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
The brain's connectome provides the scaffold for canonical neural computations. However, a comparison of connectivity studies in the mouse primary visual cortex (V1) reveals that the average number and strength of connections between specific neuron types can vary. Can variability in V1 connectivity measurements coexist with canonical neural computations? We developed a theory-driven approach to deduce V1 network connectivity from visual responses in mouse V1 and visual thalamus (dLGN). Our method revealed that the same recorded visual responses were captured by multiple connectivity configurations. Remarkably, the magnitude and selectivity of connectivity weights followed a specific order across most of the inferred connectivity configurations. We argue that this order stems from the specific shapes of the recorded contrast response functions and contrast invariance of orientation tuning. Remarkably, despite variability across connectivity studies, connectivity weights computed from individual published connectivity reports followed the order we identified with our method, suggesting that the relations between the weights, rather than their magnitudes, represent a connectivity motif supporting canonical V1 computations.
Assuntos
Córtex Visual , Animais , Camundongos , Neurônios/fisiologia , Estimulação Luminosa/métodos , Tálamo/fisiologia , Córtex Visual/fisiologia , Vias Visuais/fisiologiaRESUMO
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
The neuronal transcriptome changes dynamically to adapt to stimuli from the extracellular and intracellular environment. In this study, we adapted for the first time a click chemistry technique to label the newly synthesized RNA in cultured hippocampal neurons and intact larval zebrafish brain. Ethynyl uridine (EU) was incorporated into neuronal RNA in a time- and concentration-dependent manner. Newly synthesized RNA granules observed throughout the dendrites were colocalized with mRNA and rRNA markers. In zebrafish larvae, the application of EU to the swim water resulted in uptake and labeling throughout the brain. Using a GABA receptor antagonist, PTZ (pentylenetetrazol), to elevate neuronal activity, we demonstrate that newly transcribed RNA signal increased in specific regions involved in neurogenesis.
Assuntos
Química Click , Imagem Molecular/métodos , Neurônios/metabolismo , RNA/genética , RNA/metabolismo , Animais , Encéfalo/metabolismo , Genes de RNAr , Proteínas de Ligação a Poli(A)/metabolismo , Células Piramidais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-ZebraRESUMO
The larval zebrafish is a model organism to study the neural circuitry underlying behavior. There exist, however, few examples of robust long-term memory. Here we describe a simple, unrestrained associative place-conditioning paradigm. We show that visual access to a group of conspecifics has rewarding properties for 6- to 8-day-old larval zebrafish. We use this social reward as an unconditioned stimulus and pair it with a distinct visual environment. After training, larvae exhibited spatial preference for the location previously paired with the social reward for up to 36 h, indicating that zebrafish larvae can exhibit long-term associative memory. Furthermore, incubation with a protein synthesis inhibitor or an NMDAR-antagonist impaired memory. In future experiments, this learning paradigm could be used to study the social interactions of larval zebrafish or paired with cell-specific metabolic labeling to visualize circuits underlying memory formation.
Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Memória de Longo Prazo/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Animais , Condicionamento Clássico/efeitos dos fármacos , Larva , Biossíntese de Proteínas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Recompensa , Peixe-ZebraRESUMO
The existence of evolutionarily conserved regions in the vertebrate brain is well established. The rules and constraints underlying the evolution of neuron types, however, remain poorly understood. To compare neuron types across brain regions and species, we generated a cell type atlas of the brain of a bearded dragon and compared it with mouse datasets. Conserved classes of neurons could be identified from the expression of hundreds of genes, including homeodomain-type transcription factors and genes involved in connectivity. Within these classes, however, there are both conserved and divergent neuron types, precluding a simple categorization of the brain into ancestral and novel areas. In the thalamus, neuronal diversification correlates with the evolution of the cortex, suggesting that developmental origin and circuit allocation are drivers of neuronal identity and evolution.
Assuntos
Evolução Biológica , Córtex Cerebral , Expressão Gênica , Lagartos , Neurônios , Animais , Córtex Cerebral/citologia , Evolução Molecular , Perfilação da Expressão Gênica , Camundongos , Neurônios/citologia , Neurônios/metabolismoRESUMO
Although mRNAs are localized in the processes of excitatory neurons, it is still unclear whether interneurons also localize a large population of mRNAs. In addition, the variability in the localized mRNA population within and between cell types is unknown. Here we describe the unbiased transcriptomic characterization of the subcellular compartments of hundreds of single neurons. We separately profiled the dendritic and somatic transcriptomes of individual rat hippocampal neurons and investigated mRNA abundances in the soma and dendrites of single glutamatergic and GABAergic neurons. We found that, like their excitatory counterparts, interneurons contain a rich repertoire of ~4000 mRNAs. We observed more cell type-specific features among somatic transcriptomes than their associated dendritic transcriptomes. Finally, using celltype-specific metabolic labeling of isolated neurites, we demonstrated that the processes of glutamatergic and, notably, GABAergic neurons were capable of local translation, suggesting mRNA localization and local translation are general properties of neurons.
Assuntos
Dendritos/fisiologia , Neurônios GABAérgicos/fisiologia , Interneurônios/fisiologia , Análise de Célula Única , Transcriptoma/fisiologiaRESUMO
To accommodate their complex morphology, neurons localize messenger RNAs (mRNAs) and ribosomes near synapses to produce proteins locally. However, a relative paucity of polysomes (considered the active sites of translation) detected in electron micrographs of neuronal processes has suggested a limited capacity for local protein synthesis. In this study, we used polysome profiling together with ribosome footprinting of microdissected rodent synaptic regions to reveal a surprisingly high number of dendritic and/or axonal transcripts preferentially associated with monosomes (single ribosomes). Furthermore, the neuronal monosomes were in the process of active protein synthesis. Most mRNAs showed a similar translational status in the cell bodies and neurites, but some transcripts exhibited differential ribosome occupancy in the compartments. Monosome-preferring transcripts often encoded high-abundance synaptic proteins. Thus, monosome translation contributes to the local neuronal proteome.
Assuntos
Neurópilo/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Sinapses/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Polirribossomos/metabolismo , Proteoma/metabolismo , RNA Mensageiro/genéticaRESUMO
Gene annotation is a critical resource in genomics research. Many computational approaches have been developed to assemble transcriptomes based on high-throughput short-read sequencing, however, only with limited accuracy. Here, we combine next-generation and third-generation sequencing to reconstruct a full-length transcriptome in the rat hippocampus, which is further validated using independent 5´ and 3´-end profiling approaches. In total, we detect 28,268 full-length transcripts (FLTs), covering 6,380 RefSeq genes and 849 unannotated loci. Based on these FLTs, we discover co-occurring alternative RNA processing events. Integrating with polysome profiling and ribosome footprinting data, we predict isoform-specific translational status and reconstruct an open reading frame (ORF)-eome. Notably, a high proportion of the predicted ORFs are validated by mass spectrometry-based proteomics. Moreover, we identify isoforms with subcellular localization pattern in neurons. Collectively, our data advance our knowledge of RNA and protein isoform diversity in the rat brain and provide a rich resource for functional studies.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hipocampo/metabolismo , Proteínas/genética , RNA/genética , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Anotação de Sequência Molecular , Fases de Leitura Aberta/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/metabolismo , RNA/metabolismo , Isoformas de RNA/genética , Isoformas de RNA/metabolismo , Ratos Sprague-DawleyRESUMO
Neurons localize mRNAs near synapses where their translation can be regulated by synaptic demand and activity. Differences in the 3' UTRs of mRNAs can change their localization, stability, and translational regulation. Using 3' end RNA sequencing of microdissected rat brain slices, we discovered a huge diversity in mRNA 3' UTRs, with many transcripts showing enrichment for a particular 3' UTR isoform in either somata or the neuropil. The 3' UTR isoforms of localized transcripts are significantly longer than the 3' UTRs of non-localized transcripts and often code for proteins associated with axons, dendrites, and synapses. Surprisingly, long 3' UTRs add not only new, but also duplicate regulatory elements. The neuropil-enriched 3' UTR isoforms have significantly longer half-lives than somata-enriched isoforms. Finally, the 3' UTR isoforms can be significantly altered by enhanced activity. Most of the 3' UTR plasticity is transcription dependent, but intriguing examples of changes that are consistent with altered stability, trafficking between compartments, or local "remodeling" remain.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Células HEK293 , Hipocampo/química , Hipocampo/metabolismo , Humanos , Masculino , Neurônios/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
Computations in the mammalian cortex are carried out by glutamatergic and γ-aminobutyric acid-releasing (GABAergic) neurons forming specialized circuits and areas. Here we asked how these neurons and areas evolved in amniotes. We built a gene expression atlas of the pallium of two reptilian species using large-scale single-cell messenger RNA sequencing. The transcriptomic signature of glutamatergic neurons in reptilian cortex suggests that mammalian neocortical layers are made of new cell types generated by diversification of ancestral gene-regulatory programs. By contrast, the diversity of reptilian cortical GABAergic neurons indicates that the interneuron classes known in mammals already existed in the common ancestor of all amniotes.
Assuntos
Evolução Biológica , Rastreamento de Células/métodos , Perfilação da Expressão Gênica/métodos , Hipocampo/citologia , Neocórtex/citologia , Répteis , Análise de Célula Única/métodos , Animais , Neurônios GABAérgicos/classificação , Neurônios GABAérgicos/citologia , Neuroglia/classificação , Neuroglia/citologia , Neurônios/classificaçãoRESUMO
MicroRNAs (miRNAs) regulate gene expression by binding to target messenger RNAs (mRNAs) and preventing their translation. In general, the number of potential mRNA targets in a cell is much greater than the miRNA copy number, complicating high-fidelity miRNA-target interactions. We developed an inducible fluorescent probe to explore whether the maturation of a miRNA could be regulated in space and time in neurons. A precursor miRNA (pre-miRNA) probe exhibited an activity-dependent increase in fluorescence, suggesting the stimulation of miRNA maturation. Single-synapse stimulation resulted in a local maturation of miRNA that was associated with a spatially restricted reduction in the protein synthesis of a target mRNA. Thus, the spatially and temporally regulated maturation of pre-miRNAs can be used to increase the precision and robustness of miRNA-mediated translational repression.
Assuntos
Dendritos/metabolismo , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas/genética , Animais , Células Cultivadas , Corantes Fluorescentes/química , Hipocampo/citologia , Masculino , Clivagem do RNA , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ribonuclease III/genética , Ribonuclease III/metabolismo , Sinapses/metabolismoRESUMO
An emerging view, based on gene expression patterns, is that discrete cell types occupy different regions in the hippocampus. In this issue of Neuron, Cembrowski et al. (2016) challenge this concept by identifying gradients of gene expression that suggest a molecular continuum of excitatory neurons and provide insights for organizational motifs at hippocampal poles.
Assuntos
Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Perfilação da Expressão Gênica/métodos , Células Piramidais/fisiologia , Animais , Feminino , MasculinoRESUMO
N-glycosylation - the sequential addition of complex sugars to adhesion proteins, neurotransmitter receptors, ion channels and secreted trophic factors as they progress through the endoplasmic reticulum and the Golgi apparatus - is one of the most frequent protein modifications. In mammals, most organ-specific N-glycosylation events occur in the brain. Yet, little is known about the nature, function and regulation of N-glycosylation in neurons. Using imaging, quantitative immunoblotting and mass spectrometry, we show that hundreds of neuronal surface membrane proteins are core-glycosylated, resulting in the neuronal membrane displaying surprisingly high levels of glycosylation profiles that are classically associated with immature intracellular proteins. We report that while N-glycosylation is generally required for dendritic development and glutamate receptor surface expression, core-glycosylated proteins are sufficient to sustain these processes, and are thus functional. This atypical glycosylation of surface neuronal proteins can be attributed to a bypass or a hypo-function of the Golgi apparatus. Core-glycosylation is regulated by synaptic activity, modulates synaptic signaling and accelerates the turnover of GluA2-containing glutamate receptors, revealing a novel mechanism that controls the composition and sensing properties of the neuronal membrane.
Assuntos
Glicosilação , Canais Iônicos/metabolismo , Neurônios/química , Animais , Química Encefálica , Linhagem Celular , Immunoblotting , Mamíferos , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Imagem ÓpticaRESUMO
Circular RNAs (circRNAs) have re-emerged as an interesting RNA species. Using deep RNA profiling in different mouse tissues, we observed that circRNAs were substantially enriched in brain and a disproportionate fraction of them were derived from host genes that encode synaptic proteins. Moreover, on the basis of separate profiling of the RNAs localized in neuronal cell bodies and neuropil, circRNAs were, on average, more enriched in the neuropil than their host gene mRNA isoforms. Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons. Consistent with the idea that circRNAs might regulate synaptic function during development, many circRNAs changed their abundance abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation. Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.
Assuntos
Encéfalo/metabolismo , Dendritos/metabolismo , Plasticidade Neuronal/fisiologia , Neurópilo/metabolismo , RNA/metabolismo , Sinapses/genética , Animais , Encéfalo/crescimento & desenvolvimento , Feminino , Hipocampo/metabolismo , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , RNA Circular , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNARESUMO
The stability and dynamics of synapses rely on tight regulation of the synaptic proteome. Shank proteins, encoded by the three genes Shank1, Shank2 and Shank3 are scaffold molecules in the postsynaptic density of excitatory neurons that contribute to activity-dependent neuronal signalling. Mutations in the Shank genes are associated with neurological diseases. Using state-of-the-art technologies, we investigated the levels of expression of the Shank family messenger RNAs (mRNAs) within the synaptic neuropil of the rat hippocampus. We detected all three Shank transcripts in the neuropil of CA1 pyramidal neurons. We found Shank1 to be the most abundantly expressed among the three Shank mRNA homologues. We also examined the turnover of Shank mRNAs and predict the half-lives of Shank1, Shank2 and Shank3 mRNAs to be 18-28 h. Using 3'-end sequencing, we identified novel 3' ends for the Shank1 and Shank2 3' untranslated regions (3' UTRs) that may contribute to the diversity of alternative polyadenylation (APA) for the Shank transcripts. Our findings consolidate the view that the Shank molecules play a central role at the postsynaptic density. This study may shed light on synaptopathologies associated with disruption of local protein synthesis, perhaps linked to mutations in mRNA 3' UTRs or inappropriate 3' end processing.
Assuntos
Região CA1 Hipocampal/metabolismo , Regulação da Expressão Gênica/fisiologia , Modelos Neurológicos , Proteínas do Tecido Nervoso/metabolismo , Neurópilo/metabolismo , RNA Mensageiro/metabolismo , Sinapses/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , Meia-Vida , Immunoblotting , Hibridização In Situ , Microdissecção , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Poliadenilação , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
Brain-derived neurotrophic factor (BDNF) is a small protein of the neurotrophin family that regulates various brain functions. Although much is known about how its transcription is regulated, the abundance of endogenous BDNF mRNA and its subcellular localization pattern are matters of debate. We used next-generation sequencing and high-resolution in situ hybridization in the rat hippocampus to reexamine this question. We performed 3' end sequencing on rat hippocampal slices and detected two isoforms of Bdnf containing either a short or a long 3' untranslated region (3'UTR). Most of the Bdnf transcripts contained the short 3'UTR isoform and were present in low amounts relative to other neuronal transcripts. Bdnf mRNA was present in the somatic compartment of rat hippocampal slices or the somata of cultured rat hippocampal neurons but was rarely detected in the dendritic processes. Pharmacological stimulation of hippocampal neurons induced Bdnf expression but did not change the ratio of Bdnf isoform abundance. The findings indicate that endogenous Bdnf mRNA, although weakly abundant, is primarily localized to the somatic compartment of hippocampal neurons. Both Bdnf mRNA isoforms have shorter half-lives compared with other neuronal mRNAs. Furthermore, the findings show that using complementary high-resolution techniques can provide sensitive measures of endogenous transcript abundance.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Sequenciamento de Nucleotídeos em Larga Escala , Hipocampo/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genética , Regiões 3' não Traduzidas , Animais , Hipocampo/citologia , Hibridização In Situ , RatosRESUMO
In neurons, dendritic protein synthesis is required for many forms of long-term synaptic plasticity. The population of mRNAs that are localized to dendrites, however, remains sparsely identified. Here, we use deep sequencing to identify the mRNAs resident in the synaptic neuropil in the hippocampus. Analysis of a neuropil data set yielded a list of 8,379 transcripts of which 2,550 are localized in dendrites and/or axons. Using a fluorescent barcode strategy to label individual mRNAs, we show that their relative abundance in the neuropil varies over 3 orders of magnitude. High-resolution in situ hybridization validated the presence of mRNAs in both cultured neurons and hippocampal slices. Among the many mRNAs identified, we observed a large fraction of known synaptic proteins including signaling molecules, scaffolds and receptors. These results reveal a previously unappreciated enormous potential for the local protein synthesis machinery to supply, maintain and modify the dendritic and synaptic proteome.