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/metabolismoRESUMEN
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 SeñalRESUMEN
The neuronal Golgi apparatus (GA) localizes to the perinuclear region and dendrites as tubulo-vesicular structures designated Golgi outposts (GOPs). Current evidence suggests that GOPs shape dendrite morphology and serve as platforms for the local delivery of synaptic receptors. However, the mechanisms underlying GOP formation remain a mystery. Using live-cell imaging and confocal microscopy in cultured hippocampal neurons, we now show that GOPs destined to major "apical" dendrites are generated from the somatic GA by a sequence of events involving: (1) generation of a GA-derived tubule; (2) tubule elongation and deployment into the dendrite; (3) tubule fission; and (4) transport and condensation of the fissioned tubule. A RhoA-Rock signaling pathway involving LIMK1, PKD1, slingshot, cofilin, and dynamin regulates polarized GOP formation by controlling the tubule fission. Our observations identify a mechanism underlying polarized GOP biogenesis and provide new insights regarding involvement of RhoA in dendritic development and polarization.
Asunto(s)
Polaridad Celular/fisiología , Dendritas/metabolismo , Aparato de Golgi/metabolismo , Hipocampo/citología , Neuronas/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Células Cultivadas , Humanos , Microscopía Confocal , Neuronas/citología , Transducción de Señal/fisiologíaRESUMEN
Microtubule (MT) organization and dynamics downstream of external cues is crucial for maintaining cellular architecture and the generation of cell asymmetries. In interphase cells RhoA, Rac, and Cdc42, conspicuous members of the family of small Rho GTPases, have major roles in modulating MT stability, and hence polarized cell behaviors. However, MTs are not mere targets of Rho GTPases, but also serve as signaling platforms coupling MT dynamics to Rho GTPase activation in a variety of cellular conditions. In this article, we review some of the key studies describing the reciprocal relationship between small Rho-GTPases and MTs during migration and polarization.
Asunto(s)
Microtúbulos/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Polaridad Celular , Adhesiones Focales/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
The highly dynamic remodeling and cross talk of the microtubule and actin cytoskeleton support neuronal morphogenesis. Small RhoGTPases family members have emerged as crucial regulators of cytoskeletal dynamics. In this review we will comprehensively analyze findings that support the participation of RhoA, Rac, Cdc42, and TC10 in different neuronal morphogenetic events ranging from migration to synaptic plasticity. We will specifically address the contribution of these GTPases to support neuronal polarity and axonal elongation.
Asunto(s)
Polaridad Celular , Proteínas de Unión al GTP Monoméricas/metabolismo , Morfogénesis , Neuronas/citología , Neuronas/enzimología , Animales , Humanos , Modelos BiológicosRESUMEN
In this study, we have used a combination of biochemical and molecular biology techniques to demonstrate that the C-terminal tail domain of KIF4 directly interacts with P0, a major protein component of ribosomes. Besides, in dorsal root ganglion neurons, KIF4 and P0, as well as other ribosomal constituents, colocalize in clusters distributed along axons and neuritic tips. RNA interference suppression of KIF4 or expression of KIF4 variants lacking the tail domain or mutations of the ATP-binding site result in accumulation of P0 and other ribosomal proteins at the cell body and in their disappearance from axons. Our results also show one additional function for KIF4 involving an Ezrin-Radixin-Moesin-like domain in the second coiled-coiled region of KIF4. Expression of a KIF4 mutant lacking this domain abolishes the clustering of ribosomal constituents and prevents the anterograde translocation of the cell adhesion molecule L1. Taken together, the present results suggest that by binding to P0 through its tail domain and by using its motor activity, KIF4 is involved in the anterograde trafficking of ribosomal constituents to axons and that by means of its Ezrin-Radixin-Moesin-like domain interacts and transports L1.
Asunto(s)
Transporte Axonal , Axones/metabolismo , Cinesinas/metabolismo , Ribosomas/metabolismo , Animales , Células Cultivadas , Femenino , Ganglios Espinales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Cinesinas/genética , Masculino , Ratones , Mutación/genética , Unión Proteica , Transporte de Proteínas , Interferencia de ARN , RatasRESUMEN
Electroconvulsive shock (ECS) improves motor function in Parkinson's disease. In rats, ECS stimulates the expression of various factors some of which have been proposed to exert neuroprotective actions. We have investigated the effects of ECS on 6-hydroxydopamine (6-OHDA)-injected rats. Three weeks after a unilateral administration of 6-OHDA, 85-95% nigral dopaminergic neurons are lost. Chronic ECS prevented this cell loss, protect the nigrostriatal pathway (assessed by FloroGold retrograde labeling) and reduce motor impairment in 6-OHDA-treated animals. Injection of 6-OHDA caused loss of expression of glial cell-line derived neurotrophic factor (GDNF) in the substantia nigra. Chronic ECS completely prevented this loss of GDNF expression in 6-OHDA-treated animals. We also found that protected dopaminergic neurons co-express GDNF receptor proteins. These results strongly suggest that endogenous changes in GDNF expression may participate in the neuroprotective mechanism of ECS against 6-OHDA induced toxicity.