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1.
J Neurochem ; 157(4): 1196-1206, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33156548

RESUMO

Intracellular signaling pathways that promote axon regeneration are closely linked to the mechanism of neurite outgrowth. TC10, a signaling molecule that acts on neurite outgrowth through membrane transport, is a member of the Rho family G proteins. Axon injury increases the TC10 levels in motor neurons, suggesting that TC10 may be involved in axon regeneration. In this study, we tried to understand the roles of TC10 in the nervous system using TC10 knock-out mice. In cultured hippocampal neurons, TC10 ablation significantly reduced axon elongation without affecting ordinary polarization. We determined a role of TC10 in microtubule stabilization at the growth cone neck; therefore, we assume that TC10 limits axon retraction and promotes in vitro axon outgrowth. In addition, there were no notable differences in the size and structure of brains during prenatal and postnatal development between wild-type and TC10 knock-out mice. In motor neurons, axon regeneration after injury was strongly suppressed in mice lacking TC10 (both in conventional and injured nerve specific deletion). In retinal ganglion cells, TC10 ablation suppressed the axon regeneration stimulated by intraocular inflammation and cAMP after optic nerve crush. These results show that TC10 plays an important role in axon regeneration in both the peripheral and central nervous systems, and the role of TC10 in peripheral axon regeneration is neuron-intrinsic.


Assuntos
Axônios/metabolismo , Regeneração Nervosa/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Hipocampo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Crescimento Neuronal/fisiologia , Neurônios/metabolismo , Transdução de Sinais/fisiologia
2.
Genes Cells ; 22(11): 953-967, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29072354

RESUMO

Cyclic AMP plays a pivotal role in neurite growth. During outgrowth, a trafficking system supplies membrane at growth cones. However, the cAMP-induced signaling leading to the regulation of membrane trafficking remains unknown. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking. Recent studies have shown a role of TC10 in neurite growth in NGF-treated PC12 cells. Here, we investigated a mechanical linkage between cAMP and TC10 in neuritogenesis. Plasmalemmal TC10 activity decreased abruptly after cAMP addition in neuronal cells. TC10 was locally inactivated at extending neurite tips in cAMP-treated PC12 cells. TC10 depletion led to a decrease in cAMP-induced neurite outgrowth. Constitutively active TC10 could not rescue this growth reduction, supporting our model for a role of GTP hydrolysis of TC10 in neuritogenesis by accelerating vesicle fusion. The cAMP-induced TC10 inactivation was mediated by PKA. Considering cAMP-induced RhoA inactivation, we found that p190B, but not p190A, mediated inactivation of TC10 and RhoA. Upon cAMP treatment, p190B was recruited to the plasma membrane. STEF depletion and Rac1-N17 expression reduced cAMP-induced TC10 inactivation. Together, the PKA-STEF-Rac1-p190B pathway leading to inactivation of TC10 and RhoA at the plasma membrane plays an important role in cAMP-induced neurite outgrowth.


Assuntos
Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Crescimento Neuronal , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Animais , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Regulação para Baixo , Proteínas Ativadoras de GTPase/genética , Células HeLa , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
3.
Genes Cells ; 18(11): 1020-31, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24165023

RESUMO

In a developing nervous system, axon-dendrite formation is instructed by extrinsic cues, and the mechanism whereby a developing neuron interprets these cues using intracellular signaling is particularly important. Studies using dissociated hippocampal neurons have identified many signaling pathways underlying neuronal polarization. Among the components of these pathways, Rap1B is essential for axon specification in hippocampal cultures. However, spatiotemporal regulation of Rap1B activity in polarizing neurons and how it affects neuronal polarization remain unclear. Herein, we investigated spatiotemporal activity-change of Rap1B and its target molecules in hippocampal neurons. FRET imaging showed that specific activation of Rap1B was observed at the tip of a future axon. To dissect downstream signaling, we used three effector mutants of Rap1B. Expression of Rap1B-G12V/E37G and G12V/Y40C mutants resulted in supernumerary axons. The targets of Rap1B-G12V/E37G were RalA and Nore1A, whereas Rap1B-G12V/Y40C activated PI3-kinase. RalA was activated in the tip of stage 3 axons, and RalA-S28N expression reduced the fraction of neurons with supernumerary axons induced by Rap1B-G12V/E37G. Furthermore, Nore1A depletion reduced the number of cells without axons. These results indicate that specific activation of Rap1B contributes to neuronal polarization via interaction with RalA and Nore1A in addition to PI3-kinase.


Assuntos
Polaridade Celular , Neuritos/fisiologia , Neurônios/fisiologia , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas rap de Ligação ao GTP/genética , Animais , Axônios/fisiologia , Células Cultivadas , Dendritos/fisiologia , Hipocampo/citologia , Técnicas In Vitro , Mutação , Neurônios/ultraestrutura , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo
4.
Neurobiol Aging ; 106: 268-281, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34329965

RESUMO

Aß metabolism in the brain is mediated by endocytosis, one part of the intracellular membrane trafficking system. We previously showed that aging attenuates the interaction of dynein with dynactin, which disrupts the endosomal/lysosomal trafficking pathway involved in Aß metabolism, resulting in intracellular accumulation of Aß. Several studies have shown that in Alzheimer's disease (AD), intraneuronal accumulation of Aß precedes extracellular Aß depositions. However, it is unclear what accounts for this transition from intracellular to extracellular depositions. Accumulating evidence suggest that autophagy has an important role in AD pathology, and we observed that autophagy-related protein levels began to decrease before amyloid plaque formation in cynomolgus monkey brains. Surprisingly, experimental induction of autophagosome formation in Neuro2a cells significantly increased intracellular Aß and decreased extracellular release of Aß, accompanied by the prominent reduction of extracellular vesicle (EV) secretion. RNAi study confirmed that EV secretion affected intracellular and extracellular Aß levels, and siRNA-induced downregulation of autophagosome formation enhanced EV secretion to ameliorate intracellular Aß accumulation induced by dynein knockdown. In aged cynomolgus monkeys, Aß levels in EV/intraluminal membrane vesicle (ILV)-rich fractions isolated from temporal lobe parenchyma were drastically increased. Moreover, EV/ILV marker proteins overlapped spatially with amyloid plaques. These findings suggest that EV would be an important carrier of Aß in brain and abnormal accumulation of Aß in EVs/ILVs may be involved in the transition of age-dependent Aß pathology.


Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Vesículas Extracelulares/metabolismo , Frações Subcelulares/metabolismo , Animais , Autofagossomos/metabolismo , Autofagia , Linhagem Celular , Dineínas/metabolismo , Endocitose/fisiologia , Macaca fascicularis , Camundongos , Tecido Parenquimatoso/metabolismo , Lobo Temporal/metabolismo
6.
PLoS One ; 8(11): e79689, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24223996

RESUMO

The use of exocytosis for membrane expansion at nerve growth cones is critical for neurite outgrowth. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking to the plasma membrane. Recent studies have shown that TC10 and its effector Exo70, a component of the exocyst tethering complex, contribute to neurite outgrowth. However, the molecular mechanisms of the neuritogenesis-promoting functions of TC10 remain to be established. Here, we propose that GTP hydrolysis of vesicular TC10 near the plasma membrane promotes neurite outgrowth by accelerating vesicle fusion by releasing Exo70. Using Förster resonance energy transfer (FRET)-based biosensors, we show that TC10 activity at the plasma membrane decreased at extending growth cones in hippocampal neurons and nerve growth factor (NGF)-treated PC12 cells. In neuronal cells, TC10 activity at vesicles was higher than its activity at the plasma membrane, and TC10-positive vesicles were found to fuse to the plasma membrane in NGF-treated PC12 cells. Therefore, activity of TC10 at vesicles is presumed to be inactivated near the plasma membrane during neuronal exocytosis. Our model is supported by functional evidence that constitutively active TC10 could not rescue decrease in NGF-induced neurite outgrowth induced by TC10 depletion. Furthermore, TC10 knockdown experiments and colocalization analyses confirmed the involvement of Exo70 in TC10-mediated trafficking in neuronal cells. TC10 frequently resided on vesicles containing Rab11, which is a key regulator of recycling pathways and implicated in neurite outgrowth. In growth cones, most of the vesicles containing the cell adhesion molecule L1 had TC10. Exocytosis of Rab11- and L1-positive vesicles may play a central role in TC10-mediated neurite outgrowth. The combination of this study and our previous work on the role of TC10 in EGF-induced exocytosis in HeLa cells suggests that the signaling machinery containing TC10 proposed here may be broadly used for exocytosis.


Assuntos
Exocitose , Guanosina Trifosfato/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuritos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Exocitose/efeitos dos fármacos , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/metabolismo , Células HeLa , Humanos , Hidrólise/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
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