RESUMO
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
Super-resolution techniques have achieved localization precisions in the nanometer regime. Here we report all-optical, room temperature localization of fluorophores with precision in the Ångström range. We built on the concept of MINSTED nanoscopy where precision is increased by encircling the fluorophore with the low-intensity central region of a stimulated emission depletion (STED) donut beam while constantly increasing the absolute donut power. By blue-shifting the STED beam and separating fluorophores by on/off switching, individual fluorophores bound to a DNA strand are localized with σ = 4.7 Å, corresponding to a fraction of the fluorophore size, with only 2,000 detected photons. MINSTED fluorescence nanoscopy with single-digit nanometer resolution is exemplified by imaging nuclear pore complexes and the distribution of nuclear lamin in mammalian cells labeled by transient DNA hybridization. Because our experiments yield a localization precision σ = 2.3 Å, estimated for 10,000 detected photons, we anticipate that MINSTED will open up new areas of application in the study of macromolecular complexes in cells.
Assuntos
DNA , Corantes Fluorescentes , Animais , Microscopia de Fluorescência/métodos , MamíferosRESUMO
In neurons, autophagosome biogenesis occurs mainly in distal axons, followed by maturation during retrograde transport. Autophagosomal growth depends on the supply of membrane lipids which requires small vesicles containing ATG9, a lipid scramblase essential for macroautophagy/autophagy. Here, we show that ATG9-containing vesicles are enriched in synapses and resemble synaptic vesicles in size and density. The proteome of ATG9-containing vesicles immuno-isolated from nerve terminals showed conspicuously low levels of trafficking proteins except of the AP2-complex and some enzymes involved in endosomal phosphatidylinositol metabolism. Super resolution microscopy of nerve terminals and isolated vesicles revealed that ATG9-containing vesicles represent a distinct vesicle population with limited overlap not only with synaptic vesicles but also other membranes of the secretory pathway, uncovering a surprising heterogeneity in their membrane composition. Our results are compatible with the view that ATG9-containing vesicles function as lipid shuttles that scavenge membrane lipids from various intracellular membranes to support autophagosome biogenesis.Abbreviations: AP: adaptor related protein complex: ATG2: autophagy related 2; ATG9: autophagy related 9; DNA PAINT: DNA-based point accumulation for imaging in nanoscale topography; DyMIN STED: dynamic minimum stimulated emission depletion; EL: endosome and lysosome; ER: endoplasmic reticulum; GA: Golgi apparatus; iBAQ: intensity based absolute quantification; LAMP: lysosomal-associated membrane protein; M6PR: mannose-6-phosphate receptor, cation dependent; Minflux: minimal photon fluxes; Mito: mitochondria; MS: mass spectrometry; PAS: phagophore assembly site; PM: plasma membrane; Px: peroxisome; RAB26: RAB26, member RAS oncogene family; RAB3A: RAB3A, member RAS oncogene family; RAB5A: RAB5A, member RAS oncogene family; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment receptor; SVs: synaptic vesicles; SYP: synaptophysin; TGN: trans-Golgi network; TRAPP: transport protein particle; VTI1: vesicle transport through interaction with t-SNAREs.
RESUMO
Vesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain mammalian neurons release multiple transmitters, it is not clear whether the release occurs from the same or distinct vesicle pools at the synapse. Using quantitative single-vesicle imaging, we show that a vast majority of SVs in the rodent brain contain only one type of VT, indicating specificity for a single neurotransmitter. Interestingly, SVs containing dual transporters are highly diverse (27 types) but small in proportion (2% of all SVs), excluding the largest pool that carries VGLUT1 and ZnT3 (34%). Using VGLUT1-ZnT3 SVs, we demonstrate that the transporter colocalization influences the SV content and synaptic quantal size. Thus, the presence of diverse transporters on the same vesicle is bona fide, and depending on the VT types, this may act to regulate neurotransmitter type, content, and release in space and time.
Assuntos
Proteínas de Transporte de Neurotransmissores , Vesículas Sinápticas , Animais , Mamíferos , Proteínas de Membrana Transportadoras , Neurotransmissores , Sinapses , Vesículas Sinápticas/fisiologia , Proteína Vesicular 1 de Transporte de GlutamatoRESUMO
Fast-spiking perisomatic-inhibitory interneurons (PIIs) receive convergent excitation and mediate both feedforward and feedback inhibition in cortical microcircuits. However, it remains poorly understood how convergent excitatory inputs recruit PIIs to produce precisely timed inhibition. Here, we analyzed the interaction of inputs from the entorhinal cortex [perforant path (PP)] and from local granule cells [mossy fibers (MFs)] onto PIIs in the rat dentate gyrus (DG). PP stimulation alone activates PIIs with low temporal precision. Interestingly, when PP and MFs are coactivated with a 10 ms delay, PIIs discharge with precise timing. Moreover, repeated coactivation of the two inputs induces associative long-term potentiation (LTP) at MF synapses. Under these conditions, a single potentiated MF input is sufficient to recruit PIIs in a reliable and highly precise manner to provide feedback inhibition. MF-LTP depends on the discharge of PIIs, indicating Hebbian plasticity. However, MF-LTP is preserved when NMDA receptors are blocked but depends on transmission through Ca(2+)-permeable AMPA receptors (AMPARs). PP-PII synapses, in contrast, lack Ca(2+)-permeable AMPARs and do not show plasticity on associative activation. Thus, precise recruitment of PIIs requires excitation through MF-PII synapses during feedforward activation. We propose that associative plasticity at these synapses is a central mechanism that adjusts inhibition levels to maintain sparse activity and to improve signal-to-noise ratio in the DG network.
Assuntos
Potenciais de Ação/fisiologia , Giro Denteado/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Interneurônios/fisiologia , Plasticidade Neuronal/fisiologia , Tempo de Reação/fisiologia , Sinapses/fisiologia , Animais , Hipocampo/fisiologia , Ratos , Ratos WistarRESUMO
Axon branching is crucial for proper formation of neuronal networks. Although originally identified as an angiogenic factor, VEGF also signals directly to neurons to regulate their development and function. Here we show that VEGF and its receptor VEGFR2 (also known as KDR or FLK1) are expressed in mouse hippocampal neurons during development, with VEGFR2 locally expressed in the CA3 region. Activation of VEGF/VEGFR2 signaling in isolated hippocampal neurons results in increased axon branching. Remarkably, inactivation of VEGFR2 also results in increased axon branching in vitro and in vivo. The increased CA3 axon branching is not productive as these axons are less mature and form less functional synapses with CA1 neurons. Mechanistically, while VEGF promotes the growth of formed branches without affecting filopodia formation, loss of VEGFR2 increases the number of filopodia and enhances the growth rate of new branches. Thus, a controlled VEGF/VEGFR2 signaling is required for proper CA3 hippocampal axon branching during mouse hippocampus development.
Assuntos
Axônios/fisiologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Efrina-B2/genética , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais/genética , Sinapses/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
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
Homeostatic scaling adjusts the strength of synaptic connections up or down in response to large changes in input. To identify the landscape of proteomic changes that contribute to opposing forms of homeostatic plasticity, we examined the plasticity-induced changes in the newly synthesized proteome. Cultured rat hippocampal neurons underwent homeostatic up-scaling or down-scaling. We used BONCAT (bio-orthogonal non-canonical amino acid tagging) to metabolically label, capture, and identify newly synthesized proteins, detecting and analyzing 5,940 newly synthesized proteins using mass spectrometry and label-free quantitation. Neither up- nor down-scaling produced changes in the number of different proteins translated. Rather, up- and down-scaling elicited opposing translational regulation of several molecular pathways, producing targeted adjustments in the proteome. We discovered â¼300 differentially regulated proteins involved in neurite outgrowth, axon guidance, filopodia assembly, excitatory synapses, and glutamate receptor complexes. We also identified differentially regulated proteins that are associated with multiple diseases, including schizophrenia, epilepsy, and Parkinson's disease.
Assuntos
Hipocampo/metabolismo , Homeostase , Neurônios/metabolismo , Proteoma/metabolismo , Sinapses/metabolismo , Animais , Animais Recém-Nascidos , Anisomicina/farmacologia , Orientação de Axônios/efeitos dos fármacos , Bicuculina/farmacologia , Células Cultivadas , Cromatografia Líquida , Antagonistas de Receptores de GABA-A/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Plasticidade Neuronal , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Inibidores da Síntese de Proteínas/farmacologia , Proteoma/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Sinapses/efeitos dos fármacos , Espectrometria de Massas em TandemRESUMO
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.