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Transporting and translating mRNAs in axons is crucial for neuronal viability. Local synthesis of nuclear-encoded mitochondrial proteins protects long-lived axonal mitochondria from damage; however, the regulatory factors involved are largely unknown. We show that CLUH, which binds mRNAs encoding mitochondrial proteins, prevents peripheral neuropathy and motor deficits in the mouse. CLUH is enriched in the growth cone of developing spinal motoneurons and is required for their growth. The lack of CLUH affects the abundance of target mRNAs and the corresponding mitochondrial proteins more prominently in axons, leading to ATP deficits in the growth cone. CLUH interacts with ribosomal subunits, translation initiation, and ribosome recycling components and preserves axonal translation. Overexpression of the ribosome recycling factor ABCE1 rescues the mRNA and translation defects, as well as the growth cone size, in CLUH-deficient motoneurons. Thus, we demonstrate a role for CLUH in mitochondrial quality control and translational regulation in axons, which is essential for their development and long-term integrity and function.
Assuntos
Axônios , Mitocôndrias , Neurônios Motores , Doenças do Sistema Nervoso Periférico , Biossíntese de Proteínas , Animais , Neurônios Motores/metabolismo , Mitocôndrias/metabolismo , Axônios/metabolismo , Camundongos , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/patologia , Cones de Crescimento/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Camundongos KnockoutRESUMO
Plasma membrane-associated platforms (PMAPs) form at specific sites of plasma membrane by scaffolds including ERC1 and Liprin-α1. We identify a mechanism regulating PMAPs assembly, with consequences on motility/invasion. Silencing Ser/Thr kinase DYRK3 in invasive breast cancer cells inhibits their motility and invasive capacity. Similar effects on motility were observed by increasing DYRK3 levels, while kinase-dead DYRK3 had limited effects. DYRK3 overexpression inhibits PMAPs formation and has negative effects on stability of lamellipodia and adhesions in migrating cells. Liprin-α1 depletion results in unstable lamellipodia and impaired cell motility. DYRK3 causes increased Liprin-α1 phosphorylation. Increasing levels of Liprin-α1 rescue the inhibitory effects of DYRK3 on cell spreading, suggesting that an equilibrium between Liprin-α1 and DYRK3 levels is required for lamellipodia stability and tumor cell motility. Our results show that DYRK3 is relevant to tumor cell motility, and identify a PMAP target of the kinase, highlighting a new mechanism regulating cell edge dynamics.
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Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50-60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS.
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Within the chromatin, distal elements interact with promoters to regulate specific transcriptional programs. Histone acetylation, interfering with the net charges of the nucleosomes, is a key player in this regulation. Here, we report that the oncoprotein SET is a critical determinant for the levels of histone acetylation within enhancers. We disclose that a condition in which SET is accumulated, the severe Schinzel-Giedion Syndrome (SGS), is characterized by a failure in the usage of the distal regulatory regions typically employed during fate commitment. This is accompanied by the usage of alternative enhancers leading to a massive rewiring of the distal control of the gene transcription. This represents a (mal)adaptive mechanism that, on one side, allows to achieve a certain degree of differentiation, while on the other affects the fine and corrected maturation of the cells. Thus, we propose the differential in cis-regulation as a contributing factor to the pathological basis of SGS and possibly other the SET-related disorders in humans.
Assuntos
Elementos Facilitadores Genéticos , Histonas , Humanos , Histonas/genética , Histonas/metabolismo , Elementos Facilitadores Genéticos/genética , Diferenciação Celular/genética , Cromatina/genética , Regiões Promotoras Genéticas/genéticaRESUMO
In neurons, specific mRNAs are transported into axons, where their local translation supports essential cellular functions. Over the years, our knowledge of the molecular mechanisms underlying axonal mRNA translation has rapidly expanded. However, tools to study mRNA localization and translation in real time with high spatial precision were not available until recently. Here, we present a live imaging approach to examine axonal mRNA trafficking and translation simultaneously in Xenopus retinal ganglion cells (RGCs), using in vitro synthesized fluorescently labeled mRNAs coupled with a genetically encoded protein tagging system to visualize synthesizing peptides at single-molecule resolution. We further describe the process of image analysis in detail, thus providing a methodology that can be used to investigate new research questions in the field.
Assuntos
Axônios , Transporte de RNA , Animais , Transporte Axonal/fisiologia , Axônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Ganglionares da Retina/metabolismo , Xenopus laevis/metabolismoRESUMO
Messenger RNA (mRNA) localization allows spatiotemporal regulation of the proteome at the subcellular level. This is observed in the axons of neurons, where mRNA localization is involved in regulating neuronal development and function by orchestrating rapid adaptive responses to extracellular cues and the maintenance of axonal homeostasis through local translation. Here, we provide an overview of the key findings that have broadened our knowledge regarding how specific mRNAs are trafficked and localize to axons. In particular, we review transcriptomic studies investigating mRNA content in axons and the molecular principles underpinning how these mRNAs arrived there, including cis-acting mRNA sequences and trans-acting proteins playing a role. Further, we discuss evidence that links defective axonal mRNA localization and pathological outcomes.
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Axônios/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Sítios de Ligação , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Transporte Proteico , Transporte de RNA , Proteínas de Ligação a RNA/metabolismo , Elementos de Resposta , TranscriptomaRESUMO
Ribosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons.
Assuntos
Axônios/metabolismo , Neurogênese , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Animais , Axônios/ultraestrutura , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Células Cultivadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Ribonucleico , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Xenopus laevisRESUMO
Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.
Assuntos
Endossomos/fisiologia , Biossíntese de Proteínas/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Axônios/metabolismo , Endossomos/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/fisiologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/fisiologia , Ribossomos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/fisiologia , proteínas de unión al GTP Rab7RESUMO
During amniote peripheral nervous system development, segmentation ensures the correct patterning of the spinal nerves relative to the vertebral column. Along the antero-posterior (rostro-caudal) axis, each somite-derived posterior half-sclerotome expresses repellent molecules to restrict axon growth and neural crest migration to the permissive anterior half-segment. To identify novel regulators of spinal nerve patterning, we investigated the differential gene expression of anterior and posterior half-sclerotomes in the chick embryo by RNA-sequencing. Several genes encoding extracellular matrix proteins were found to be enriched in either anterior (e.g. Tenascin-C, Laminin alpha 4) or posterior (e.g. Fibulin-2, Fibromodulin, Collagen VI alpha 2) half-sclerotomes. Among them, the extracellular matrix protein Fibulin-2 was found specifically restricted to the posterior half-sclerotome. By using in ovo ectopic expression in chick somites, we found that Fibulin-2 modulates spinal axon growth trajectories in vivo. While no intrinsic axon repellent activity of Fibulin-2 was found, we showed that it enhances the growth cone repulsive activity of Semaphorin 3A in vitro. Some molecules regulating axon growth during development are found to be upregulated in the adult central nervous system (CNS) following traumatic injury. Here, we found increased Fibulin-2 protein levels in reactive astrocytes at the lesion site of a mouse model of CNS injury. Together, these results suggest that the developing vertebral column and the adult CNS share molecular features that control axon growth and plasticity, which may open up the possibility for the identification of novel therapeutic targets for brain and spinal cord injury.
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Proteínas de Ligação ao Cálcio/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Nervos Espinhais/embriologia , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Axônios/fisiologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/fisiologia , Embrião de Galinha , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Camundongos , Crista Neural/metabolismo , Crista Neural/fisiologia , Semaforina-3A/metabolismo , Somitos/fisiologia , Medula Espinal/metabolismo , Medula Espinal/fisiologiaRESUMO
The tips of axons are often far away from the cell soma where most proteins are synthesized. Recent work has revealed that axonal mRNA transport and localised translation are key regulatory mechanisms that allow these distant outposts of the cell to respond rapidly to extrinsic factors and maintain axonal homeostasis. Here, we review recent evidence pointing to an increasingly broad role for local protein synthesis in controlling axon shape, synaptogenesis and axon survival by regulating diverse cellular processes such as vesicle trafficking, cytoskeletal remodelling and mitochondrial integrity. We further highlight current research on the regulatory mechanisms that coordinate the localization and translation of functionally linked mRNAs in axons.
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Transporte Axonal/fisiologia , Axônios/fisiologia , Biossíntese de Proteínas/fisiologia , Transporte Proteico/fisiologia , Animais , Transporte Proteico/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The axons of retinal ganglion cells (RGCs) are topographically sorted before they arrive at the optic tectum. This pre-target sorting, typical of axon tracts throughout the brain, is poorly understood. Here, we show that cytoplasmic FMR1-interacting proteins (CYFIPs) fulfill non-redundant functions in RGCs, with CYFIP1 mediating axon growth and CYFIP2 specifically involved in axon sorting. We find that CYFIP2 mediates homotypic and heterotypic contact-triggered fasciculation and repulsion responses between dorsal and ventral axons. CYFIP2 associates with transporting ribonucleoprotein particles in axons and regulates translation. Axon-axon contact stimulates CYFIP2 to move into growth cones where it joins the actin nucleating WAVE regulatory complex (WRC) in the periphery and regulates actin remodeling and filopodial dynamics. CYFIP2's function in axon sorting is mediated by its binding to the WRC but not its translational regulation. Together, these findings uncover CYFIP2 as a key regulatory link between axon-axon interactions, filopodial dynamics, and optic tract sorting.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Axônios/metabolismo , Comunicação Celular/fisiologia , Trato Óptico/metabolismo , Vias Visuais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Animais , Animais Geneticamente Modificados , Axônios/química , Feminino , Masculino , Trato Óptico/química , Trato Óptico/citologia , Células Ganglionares da Retina/química , Células Ganglionares da Retina/metabolismo , Colículos Superiores/química , Colículos Superiores/metabolismo , Vias Visuais/química , Vias Visuais/citologia , Xenopus laevis , Peixe-ZebraRESUMO
Nascent proteins can be positioned rapidly at precise subcellular locations by local protein synthesis (LPS) to facilitate localized growth responses. Axon arbor architecture, a major determinant of synaptic connectivity, is shaped by localized growth responses, but it is unknown whether LPS influences these responses in vivo. Using high-resolution live imaging, we examined the spatiotemporal dynamics of RNA and LPS in retinal axons during arborization in vivo. Endogenous RNA tracking reveals that RNA granules dock at sites of branch emergence and invade stabilized branches. Live translation reporter analysis reveals that de novo ß-actin hotspots colocalize with docked RNA granules at the bases and tips of new branches. Inhibition of axonal ß-actin mRNA translation disrupts arbor dynamics primarily by reducing new branch emergence and leads to impoverished terminal arbors. The results demonstrate a requirement for LPS in building arbor complexity and suggest a key role for pre-synaptic LPS in assembling neural circuits.
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Axônios/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , RNA/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Anisomicina/farmacologia , Biotina/metabolismo , Blastômeros , Carbocianinas/metabolismo , Cicloeximida/farmacologia , Nucleotídeos de Desoxiuracil/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas In Vitro , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mitocôndrias/metabolismo , Morfolinos/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Técnicas de Cultura de Órgãos , Inibidores da Síntese de Proteínas/farmacologia , RNA/genética , Retina/citologia , Xenopus laevisRESUMO
The establishment of precise topographic maps during neural development is facilitated by the presorting of axons in the pathway before they reach their targets. In the vertebrate visual system, such topography is seen clearly in the optic tract (OT) and in the optic radiations. However, the molecular mechanisms involved in pretarget axon sorting are poorly understood. Here, we show in zebrafish that the RNA-binding protein Hermes, which is expressed exclusively in retinal ganglion cells (RGCs), is involved in this process. Using a RiboTag approach, we show that Hermes acts as a negative translational regulator of specific mRNAs in RGCs. One of these targets is the guidance cue receptor Neuropilin 1 (Nrp1), which is sensitive to the repellent cue Semaphorin 3A (Sema3A). Hermes knock-down leads to topographic missorting in the OT through the upregulation of Nrp1. Restoring Nrp1 to appropriate levels in Hermes-depleted embryos rescues this effect and corrects the axon-sorting defect in the OT. Our data indicate that axon sorting relies on Hermes-regulated translation of Nrp1. SIGNIFICANCE STATEMENT: An important mechanism governing the formation of the mature neural map is pretarget axon sorting within the sensory tract; however, the molecular mechanisms involved in this process remain largely unknown. The work presented here reveals a novel function for the RNA-binding protein Hermes in regulating the topographic sorting of retinal ganglion cell (RGC) axons in the optic tract and tectum. We find that Hermes negatively controls the translation of the guidance cue receptor Neuropilin-1 in RGCs, with Hermes knock-down resulting in aberrant growth cone cue sensitivity and axonal topographic misprojections. We characterize a novel RNA-based mechanism by which axons restrict their translatome developmentally to achieve proper targeting.
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Axônios/fisiologia , Neuropilina-1/fisiologia , Proteínas de Ligação a RNA/fisiologia , Vias Visuais/fisiologia , Proteínas de Xenopus/fisiologia , Animais , Embrião não Mamífero , Técnicas de Silenciamento de Genes , Cones de Crescimento , Neuropilina-1/genética , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas de Ligação a RNA/genética , Células Ganglionares da Retina/metabolismo , Semaforina-3A/genética , Semaforina-3A/fisiologia , Colículos Superiores/anatomia & histologia , Colículos Superiores/fisiologia , Proteínas de Xenopus/genética , Xenopus laevis , Peixe-ZebraRESUMO
Subcellular target recognition in the CNS is the culmination of a multiple-step program including axon guidance, target recognition, and synaptogenesis. In cerebellum, basket cells (BCs) innervate the soma and axon initial segment (AIS) of Purkinje cells (PCs) to form the pinceau synapse, but the underlying mechanisms remain incompletely understood. Here, we demonstrate that neuropilin-1 (NRP1), a Semaphorin receptor expressed in BCs, controls both axonal guidance and subcellular target recognition. We show that loss of Semaphorin 3A function or specific deletion of NRP1 in BCs alters the stereotyped organization of BC axon and impairs pinceau synapse formation. Further, we identified NRP1 as a trans-synaptic binding partner of the cell adhesion molecule neurofascin-186 (NF186) expressed in the PC AIS during pinceau synapse formation. These findings identify a dual function of NRP1 in both axon guidance and subcellular target recognition in the construction of GABAergic circuitry.
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Orientação de Axônios/fisiologia , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Neurônios GABAérgicos/fisiologia , Neuropilina-1/fisiologia , Animais , Células CHO , Moléculas de Adesão Celular/metabolismo , Técnicas de Cocultura , Cricetulus , Humanos , Fatores de Crescimento Neural/metabolismo , Neurogênese/fisiologia , Células de Purkinje/fisiologia , Semaforina-3A/fisiologia , Sinapses/fisiologiaRESUMO
The establishment of precise neural circuits during development involves a variety of contact-mediated and secreted guidance molecules that are expressed in a complementary fashion by different cell types. To build a functional circuit, each cell type must first trigger an intrinsic genetic program that is led by their environment at a key time point. It is therefore essential to identify the different cell-specific and stage-specific transcriptional profiles expressed by neurons. However, very few studies have been done to address this issue thus far. Herein, we have carried out a large-scale quantitative real-time PCR analysis of all classical axon guidance molecules (i.e., Semaphorins, Netrins, Ephrins, and Slits) and their receptors expressed by Purkinje cells (PCs) at specific stages of postnatal cerebellar development in vivo. Most cerebellar connections are setup in a well-characterized sequential manner during postnatal development and lead to the fine regulation of the PC, the sole output of the structure. Our analysis of the relative expression of these guidance cues has uncovered a dynamic expression pattern corresponding to specific stages of cerebellar development, thus providing a starting point for studying the role of these axon guidance molecules in cerebellar wiring.
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Axônios , Cerebelo/crescimento & desenvolvimento , Expressão Gênica , Rede Nervosa/crescimento & desenvolvimento , Neurônios/citologia , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Animais , Axônios/metabolismo , Expressão Gênica/fisiologia , Camundongos , Análise em Microsséries/métodos , Fatores de Crescimento Neural/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/metabolismoRESUMO
BACKGROUND: GABAergic interneurons regulate the balance and dynamics of neural circuits, in part, by elaborating their strategically placed axon branches that innervate specific cellular and subcellular targets. However, the molecular mechanisms that regulate target-directed GABAergic axon branching are not well understood. RESULTS: Here we show that the secreted axon guidance molecule, SEMA3A, expressed locally by Purkinje cells, regulates cerebellar basket cell axon branching through its cognate receptor Neuropilin-1 (NRP1). SEMA3A was specifically localized and enriched in the Purkinje cell layer (PCL). In sema3A(-/-) and nrp1(sema-/sema-) mice lacking SEMA3A-binding domains, basket axon branching in PCL was reduced. We demonstrate that SEMA3A-induced axon branching was dependent on local recruitment of soluble guanylyl cyclase (sGC) to the plasma membrane of basket cells, and sGC subcellular trafficking was regulated by the Src kinase FYN. In fyn-deficient mice, basket axon terminal branching was reduced in PCL, but not in the molecular layer. CONCLUSIONS: These results demonstrate a critical role of local SEMA3A signaling in layer-specific axonal branching, which contributes to target innervation.