RESUMEN

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.

Asunto(s)

Axones , Conos de Crecimiento , Microtúbulos , Transducción de Señal , Proteína de Unión al GTP rhoA , Microtúbulos/metabolismo , Animales , Proteína de Unión al GTP rhoA/metabolismo , Axones/metabolismo , Conos de Crecimiento/metabolismo , Quinasas Asociadas a rho/metabolismo , Hipocampo/metabolismo , Hipocampo/citología , Ratas , Forminas/metabolismo , Células Cultivadas , Neuronas/metabolismo

RESUMEN

Axon-dendrite formation is a crucial milestone in the life history of neurons. During this process, historically referred as "the establishment of polarity," newborn neurons undergo biochemical, morphological and functional transformations to generate the axonal and dendritic domains, which are the basis of neuronal wiring and connectivity. Since the implementation of primary cultures of rat hippocampal neurons by Gary Banker and Max Cowan in 1977, the community of neurobiologists has made significant achievements in decoding signals that trigger axo-dendritic specification. External and internal cues able to switch on/off signaling pathways controlling gene expression, protein stability, the assembly of the polarity complex (i.e., PAR3-PAR6-aPKC), cytoskeleton remodeling and vesicle trafficking contribute to shape the morphology of neurons. Currently, the culture of hippocampal neurons coexists with alternative model systems to study neuronal polarization in several species, from single-cell to whole-organisms. For instance, in vivo approaches using C. elegans and D. melanogaster, as well as in situ imaging in rodents, have refined our knowledge by incorporating new variables in the polarity equation, such as the influence of the tissue, glia-neuron interactions and three-dimensional development. Nowadays, we have the unique opportunity of studying neurons differentiated from human induced pluripotent stem cells (hiPSCs), and test hypotheses previously originated in small animals and propose new ones perhaps specific for humans. Thus, this article will attempt to review critical mechanisms controlling polarization compiled over decades, highlighting points to be considered in new experimental systems, such as hiPSC neurons and human brain organoids.

RESUMEN

Neurons are highly polarized cells requiring precise regulation of trafficking and targeting of membrane proteins to generate and maintain different and specialized compartments, such as axons and dendrites. Disruption of the Golgi apparatus (GA) secretory pathway in developing neurons alters axon/dendritic formation. Therefore, detailed knowledge of the mechanisms underlying vesicles exiting from the GA is crucial for understanding neuronal polarity. In this study, we analyzed the role of Brefeldin A-Ribosylated Substrate (CtBP1-S/BARS), a member of the C-terminal-binding protein family, in the regulation of neuronal morphological polarization and the exit of membrane proteins from the Trans Golgi Network. Here, we show that BARS is expressed during neuronal development in vitro and that RNAi suppression of BARS inhibits axonal and dendritic elongation in hippocampal neuronal cultures as well as largely perturbed neuronal migration and multipolar-to-bipolar transition during cortical development in situ. In addition, using plasma membrane (PM) proteins fused to GFP and engineered with reversible aggregation domains, we observed that expression of fission dominant-negative BARS delays the exit of dendritic and axonal membrane protein-containing carriers from the GA. Taken together, these data provide the first set of evidence suggesting a role for BARS in neuronal development by regulating post-Golgi membrane trafficking.

Asunto(s)

Aparato de Golgi , Neuronas , Axones/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/fisiología , Red trans-Golgi/metabolismo

RESUMEN

Förster resonance energy transfer (FRET) imaging methods provide unique insight into the spatial distribution of energy transfer and (bio)molecular interaction events, though they deliver average information for an ensemble of events included in a diffraction-limited volume. Coupling super-resolution fluorescence microscopy and FRET has been a challenging and elusive task. Here, we present STED-FRET, a method of general applicability to obtain super-resolved energy transfer images. In addition to higher spatial resolution, STED-FRET provides a more accurate quantification of interaction and has the capacity of suppressing contributions of noninteracting partners, which are otherwise masked by averaging in conventional imaging. The method capabilities were first demonstrated on DNA-origami model systems, verified on uniformly double-labeled microtubules, and then utilized to image biomolecular interactions in the membrane-associated periodic skeleton (MPS) of neurons.

RESUMEN

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

Asunto(s)

Fluorescencia , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Imagen Individual de Molécula/métodos , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , ADN/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Microtúbulos/metabolismo , Fotometría/métodos

RESUMEN

Mechanisms supporting axon growth and the establishment of neuronal polarity have remained largely disconnected from their genetic and epigenetic fundamentals. Recently, post-transcriptional modifications of histones involved in chromatin folding and transcription, and microRNAs controlling translation have emerged as regulators of axonal specification, growth, and guidance. In this article, we review novel evidence supporting the concept that epigenetic mechanisms work at both transcriptional and post-transcriptional levels to shape axons. We also discuss the role of splicing on axonal growth, as one of the most (if not the most) powerful post-transcriptional mechanism to diversify genetic information. Overall, we think exploring the gap between epigenetics and axonal growth raises new questions and perspectives to the development of axons in physiological and pathological contexts.

Asunto(s)

Epigénesis Genética/genética , Histonas/genética , MicroARNs/genética , Animales , Humanos

RESUMEN

The establishment of polarity is crucial for the physiology and wiring of neurons. Therefore, monitoring the axo-dendritic specification allows the mechanisms and signals associated with development, growth, and disease to be explored. Here, we describe major and minor steps to study polarity acquisition, using primary cultures of hippocampal neurons isolated from embryonic rat hippocampi, for in vitro monitoring. Furthermore, we use in utero electroporated, GFP-expressing embryonic mouse brains for visualizing cortical neuron migration and polarization in situ. Some underreported after-protocol steps are also included. For complete details on the use and execution of this protocol, please refer to Wilson et al. (2020).

Asunto(s)

Polaridad Celular/fisiología , Neuronas/metabolismo , Cultivo Primario de Células/métodos , Animales , Axones/fisiología , Células Cultivadas , Dendritas/fisiología , Electroporación , Hipocampo/metabolismo , Ratones , Neurogénesis , Neuronas/fisiología , Ratas

RESUMEN

Rotenone, a broad-spectrum insecticide, piscicide and pesticide, produces a complete and selective suppression of axonogenesis in cultured hippocampal neurons. This effect is associated with an inhibition of actin dynamics through activation of Ras homology member A (RhoA) activity. However, the upstream signaling mechanisms involved in rotenone-induced RhoA activation were unknown. We hypothesized that rotenone might inhibit axon growth by the activation of RhoA/ROCK pathway because of the changes in microtubule (MT) dynamics and the concomitant release of Lfc, a MT-associated Guanine Nucleotide Exchange Factor (GEF) for RhoA. In this study, we demonstrate that rotenone decreases MT stability in morphologically unpolarized neurons. Taxol (3 nM), a drug that stabilizes MT, attenuates the inhibitory effect of rotenone (0.1 µM) on axon formation. Radiometric Forster Resonance Energy Transfer, revealed that this effect is associated with inhibition of rotenone-induced RhoA and ROCK activation. Interestingly, silencing of Lfc, but not of the RhoA GEF ArhGEF1, prevents the inhibitory effect of rotenone on axon formation. Our results suggest that rotenone-induced MT de-stabilization releases Lfc from MT thereby promoting RhoA and ROCK activities and the consequent inhibition of axon growth. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Asunto(s)

Factores de Intercambio de Guanina Nucleótido/metabolismo , Insecticidas/uso terapéutico , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Rotenona/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Células Cultivadas , Embrión de Mamíferos , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Hipocampo/citología , Fosforilación/efectos de los fármacos , Embarazo , Ratas , Transducción Genética , Tubulina (Proteína)/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteínas tau/metabolismo

RESUMEN

Estrogen receptor α (ERα) is expressed in tissues as diverse as brains and mammary glands. In breast cancer, ERα is a key regulator of tumor progression. Therefore, understanding what activates ERα is critical for cancer treatment in particular and cell biology in general. Using biochemical approaches and superresolution microscopy, we show that estrogen drives membrane ERα into endosomes in breast cancer cells and that its fate is determined by the presence of fibronectin (FN) in the extracellular matrix; it is trafficked to lysosomes in the absence of FN and avoids the lysosomal compartment in its presence. In this context, FN prolongs ERα half-life and strengthens its transcriptional activity. We show that ERα is associated with ß1-integrin at the membrane, and this integrin follows the same endocytosis and subcellular trafficking pathway triggered by estrogen. Moreover, ERα+ vesicles are present within human breast tissues, and colocalization with ß1-integrin is detected primarily in tumors. Our work unravels a key, clinically relevant mechanism of microenvironmental regulation of ERα signaling.

Asunto(s)

Receptor alfa de Estrógeno/metabolismo , Fibronectinas/fisiología , Lisosomas/metabolismo , Línea Celular Tumoral , Endosomas/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Integrina beta1/metabolismo , Células MCF-7 , Modelos Biológicos , Transporte de Proteínas , Proteolisis , Microambiente Tumoral

RESUMEN

Neurons are the most asymmetric cell types, with their axons commonly extending over lengths that are thousand times longer than the diameter of the cell soma. Fluorescence nanoscopy has recently unveiled that actin, spectrin and accompanying proteins form a membrane-associated periodic skeleton (MPS) that is ubiquitously present in mature axons from all neuronal types evaluated so far. The MPS is a regular supramolecular protein structure consisting of actin "rings" separated by spectrin tetramer "spacers". Although the MPS is best organized in axons, it is also present in dendrites, dendritic spine necks and thin cellular extensions of non-neuronal cells such as oligodendrocytes and microglia. The unique organization of the actin/spectrin skeleton has raised the hypothesis that it might serve to support the extreme physical and structural conditions that axons must resist during the lifespan of an organism. Another plausible function of the MPS consists of membrane compartmentalization and subsequent organization of protein domains. This review focuses on what we know so far about the structure of the MPS in different neuronal subdomains, its dynamics and the emerging evidence of its impact in axonal biology.

RESUMEN

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

RESUMEN

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.

Asunto(s)

Actinas/metabolismo , Axones/patología , Membrana Celular/metabolismo , Conos de Crecimiento/patología , Plasticidad Neuronal , Degeneración Retrógrada , Espectrina/metabolismo , Citoesqueleto de Actina , Animales , Axones/metabolismo , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Microtúbulos/metabolismo , Ratas , Ratas Wistar

RESUMEN

Here, will review current evidence regarding the signaling pathways and mechanisms underlying membrane addition at sites of active growth during axon formation. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 170-180, 2018.

Asunto(s)

Axones/metabolismo , Membrana Celular/metabolismo , Animales , Aumento de la Célula , Transducción de Señal

RESUMEN

Fluorescence nanoscopy imaging permits the observation of periodic supramolecular protein structures in their natural environment, as well as the unveiling of previously unknown protein periodic structures. Deciphering the biological functions of such protein nanostructures requires systematic and quantitative analysis of large number of images under different experimental conditions and specific stimuli. Here we present a method and an open source software for the automated quantification of protein periodic structures in super-resolved images. Its performance is demonstrated by analyzing the abundance and regularity of the spectrin membrane-associated periodic skeleton (MPS) in hippocampal neurons of 2 to 40 days in vitro, imaged by STED and STORM nanoscopy. The automated analysis reveals that both the abundance and the regularity of the MPS increase over time and reach maximum plateau values after 14 DIV. A detailed analysis of the distributions of correlation coefficients provides indication of dynamical assembly and disassembly of the MPS.

Asunto(s)

Membrana Celular/metabolismo , Hipocampo/metabolismo , Microscopía Fluorescente/métodos , Espectrina/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Ratones , Neuronas/metabolismo

RESUMEN

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein better known for its participation in the physiopathology of Alzheimer disease as the source of the beta amyloid fragment. However, the physiological functions of the full length protein and its proteolytic fragments have remained elusive. APP was first described as a cell-surface receptor; nevertheless, increasing evidence highlighted APP as a cell adhesion molecule. In this review, we will focus on the current knowledge of the physiological role of APP as a cell adhesion molecule and its involvement in key events of neuronal development, such as migration, neurite outgrowth, growth cone pathfinding, and synaptogenesis. Finally, since APP is over-expressed in Down syndrome individuals because of the extra copy of chromosome 21, in the last section of the review, we discuss the potential contribution of APP to the neuronal and synaptic defects described in this genetic condition. Read the Editorial Highlight for this article on page 9. Cover Image for this issue: doi. 10.1111/jnc.13817.

Asunto(s)

Precursor de Proteína beta-Amiloide/fisiología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Moléculas de Adhesión Celular/fisiología , Neurogénesis/fisiología , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/química , Animales , Moléculas de Adhesión Celular/química , Movimiento Celular/fisiología , Síndrome de Down/metabolismo , Humanos , Neuronas/fisiología

RESUMEN

Here we describe the use of confocal microscopy in combination with antibodies specific to Golgi proteins to visualize dendritic Golgi outposts (GOPs) in cultured hippocampal pyramidal neurons. We also describe the use of spinning disk confocal microscopy, in combination with ectopically expressed glycosyltransferases fused to GFP variants, to visualize GOPs in living neurons.

Asunto(s)

Anticuerpos/química , Aparato de Golgi/metabolismo , Células Piramidales/citología , Células Piramidales/metabolismo , Animales , Humanos , Microscopía Confocal/métodos

RESUMEN

Several reports have linked the presence of high titers of anti-Gg Abs with delayed recovery/poor prognosis in GBS. In most cases, failure to recover is associated with halted/deficient axon regeneration. Previous work identified that monoclonal and patient-derived anti-Gg Abs can act as inhibitory factors in an animal model of axon regeneration. Further studies using primary dorsal root ganglion neuron (DRGn) cultures demonstrated that anti-Gg Abs can inhibit neurite outgrowth by targeting gangliosides via activation of the small GTPase RhoA and its associated kinase (ROCK), a signaling pathway common to other established inhibitors of axon regeneration. We aimed to study the molecular basis of the inhibitory effect of anti-Gg abs on neurite outgrowth by dissecting the molecular dynamics of growth cones (GC) cytoskeleton in relation to the spatial-temporal analysis of RhoA activity. We now report that axon growth inhibition in DRGn induced by a well characterized mAb targeting gangliosides GD1a/GT1b involves: i) an early RhoA/ROCK-independent collapse of lamellipodia; ii) a RhoA/ROCK-dependent shrinking of filopodia; and iii) alteration of GC microtubule organization/and presumably dynamics via RhoA/ROCK-dependent phosphorylation of CRMP-2 at threonine 555. Our results also show that mAb 1B7 inhibits peripheral axon regeneration in an animal model via phosphorylation/inactivation of CRMP-2 at threonine 555. Overall, our data may help to explain the molecular mechanisms underlying impaired nerve repair in GBS. Future work should define RhoA-independent pathway/s and effectors regulating actin cytoskeleton, thus providing an opportunity for the design of a successful therapy to guarantee an efficient target reinnervation.

Asunto(s)

Anticuerpos/farmacología , Microtúbulos/patología , Regeneración Nerviosa/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Polisacáridos/inmunología , Proteína de Unión al GTP rhoA/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular , Microtúbulos/efectos de los fármacos , Regeneración Nerviosa/fisiología , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Wistar , Neuropatía Ciática/metabolismo , Neuropatía Ciática/patología , Transducción de Señal

RESUMEN

Objetivo: comparar la edad gestacional obtenida por la mediación del diámetro transverso del cerebelo y la longitud de fémur para la estimación de la edad gestacional en el segundo trimestre. Métodos: el estudio es de tipo observacional descriptivo, retrospectivo y de corte transversal. El área de estudio fue el sanatorio Galenos, servicio de imágenes, de ciudad del Este. La población estuvo conformada por 74 embarazadas de 15 a 41 años, del segundo trimestre, específicamente entre las 19.3 y 34.1 semanas de gestación que asistieron al servicio para realizarse el estudio durante el periodo comprendido del 11 de febrero al 26 de septiembre del 2016. Resultados: se estudiaron en total 74 gestantes, las cuales cumplieron los criterios de inclusión y exclusión. Se realizó el contraste de hipótesis tomando la mediana de las diferencias significativas entre la edad gestacional estimada por LF. Conclusión: la edad gestacional obtenida por la biometría del diámetro transverso del cerebro similar a la obtenida por la biometría del diámetro transverso del cerebelo es similar a la obtenida por la longitud de fémur y por lo tanto constituye un parámetro valido para la determinación de la edad gestacional

Asunto(s)

Embarazo , Diagnóstico Prenatal , Cerebelo , Edad Gestacional , Mujeres Embarazadas , Desarrollo Fetal , Fémur , Atención Prenatal , Ultrasonografía

RESUMEN

Paenibacillus sp. A59 was isolated from decaying forest soil in Argentina and characterized as a xylanolytic strain. We report the draft genome sequence of this isolate, with an estimated genome size of 7 Mb which harbor 6,424 coding sequences. Genes coding for hydrolytic enzymes involved in lignocellulose deconstruction were predicted.