RESUMO
Binding of bacterial lipopolysaccharides (LPS) to toll-like receptor 4 (TLR4) triggers an innate immunoresponse associated with pain and inflammation. The expression, and to a greater extent the regulation of TLR4 and its auxiliary proteins (myeloid differentiation protein 1 (MD1), myeloid differentiation protein 2 (MD2) and cluster of differentiation 14 (CD14)), are both poorly understood in trigeminal and nodose neurons. We used a combination of Western blotting, semi-quantitative polymerase chain reaction (PCR), pharmacological manipulation and immunohistochemistry. The expression pattern and regulation by LPS and trophic factors of TLR4/MD2/CD14 and radioprotective protein of 105kDa (RP105)/MD1 were determined in neonatal trigeminal and nodose mice neurons. We found that all these proteins were expressed in both trigeminal and nodose neurons. The trophic factors Artemin and nerve growth factor (NGF) up-regulated MD2 and RP105 mRNA levels in trigeminal neurons. In nodose neurons the trophic factors brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) up-regulated MD1 and RP105 mRNA levels. Also we observed that in both neuronal types LPS acutely (within 20 min) down-regulated CD14 and MD2 mRNAs. In addition, LPS increased significantly the proportion of trigeminal and nodose neurons expressing nociceptin/orphanin FQ in culture probably acting via TLR4/MD2. Although the exact mechanisms underlying the regulation by trophic factors and LPS require further elucidation, the findings of this study indicate that LPS acts through its archetypical receptor in trigeminal and nodose neurons.
Assuntos
Lipopolissacarídeos/metabolismo , Neurônios/metabolismo , Gânglio Nodoso/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Superfície/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Fator Neurotrófico Ciliar/metabolismo , Fator Inibidor de Leucemia/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
In cultured neurons, axon formation is preceded by the appearance in one of the multiple neurites of a large growth cone containing a labile actin network and abundant dynamic microtubules. The invasion-inducing T-lymphoma and metastasis 1 (Tiam1) protein that functions as a guanosine nucleotide exchange factor for Rac1 localizes to this neurite and its growth cone, where it associates with microtubules. Neurons overexpressing Tiam1 extend several axon-like neurites, whereas suppression of Tiam1 prevents axon formation, with most of the cells failing to undergo changes in growth cone size and in cytoskeletal organization typical of prospective axons. Cytochalasin D reverts this effect leading to multiple axon formation and penetration of microtubules within neuritic tips devoid of actin filaments. Taken together, these results suggest that by regulating growth cone actin organization and allowing microtubule invasion within selected growth cones, Tiam1 promotes axon formation and hence participates in neuronal polarization.
Assuntos
Axônios/fisiologia , Proteínas/fisiologia , Células Piramidais/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Polaridade Celular/fisiologia , Células Cultivadas , Imunofluorescência , Cones de Crescimento/fisiologia , Fatores de Troca do Nucleotídeo Guanina , Microtúbulos/fisiologia , Proteínas de Neoplasias , Ratos , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células TRESUMO
Cultures of cerebellar macroneurons were used to study the pattern of expression, subcellular localization, and function of the neuronal cdk5 activator p35 during laminin-enhanced axonal growth. The results obtained indicate that laminin, an extracellular matrix molecule capable of selectively stimulating axonal extension and promoting MAP1B phosphorylation at a proline-directed protein kinase epitope, selectively stimulates p35 expression, increases its association with the subcortical cytoskeleton, and accelerates its redistribution to the axonal growth cones. Besides, suppression of p35, but not of a highly related isoform designated as p39, by antisense oligonucleotide treatment selectively reduces cdk5 activity, laminin-enhanced axonal elongation, and MAP1b phosphorylation. Taken collectively, the present results suggest that cdk5/p35 may serve as an important regulatory linker between environmental signals (e.g., laminin) and constituents of the intracellular machinery (e.g., MAP1B) involved in axonal elongation.
Assuntos
Axônios/fisiologia , Quinases Ciclina-Dependentes , Laminina/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Anticorpos , Elementos Antissenso (Genética) , Axônios/química , Células Cultivadas , Cerebelo/citologia , Quinase 5 Dependente de Ciclina , Epitopos/fisiologia , Imunofluorescência , Regulação Enzimológica da Expressão Gênica/fisiologia , Laminina/análise , Laminina/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/citologia , Neurônios/ultraestrutura , Fosforilação , RNA Mensageiro/análise , CoelhosRESUMO
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
GD3 and GM2 synthases act on ganglioside GM3 at the branching point of the pathway of synthesis of gangliosides in which the "a", "b" and "c" families are produced. The relative activities of these enzymes are important for regulating the ganglioside composition of a given tissue. In the present work, we report the cloning and characterization of a chick GD3 synthase cDNA. The cloned cDNA directed the synthesis of a functionally active enzyme in transiently transfected CHO-K1 cells and was highly homologous to mammalian GD3 synthases. In Northern blot experiments the cDNA detected a single specific GD3 synthase mRNA of about 9.0 kb both in the chicken brain and retina. The abundance of the specific mRNA transcript declined steadily from E7-E9 to very low values around PN2. The levels of enzyme activities measured at the same developmental stages roughly followed the changes of specific mRNA levels in both tissues. In situ hybridization of embryonic neural retina cells in culture showed that both glial- and neuron-like cells expressed the specific GD3 synthase mRNA, although with different intensities. Results indicate that transcription and/or stability of the specific GD3 synthase mRNA constitute a level of control of the expression of GD3 synthase and indirectly of the ganglioside composition in the developing chicken central nervous system (CNS).