zebrafish. Transcription of pth2 exhibited bidirectional dynamics; following the acute isolation of socially reared fish, a rapid reduction in the levels of pth2 was observed. The expression of pth2 tracked not only the presence of other fish but also the density of the group. The sensory modality that controls the expression of pth2 was neither visual nor chemosensory in origin but instead was mechanical, induced by the movements of neighbouring fish. Chemical ablation of the mechanosensitive neuromast cells within the lateral line of fish prevented the rescue of pth2 levels that was induced by the social environment. In addition, mechanical perturbation of the water at frequencies similar to the movements of the zebrafish tail was sufficient to rescue the levels of pth2 in previously isolated fish. These data indicate a previously underappreciated role for the relatively unexplored neuropeptide Pth2 in both tracking and responding to the population density of the social environment of an animal.
Assuntos
Mecanotransdução Celular , Hormônio Paratireóideo/metabolismo , Peixe-Zebra/metabolismo , Animais , Feminino , Masculino , Hormônio Paratireóideo/genética , Isolamento Social , Transcrição Gênica , Peixe-Zebra/genéticaRESUMO
Angelman syndrome is a neurodevelopmental disorder caused by mutations in the maternally inherited UBE3A gene, which encodes a ubiquitin ligase. Greer et al. (2010) now identify a UBE3A substrate called Arc that promotes endocytosis of neuronal AMPA receptors, providing insight into synaptic defects that may underlie the cognitive deficits in Angelman syndrome.
RESUMO
Decades of work have demonstrated that messenger RNAs (mRNAs) are localized and translated within neuronal dendrites and axons to provide proteins for remodeling and maintaining growth cones or synapses. It remains unknown, however, whether specific forms of plasticity differentially regulate the dynamics and translation of individual mRNA species. To address this, we targeted three individual synaptically localized mRNAs, CamkIIa, ß-actin, Psd95, and used molecular beacons to track endogenous mRNA movements. We used reporters and CRISPR/Cas9 gene editing to track mRNA translation in cultured neurons. We found alterations in mRNA dynamic properties occurred during two forms of synaptic plasticity, long-term potentiation (cLTP) and depression (mGluR-LTD). Changes in mRNA dynamics following either form of plasticity resulted in an enrichment of mRNA in the vicinity of dendritic spines. Both the reporters and tagging of endogenous proteins revealed the transcript-specific stimulation of protein synthesis following cLTP or mGluR-LTD. As such, the plasticity-induced enrichment of mRNA near synapses could be uncoupled from its translational status. The enrichment of mRNA in the proximity of spines allows for localized signaling pathways to decode plasticity milieus and stimulate a specific translational profile, resulting in a customized remodeling of the synaptic proteome.
Assuntos
Potenciação de Longa Duração/genética , Depressão Sináptica de Longo Prazo/genética , Neurônios/fisiologia , RNA Mensageiro/metabolismo , Sinapses/fisiologia , Animais , Células Cultivadas , Hipocampo/citologia , Microscopia Intravital , Cultura Primária de Células , Biossíntese de Proteínas , RatosRESUMO
To form synaptic connections and store information, neurons continuously remodel their proteomes. The impressive length of dendrites and axons imposes logistical challenges to maintain synaptic proteins at locations remote from the transcription source (the nucleus). The discovery of thousands of messenger RNAs (mRNAs) near synapses suggested that neurons overcome distance and gain autonomy by producing proteins locally. It is not generally known, however, if, how, and when localized mRNAs are translated into protein. To investigate the translational landscape in neuronal subregions, we performed simultaneous RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) from microdissected rodent brain slices to identify and quantify the transcriptome and translatome in cell bodies (somata) as well as dendrites and axons (neuropil). Thousands of transcripts were differentially translated between somatic and synaptic regions, with many scaffold and signaling molecules displaying increased translation levels in the neuropil. Most translational changes between compartments could be accounted for by differences in RNA abundance. Pervasive translational regulation was observed in both somata and neuropil influenced by specific mRNA features (e.g., untranslated region [UTR] length, RNA-binding protein [RBP] motifs, and upstream open reading frames [uORFs]). For over 800 mRNAs, the dominant source of translation was the neuropil. We constructed a searchable and interactive database for exploring mRNA transcripts and their translation levels in the somata and neuropil [MPI Brain Research, The mRNA translation landscape in the synaptic neuropil. https://public.brain.mpg.de/dashapps/localseq/ Accessed 5 October 2021]. Overall, our findings emphasize the substantial contribution of local translation to maintaining synaptic protein levels and indicate that on-site translational control is an important mechanism to control synaptic strength.
Assuntos
Axônios/metabolismo , Corpo Celular/metabolismo , Dendritos/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas , Análise de Sequência de RNA/métodos , Animais , Proteoma , RNA Mensageiro/genética , TranscriptomaRESUMO
Research in the past twenty years or so has revealed that neurons synthesize and degrade proteins at their synapses to enable synaptic proteome remodelling on demand and in real-time. Here we provide a quantitative overview of the decentralized neuronal protein-turnover logistics. We first analyse the huge neuronal protein turnover demand that arises from subcellular compartments outside the cell body, followed by an overview of key quantities and modulation strategies in neuronal protein turnover logistics. In the end, we briefly review recent progress in neuronal local protein synthesis and summarize diverse protein-degradation mechanisms that are found near synapses.
Assuntos
Neurônios , Sinapses , Proteólise , ProteomaRESUMO
Chemical inhibitors have revealed requirements for protein synthesis that drive cellular plasticity. We developed a genetically encodable protein synthesis inhibitor (gePSI) to achieve cell-type-specific temporal control of protein synthesis. Controlled expression of the gePSI in neurons or glia resulted in rapid, potent and reversible cell-autonomous inhibition of protein synthesis. Moreover, gePSI expression in a single neuron blocked the structural plasticity induced by single-synapse stimulation.
Assuntos
Engenharia Genética , Hipocampo/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/metabolismo , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas/farmacologia , Sinapses/metabolismo , Animais , Células Cultivadas , Células HeLa , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Humanos , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Inibidores da Síntese de Proteínas/química , Ratos , Sinapses/efeitos dos fármacosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Secreted amyloid precursor protein-alpha (sAPPα) has growth factor-like properties and can modulate long-term potentiation (LTP) and memory. Here, we demonstrate that exposure to sAPPα converts short-lasting LTP into protein-synthesis-dependent late LTP in hippocampal slices from male rats. sAPPß had no discernable effect. We hypothesized that sAPPα facilitated LTP via regulated glutamate receptor trafficking and de novo protein synthesis. We found using a linear mixed model that sAPPα stimulated trafficking of GluA2-lacking AMPARs, as well as NMDARs to the extrasynaptic cell surface, in a calcium/calmodulin-dependent kinase II and protein kinase G-dependent manner. Both cell surface receptor accumulation and LTP facilitation were present even after sAPPα washout and inhibition of receptor trafficking or protein synthesis prevented all these effects. Direct visualization of newly synthesized proteins (FUNCAT-PLA) confirmed the ability of sAPPα to stimulate de novo protein synthesis and revealed GluA1 as one of the upregulated proteins. Therefore, sAPPα generates a coordinated synthesis and trafficking of glutamate receptors to the cell surface that facilitate LTP.SIGNIFICANCE STATEMENT Secreted amyloid precursor protein-alpha (sAPPα) is a neurotrophic and neuroprotective protein that can promote synaptic plasticity and memory, yet the molecular mechanisms underlying these effects are still not well understood. Here, we show that sAPPα facilitates long-term potentiation (LTP) in a concentration-dependent fashion through cellular processes involving de novo protein synthesis and trafficking of both GluA2-lacking AMPARs and NMDARs to the extrasynaptic cell surface. sAPPα also enhances GluA1, but not GluA2, synthesis. The trafficking effects, along with the LTP facilitation, persist after sAPPα washout, revealing a metaplastic capability of exogenous sAPPα administration. sAPPα thus facilitates LTP through coordinated activation of protein synthesis and trafficking of glutamate receptors to the cell surface, where they are positioned for priming LTP.
Assuntos
Precursor de Proteína beta-Amiloide/farmacologia , Hipocampo/fisiologia , Potenciação de Longa Duração/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Biossíntese de Proteínas/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Long-term memory has been associated with morphological changes in the brain, which in turn tightly correlate with changes in synaptic efficacy. Such plasticity is proposed to rely on dendritic spines as a neuronal canvas on which these changes can occur. Given the key role of actin cytoskeleton dynamics in spine morphology, major regulating factors of this process such as Cofilin 1 (Cfl1) and LIM kinase (LIMK), an inhibitor of Cfl1 activity, are prime molecular targets that may regulate dendritic plasticity. Using a contextual fear conditioning paradigm in mice, we found that pharmacological induction of depolymerization of actin filaments through the inhibition of LIMK causes an impairment in memory reconsolidation, as well as in memory consolidation. On top of that, Cfl1 activity is inhibited and its mRNA is downregulated in CA1 neuropil after re-exposure to the training context. Moreover, by pharmacological disruption of actin cytoskeleton dynamics, the process of memory extinction can either be facilitated or impaired. Our results lead to a better understanding of the role of LIMK, Cfl1 and actin cytoskeleton dynamics in the morphological and functional changes underlying the synaptic plasticity of the memory trace.
Assuntos
Actinas/metabolismo , Cofilina 1/metabolismo , Medo/fisiologia , Hipocampo/metabolismo , Quinases Lim/metabolismo , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Masculino , Consolidação da Memória/fisiologia , CamundongosRESUMO
Adaptation to the environment during development influences the life-long survival of an animal. While brain-wide proteomic changes are expected to underlie such experience-driven physiological and behavioral flexibility, a comprehensive overview of the nature and extent of the proteomic regulation following an environmental challenge during development is currently lacking. In this study, the brain proteome of larval zebrafish is identified and it is determined how it is altered by an exposure to a natural and physical environmental challenge, namely prolonged exposure to strong water currents. A comprehensive larval zebrafish brain proteome is presented here. Furthermore, 57 proteins that are regulated by the exposure to an environmental challenge are identified, which cover multiple functions including neuronal plasticity, the stress response, axonal growth and guidance, spatial learning, and energy metabolism. These represent candidate proteins that may play crucial roles for the adaption to an environmental challenge during development.
Assuntos
Encéfalo/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Animais , Larva , Peixe-ZebraRESUMO
Efficient neuronal function depends on the continued modulation of the local neuronal proteome. Local protein synthesis plays a central role in tuning the neuronal proteome at specific neuronal regions. Various aspects of translation such as the localization of translational machinery, spatial spread of the newly translated proteins, and their site of action are carried out in specialized neuronal subcompartments to result in a localized functional outcome. In this review, we focus on the various aspects of these local translation compartments such as size, biochemical and organelle composition, structural boundaries, and temporal dynamics. We also discuss the apparent absence of definitive components of translation in these local compartments and the emerging state-of-the-art tools that could help dissecting these conundrums in greater detail in the future.
Assuntos
Plasticidade Neuronal , Neurônios/fisiologia , Biossíntese de Proteínas , Animais , Dendritos/fisiologia , Neurônios/citologia , Organelas/fisiologia , Proteoma , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer inhibitor, widely used in cell biology for tagging newly synthesized proteins. Here, we report synthesis and application of an optimized puromycin carrying a photolabile protecting group as a powerful tool for tagging nascent proteins with high spatiotemporal resolution. The photocaged 7-N,N-(diethylaminocumarin-4-yl)-methoxycarbonyl-puromycin (DEACM-puromycin) was synthesized and compared with the previously developed 6-nitroveratryloxycarbonyl puromycin (NVOC-puromycin). The photochemical behavior as well as the effectiveness in controlling puromycylation in living hippocampal neurons using two-photon excitation is superior to the previously used NVOCpuromycin. We further report on the application of light-controlled puromycylation to visualize new translated proteins in neurons.
Assuntos
Cumarínicos/química , Sondas Moleculares/química , Neurônios/metabolismo , Proteínas/química , Puromicina/análogos & derivados , Animais , Sobrevivência Celular/efeitos da radiação , Cumarínicos/síntese química , Cumarínicos/efeitos da radiação , Hipocampo/citologia , Sondas Moleculares/síntese química , Sondas Moleculares/efeitos da radiação , Puromicina/síntese química , Puromicina/efeitos da radiação , Ratos , Raios UltravioletaRESUMO
Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ.
Assuntos
Fibroblastos/metabolismo , Proteínas/química , Proteínas/metabolismo , Animais , Anticorpos , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Hipocampo/citologia , Camundongos , Neurônios/metabolismo , Ratos , Coloração e RotulagemRESUMO
Like all cells, neurons are made of proteins that have characteristic synthesis and degradation profiles. Unlike other cells, however, neurons have a unique multipolar architecture that makes â¼10,000 synaptic contacts with other neurons. Both the stability and modifiability of the neuronal proteome are crucial for its information-processing, storage and plastic properties. The cell biological mechanisms that synthesize, modify, deliver and degrade dendritic and synaptic proteins are not well understood but appear to reflect unique solutions adapted to the particular morphology of neurons.
Assuntos
Compartimento Celular/fisiologia , Dendritos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Príons/metabolismo , Sinapses/metabolismo , Animais , Humanos , Proteínas do Tecido Nervoso/biossíntese , Neurônios/ultraestruturaRESUMO
Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.