RESUMO
To determine the role of Dp71 in neuronal cells, we generated PC12 cell lines in which Dp71 protein levels were controlled by stable transfection with either antisense or sense constructs. Cells expressing the antisense Dp71 RNA (antisense-Dp71 cells) contained reduced amounts of the two endogenous Dp71 isoforms. Antisense-Dp71 cells exhibited a marked suppression of neurite outgrowth upon the induction with NGF or dibutyryl cyclic AMP. Early responses to NGF-induced neuronal differentiation, such as the cessation of cell division and the activation of ERK1/2 proteins, were normal in the antisense-Dp71 cells. On contrary, the induction of MAP2, a late differentiation marker, was disturbed in these cells. Additionally, the deficiency of Dp71 correlated with an altered expression of the dystrophin-associated protein complex (DAPC) members alpha and beta dystrobrevins. Our results indicate that normal expression of Dp71 is essential for neurite outgrowth in PC12 cells and constitute the first direct evidence implicating Dp71 in a neuronal function.
Assuntos
Distrofina/análogos & derivados , Distrofina/fisiologia , Neuritos/ultraestrutura , Células PC12/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , DNA Antissenso/farmacologia , Distrofina/genética , Humanos , Cinética , Proteínas Associadas aos Microtúbulos/análise , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuritos/química , Neuritos/efeitos dos fármacos , Neurônios/citologia , Neurônios/ultraestrutura , Neuropeptídeos/análise , Isoformas de Proteínas/análise , Ratos , TransfecçãoRESUMO
In the adult central nervous system (CNS), prominent reactive astrocytosis is seen in acute traumatic brain injury, neurodegenerative diseases and a variety of viral infections. Reactive astrocytes synthesize a number of factors that could play different roles in neuronal regeneration. In this study, the effects of thermal stress were evaluated on nuclear factor-kappaB (NF-kappaB) activation and proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) secretion in primary astrocytic cultures. The ability of HSP70-positive astrocytes to support or inhibit neurite outgrowth was investigated in neuron-astrocyte cocultures. Cultured astrocytes from cerebral cortex of rats were exposed to transient hyperthermia (42 degrees C/30 min) and incubated at 37 degrees C for different periods of recovery. During HSP70 accumulation, astrocytes extended large and thick processes associated to rearrangement of glial fibrillary acidic protein (GFAP) filaments and an increase in protein synthesis and GFAP, suggesting an astrogliosis event. A delay of NF-kappaB activation appeared closely related to TNF-alpha secretion by HSP70-positive astrocytes. These cells demonstrated a functional shift from neurite growth-promoting to non-permissive substrate. We also found that gliotoxin, a specific NF-kappaB inhibitor, partially abrogated the inhibitory ability of reactive astrocytes. These findings may suggest a involvement of NF-kappaB and TNF-alpha in modulating the failure of HSP70-positive astrocytes to provide functional support to neuritic outgrowth.
Assuntos
Astrócitos/patologia , Proteínas de Choque Térmico HSP70/biossíntese , NF-kappa B/metabolismo , Neuritos/patologia , Animais , Astrócitos/química , Astrócitos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Proteínas de Choque Térmico HSP70/análise , Temperatura Alta/efeitos adversos , NF-kappa B/análise , Neuritos/química , Neuritos/metabolismo , Ratos , Ratos WistarRESUMO
In this study we have examined the cellular functions of ERM proteins in developing neurons. The results obtained indicate that there is a high degree of spatial and temporal correlation between the expression and subcellular localization of radixin and moesin with the morphological development of neuritic growth cones. More importantly, we show that double suppression of radixin and moesin, but not of ezrin-radixin or ezrin-moesin, results in reduction of growth cone size, disappearance of radial striations, retraction of the growth cone lamellipodial veil, and disorganization of actin filaments that invade the central region of growth cones where they colocalize with microtubules. Neuritic tips from radixin-moesin suppressed neurons displayed high filopodial protrusive activity; however, its rate of advance is 8-10 times slower than the one of growth cones from control neurons. Radixin-moesin suppressed neurons have short neurites and failed to develop an axon-like neurite, a phenomenon that appears to be directly linked with the alterations in growth cone structure and motility. Taken collectively, our data suggest that by regulating key aspects of growth cone development and maintenance, radixin and moesin modulate neurite formation and the development of neuronal polarity.
Assuntos
Proteínas Sanguíneas/genética , Proteínas do Citoesqueleto , Cones de Crescimento/fisiologia , Proteínas de Membrana/genética , Proteínas dos Microfilamentos , Proteínas/genética , Células Piramidais/citologia , Actinas/metabolismo , Animais , Elementos Antissenso (Genética) , Proteínas Sanguíneas/metabolismo , Polaridade Celular/fisiologia , Células Cultivadas , Citoesqueleto/fisiologia , Expressão Gênica/fisiologia , Cones de Crescimento/química , Hipocampo/citologia , Proteínas de Membrana/metabolismo , Neuritos/química , Neuritos/fisiologia , Proteínas/metabolismo , Células Piramidais/química , Células Piramidais/ultraestrutura , Ratos , Frações Subcelulares/química , TionucleotídeosRESUMO
We have investigated the role of 9-O-acetylated gangliosides identified by the Jones monoclonal antibody (Jones mAb) in the elongation of neurites extended by neurons of embryonic rat dorsal root ganglia (DRG) explants grown on laminin substratum. The behavior of individual growth cones was recorded using a time-lapse video-enhanced imaging system before and after the addition of antibodies that recognize specific gangliosides known to be expressed on these growth cones. It was possible to demonstrate that the advance of growth cones on laminin was halted in the presence of Jones mAb. The onset of effects was rapid and signaled by an immediate cessation of elongation, a loss of lamellipodia and a retrieval of axoplasm. This effect was partially reverted by washing the explants for several minutes with culture medium. mAb A2B5 which also recognizes gangliosides expressed on these growth cones does not induce any change on the growth rate. Our findings show that 9-O-acetylated gangliosides may play an important role on the extension of growth cones and consequently influence navigation and pathway finding during development.