RESUMEN
Semaphorin3A (Sema3A) is a secreted type of axon guidance molecule that regulates axon wiring through complexes of neuropilin-1 (NRP1) with PlexinA protein receptors. Sema3A regulates the dendritic branching through tetrodotoxin (TTX)-sensitive retrograde axonal transport of PlexA proteins and tropomyosin-related kinase A (TrkA) complex. We here demonstrate that Nav1.7 (encoded by SCN9A), a TTX-sensitive Na+ channel, by coupling with collapsin response mediator protein 1 (CRMP1), mediates the Sema3A-induced retrograde transport. In mouse dorsal root ganglion (DRG) neurons, Sema3A increased co-localization of PlexA4 and TrkA in the growth cones and axons. TTX treatment and RNAi knockdown of Nav1.7 sustained Sema3A-induced colocalized signals of PlexA4 and TrkA in growth cones and suppressed the subsequent localization of PlexA4 and TrkA in distal axons. A similar localization phenotype was observed in crmp1-/- DRG neurons. Sema3A induced colocalization of CRMP1 and Nav1.7 in the growth cones. The half maximal voltage was increased in crmp1-/- neurons when compared to that in wild type. In HEK293 cells, introduction of CRMP1 lowered the threshold of co-expressed exogenous Nav1.7. These results suggest that Nav1.7, by coupling with CRMP1, mediates the axonal retrograde signaling of Sema3A.
Asunto(s)
Orientación del Axón , Ganglios Espinales/citología , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Semaforina-3A/metabolismo , Transducción de Señal , Animales , Células HEK293 , Humanos , Ratones , Ratones Endogámicos , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.7/genética , Red Nerviosa , Proteínas del Tejido Nervioso/genética , Neuropilina-1/metabolismo , Unión Proteica , ARN Interferente Pequeño/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Semaphorin3A (Sema3A) exerts a wide variety of biological functions by regulating reorganization of actin and tubulin cytoskeletal proteins through signaling pathways including sequential phosphorylation of collapsin response mediator protein 1 (CRMP1) and CRMP2 by cyclin-dependent kinase-5 and glycogen synthase kinase-3ß (GSK3ß). To delineate how GSK3ß mediates Sema3A signaling, we here determined the substrates of GSK3ß involved. Introduction of either GSK3ß mutants, GSK3ß-R96A, L128A, or K85M into chick dorsal root ganglion (DRG) neurons suppressed Sema3A-induced growth cone collapse, thereby suggesting that unprimed as well as primed substrates are involved in Sema3A signaling. Axin-1, a key player in Wnt signaling, is an unprimed substrate of GSK3ß. The phosphorylation of Axin-1 by GSK3ß accelerates the association of Axin-1 with ß-catenin. Immunocytochemical studies revealed that Sema3A induced an increase in the intensity levels of ß-catenin in the DRG growth cones. Axin-1 siRNA knockdown suppressed Sema3A-induced growth cone collapse. The reintroduction of RNAi-resistant Axin-1 (rAxin-1)-wt rescued the responsiveness to Sema3A, while that of nonphosphorylated mutants, rAxin S322A/S326A/S330A and T485A/S490A/S497A, did not. Sema3A also enhanced the colocalization of GSK3ß, Axin-1, and ß-catenin in the growth cones. The increase of ß-catenin in the growth cones was suppressed by the siRNA knockdown of Axin-1. Furthermore, either Axin-1 or ß-catenin RNAi knockdown suppressed the internalization of Sema3A. These results suggest that Sema3A induces the formation of GSK3ß/Axin-1/ß-catenin complex, which regulates signaling cascade of Sema3A via an endocytotic mechanism. This finding should provide clue for understanding of mechanisms of a wide variety of biological functions of Sema3A.
Asunto(s)
Proteína Axina/fisiología , Glucógeno Sintasa Quinasa 3/fisiología , Semaforina-3A/fisiología , Transducción de Señal/fisiología , beta Catenina/fisiología , Animales , Proteína Axina/genética , Células Cultivadas , Embrión de Pollo , Endocitosis/genética , Femenino , Ganglios Espinales/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Masculino , Ratones , Mutagénesis Sitio-Dirigida , Técnicas de Cultivo de Órganos , Fosforilación/genética , Ratas , Semaforina-3A/genética , Transducción de Señal/genética , Especificidad por Sustrato/genética , beta Catenina/genéticaRESUMEN
Axonal transport plays a crucial role in neuronal morphogenesis, survival and function. Despite its importance, however, the molecular mechanisms of axonal transport remain mostly unknown because a simple and quantitative assay system for monitoring this cellular process has been lacking. In order to better characterize the mechanisms involved in axonal transport, we formulate a novel computer-assisted monitoring system of axonal transport. Potential uses of this system and implications for future studies will be discussed.
Asunto(s)
Transporte Axonal/fisiología , Modelos Neurológicos , Animales , Transporte Axonal/efectos de los fármacos , Supervivencia Celular , Embrión de Pollo , Biología Computacional , Procesamiento de Imagen Asistido por Computador , Factores de Crecimiento Nervioso/fisiología , Red Nerviosa/crecimiento & desarrollo , Red Nerviosa/fisiología , Neurogénesis/fisiología , Plasticidad Neuronal , Neuronas/citología , Neuronas/fisiología , Neurotoxinas/toxicidadRESUMEN
Axonal transport plays a crucial role in neuronal morphogenesis, survival, and function. Despite its importance, however, the molecular mechanisms of axonal transport remain mostly unknown because a simple and quantitative assay system for axonal transport has been lacking. In order to better characterize the molecular mechanisms involved in axonal transport, we here developed a computer-assisted monitoring system. Using lipophilic fluorochrome chloromethylbenzamido dialkylcarbocyanine (CM-DiI) as a labeling dye, we have successfully labeled membranous organelles in cultured chick dorsal root ganglia neurons. We confirmed that sodium azide, an ATPase inhibitor, and nocodazole, a microtubule-destabilizing agent, markedly suppressed anterograde and retrograde axonal transport of CM-DiI-labeled particles. We further tested the effects of several anti-neoplastic drugs on axonal transport. Paclitaxel, vincristine, cisplatin, and oxaliplatin, all of which are known to be neurotoxic and to cause neurological symptoms, suppressed anterograde and retrograde axonal transport. Another series of anti-neoplastic drugs, including methotrexate and 5-fluorouracil, did not affect the axonal transport. This is the first report of an automated monitoring system for axonal transport. This system will be useful for toxicity assays, characterizing axonal transport, or screening drugs that may modify neuronal functions.
Asunto(s)
Antineoplásicos/farmacología , Transporte Axonal/efectos de los fármacos , Animales , Transporte Axonal/fisiología , Carbocianinas/metabolismo , Células Cultivadas , Embrión de Pollo , Cisplatino/farmacología , Biología Computacional , Colorantes Fluorescentes/metabolismo , Fluorouracilo/farmacología , Ganglios Espinales/citología , Neuronas/efectos de los fármacos , Nocodazol/farmacología , Orgánulos/metabolismo , Compuestos Organoplatinos/farmacología , Oxaliplatino , Paclitaxel/farmacología , Azida Sódica/farmacología , Programas Informáticos , Vincristina/farmacologíaRESUMEN
Mutations in human ß3-tubulin (TUBB3) cause an ocular motility disorder termed congenital fibrosis of the extraocular muscles type 3 (CFEOM3). In CFEOM3, the oculomotor nervous system develops abnormally due to impaired axon guidance and maintenance; however, the underlying mechanism linking TUBB3 mutations to axonal growth defects remains unclear. Here, we investigate microtubule (MT)-based motility in vitro using MTs formed with recombinant TUBB3. We find that the disease-associated TUBB3 mutations R262H and R262A impair the motility and ATPase activity of the kinesin motor. Engineering a mutation in the L12 loop of kinesin surprisingly restores a normal level of motility and ATPase activity on MTs carrying the R262A mutation. Moreover, in a CFEOM3 mouse model expressing the same mutation, overexpressing the suppressor mutant kinesin restores axonal growth in vivo. Collectively, these findings establish the critical role of the TUBB3-R262 residue for mediating kinesin interaction, which in turn is required for normal axonal growth and brain development.
Asunto(s)
Axones/patología , Cinesinas/metabolismo , Microtúbulos/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/metabolismo , Femenino , Fibrosis/metabolismo , Inmunohistoquímica , Ratones , Mutación , EmbarazoRESUMEN
The establishment of neuronal polarity is necessary for proper neuronal wiring. Semaphorin3A (Sema3A), originally identified as a repulsive axon guidance molecule, exerts a wide variety of biological functions through signaling pathways including sequential phosphorylation of collapsin response mediator protein by cyclin-dependent kinase-5 (Cdk5) and glycogen synthase kinase-3ß (GSK3ß). Sema3A acts on its receptor neuropilin-1 to regulate axonal transport. To delineate mechanism by which Sema3A induces axonal transport, we investigate whether GSK3ß is involved in mediating Sema3A-induced axonal transport. 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, an inhibitor of GSK3ß, suppressed Sema3A-induced antero- and retrograde axonal transport. Introduction of either GSK3ß mutants, GSK3ß-L128A or K85M, suppressed Sema3A-induced axonal transport. On the other hand, introduction of GSK3ß-R96A did not affect the Sema3A effect, suggesting that unprimed substrates are primarily involved in Sema3A-induced axonal transport. Overexpression of a partial fragment of frequently rearranged in advanced T-cell lymphomas 1 (FRATtide), which interferes the interaction between GSK3ß and Axis inhibitor-1 (Axin-1), also suppressed Sema3A-induced transport. siRNA knockdown of Axin-1, an unprimed substrate of GSK3ß, suppressed Sema3A-induced antero- and retrograde axonal transport. These results indicate that GSK3ß and Axin-1 are involved in Sema3A-induced bidirectional axonal transport. This finding should provide a clue for understanding of mechanisms of a wide variety of biological activities of Sema3A.
Asunto(s)
Proteínas Aviares/metabolismo , Proteína Axina/metabolismo , Transporte Axonal/fisiología , Glucógeno Sintasa Quinasa 3/metabolismo , Semaforina-3A/metabolismo , Animales , Proteínas Aviares/genética , Proteína Axina/genética , Transporte Axonal/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Mutación , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Tiadiazoles/farmacología , Técnicas de Cultivo de Tejidos , Vesículas Transportadoras/efectos de los fármacos , Vesículas Transportadoras/metabolismoRESUMEN
AIM: To assess the efficacy of topical Semaphorin-3A (SEMA3A) in the treatment of allergic conjunctivitis. METHODS: Experimental allergic conjunctivitis (EAC) mice model induced by short ragweed pollen (SRW) in 4-week-old of BALB/c mice, mice were evaluated using haematoxylin and eosin (H&E) staining, immunofluorescence and light microscope photographs. Early phase took the samples in 24h after instillation and late phase took the samples between 4 to 14d after the start of treatment. The study use of topical SEMA3A (10 U, 100 U, 1000 U) eye drops and subconjunctival injection of SEMA3A with same concentration. For comparison, five types of allergy eyedrops were quantified using clinical characteristics. RESULTS: Clinical score of composite ocular symptoms of the mice treated with SEMA3A were significantly decreased both in the immediate phase and the late phase compared to those treated with commercial ophthalmic formulations and non-treatment mice. SEMA3A treatment attenuates infiltration of eosinophils entering into conjunctiva in EAC mice. The score of eosinophil infiltration in the conjunctiva of SEMA3A 1000 U-treated group were significantly lower than low-concentration of SEMA3A treated groups and non-treated group. SEMA3A treatment also suppressed T-cell proliferation in vitro and decreased serum total IgE levels in EAC mice. Moreover, Treatment of SEMA3A suppressed Th2-related cytokines (IL-5, IL-13 and IL-4) and pro-inflammatory cytokines (IFN-γ, IL-17 and TNF-α) release, but increased regulatory cytokine IL-10 concentration in the conjunctiva of EAC mice. CONCLUSIONS: SEMA3A as a biological agent, showed the beneficial activity in ocular allergic processes with the less damage to the intraocular tissue. It is expected that SEMA3A may be contributed in patients with a more severe spectrum of refractory ocular allergic diseases including allergic conjunctivitis in the near future.
RESUMEN
The dendritic targeting of neurotransmitter receptors is vital for dendritic development and function. However, how such localization is established remains unclear. Here we show that semaphorin 3A (Sema3A) signalling at the axonal growth cone is propagated towards the cell body by retrograde axonal transport and drives AMPA receptor GluA2 to the distal dendrites, which regulates dendritic development. Sema3A enhances glutamate receptor interacting protein 1-dependent localization of GluA2 in dendrites, which is blocked by knockdown of cytoplasmic dynein heavy chain. PlexinA (PlexA), a receptor component for Sema3A, interacts with GluA2 at the immunoglobulin-like Plexin-transcription-factor domain (PlexA-IPT) in somatodendritic regions. Overexpression of PlexA-IPT suppresses dendritic localization of GluA2 and induces aproximal bifurcation phenotype in the apical dendrites of CA1 hippocampal neurons. Thus, we propose a control mechanism by which retrograde Sema3A signalling regulates the glutamate receptor localization through trafficking of cis-interacting PlexA with GluA2 along dendrites.
Asunto(s)
Dendritas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores AMPA/metabolismo , Receptores de Superficie Celular/metabolismo , Semaforina-3A/metabolismo , Transducción de Señal , Animales , Axones/metabolismo , Western Blotting , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/embriología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Femenino , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Microscopía Confocal , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/metabolismo , Interferencia de ARN , Ratas , Ratas Wistar , Receptores AMPA/genética , Receptores de Superficie Celular/genética , Semaforina-3A/genética , Imagen de Lapso de Tiempo/métodosRESUMEN
Reorganization of the actin cytoskeleton is an early cellular response to various extracellular signals. Sema3A, a repulsive axon guidance molecule, induces the reorganization of actin cytoskeleton in the growth cones. Collapsin response mediator protein 1 (CRMP1) mediates the intracellular Sema3A signalling through its Ser522 phosphorylation. Here we show that UNC-33, CRMP1 C. elegans homologue, interacts with FLN-1, an actin-binding Filamin-A orthologue. In nematodes, this interaction participates in the projection of DD/VD motor neurons. CRMP1 binds both the actin-binding domain and the last immunoglobulin-like repeat of Filamin-A. The alanine mutants of Filamin-A or CRMP1 in their interacting residues suppress the Sema3A repulsion in neurons. Conversely, a phosphor-mimicking mutant CRMP1(Ser522Asp) enhances the Sema3A response. Atomic-force microscopy analysis reveals that the V-shaped Filamin-A changes to a condensed form with CRMP1(Ser522Asp). CRMP1(Ser522Asp) weakens the F-actin gelation crosslinked by Filamin-A. Thus, phosphorylated CRMP1 may remove Filamin-A from the actin cytoskeleton to facilitate its remodelling.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Filaminas/metabolismo , Conos de Crecimiento/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Actinas/metabolismo , Animales , Caenorhabditis elegans/genética , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Ratas Wistar , Semaforina-3A/metabolismoRESUMEN
Topical steroids and antihistamines are commonly used for the treatment of atopic dermatitis (AD). However, in a substantial number of patients with AD, these treatments are not sufficiently effective. In AD patients, C-fibers in the epidermis increase and sprout, inducing hypersensitivity, which is considered to aggravate the disease. Semaphorin3A (Sema3A), an axon guidance molecule, is a potent inhibitor of neurite outgrowth of sensory neurons. To investigate the effect of Sema3A on AD, we administered recombinant Sema3A intracutaneously into the skin lesions of NC/Nga mice, an animal model of AD. Sema3A dose-dependently improved skin lesions and attenuated the scratching behavior in NC/Nga mice. Histological examinations revealed a decrease in: (a) epidermal thickness; (b) the density of invasive nerve fibers in the epidermis; (c) inflammatory infiltrates, including mast cells and CD4+ T cells; and (d) the production of IL-4 in the Sema3A-treated lesions. Because the interruption of the itch-scratch cycle likely contributes to the improvement of the AD-like skin lesions, Sema3A is promising in the treatment of patients with refractory AD, as well as overall itching dermatosis.