RESUMO
We demonstrate that EphB3 receptors mediate oligodendrocyte (OL) cell death in the injured spinal cord through dependence receptor mechanism. OLs in the adult spinal cord express EphB3 as well as other members of the Eph receptor family. Spinal cord injury (SCI) is associated with tissue damage, cellular loss and disturbances in EphB3-ephrinB3 protein balance acutely (days) after the initial impact creating an environment for a dependence receptor-mediated cell death to occur. Genetic ablation of EphB3 promotes OL survival associated with increased expression of myelin basic protein and improved locomotor function in mice after SCI. Moreover, administration of its ephrinB3 ligand to the spinal cord after injury also promotes OL survival. Our in vivo findings are supported by in vitro studies showing that ephrinB3 administration promotes the survival of both oligodendroglial progenitor cells and mature OLs cultured under pro-apoptotic conditions. In conclusion, the present study demonstrates a novel dependence receptor role of EphB3 in OL cell death after SCI, and supports further development of ephrinB3-based therapies to promote recovery.
Assuntos
Apoptose , Oligodendroglia/fisiologia , Receptor EphB3/fisiologia , Traumatismos da Medula Espinal/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Efrina-B3/farmacologia , Efrina-B3/uso terapêutico , Feminino , Camundongos Knockout , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologiaRESUMO
The central nervous system (CNS) of higher organisms is bilaterally-symmetric. The transfer of information between the two sides of the nervous system occurs through commissures formed by neurons that project axons across the midline to the contralateral side of the CNS. Interestingly, these axons cross the midline only once. Other neurons extend axons that never cross the midline; they project exclusively on their own (ipsilateral) side of the CNS. Thus, the midline is an important choice point for several classes of pathfinding axons. Recent studies demonstrate that specialized midline cells play critical roles in regulating the guidance of both crossing and non-crossing axons at the ventral midline of the developing vertebrate spinal cord and the Drosophila ventral nerve cord. For example, these cells secrete attractive cues that guide commissural axons over long distances to the midline of the CNS. Furthermore, short-range interactions between guidance cues present on the surfaces of midline cells, and their receptors expressed on the surfaces of pathfinding axons, allow commissural axons to cross the midline only once and prevent ipsilaterally-projecting axons from entering the midline. Remarkably, the molecular composition of commissural axon surfaces is dynamically-altered as they cross the midline. Consequently, commissural axons become responsive to repulsive midline guidance cues that they had previously ignored on the ipsilateral side of the midline. Concomitantly, commissural axons lose responsiveness to attractive guidance cues that had initially attracted them to the midline. Thus, these exquisitely regulated guidance systems prevent commissural axons from lingering within the confines of the midline and allow them to pioneer an appropriate pathway on the contralateral side of the CNS. Many aspects of midline guidance are controlled by mechanistically and evolutionarily-conserved ligand-receptor systems. Strikingly, recent studies demonstrate that these receptors are modular; the ectodomains determine ligand recognition and the cytoplasmic domains specify the response of an axon to a given guidance cue. Despite rapid and dramatic progress in elucidating the molecular mechanisms that control midline guidance, many questions remain.
Assuntos
Axônios/metabolismo , Axônios/fisiologia , Sistema Nervoso Central/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Caenorhabditis elegans , Drosophila , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Mutação , Fatores de Crescimento Neural/metabolismo , Netrina-1 , Fenótipo , Medula Espinal/embriologia , Proteínas Supressoras de TumorRESUMO
Bilaterally symmetric animals must be capable of transmitting information between the left and right sides of their body to integrate sensory input and to coordinate motor control. Thus, many neurons in the central nervous system (CNS) of a wide variety of higher organisms project so-called commissural axons across the midline. Interestingly, these axons are never observed to re-cross the midline. On the other hand, some neurons project axons that remain on their own (ipsilateral) side of the CNS, without ever crossing the midline. Recent studies demonstrate that specialized cells which reside at the ventral midline of the developing vertebrate spinal cord and Drosophila ventral nerve cord play critical roles in regulating the guidance of both crossing and non-crossing axons. For example, these cells secrete positively-acting guidance cues that attract commissural axons over long distances to the midline of the CNS. Furthermore, short-range interactions between guidance cues present on the surfaces of midline cells, and their receptors expressed on the surfaces of pathfinding axons, allow commissural axons to cross the midline and prevent ipsilaterally projecting axons from entering the midline. Remarkably, as commissural axons cross over to the opposite side of the CNS, the molecular composition of their surfaces is dynamically altered so that they become responsive to repulsive midline guidance cues that they had previously ignored. Thus, this exquisitely controlled guidance system prevents commissural axons from crossing the midline more than once. Strikingly, many of the molecular mechanisms that control midline guidance appear to be evolutionarily conserved.
Assuntos
Axônios/fisiologia , Sistema Nervoso Central/crescimento & desenvolvimento , Proteínas de Drosophila , Animais , Axônios/metabolismo , Padronização Corporal , Caenorhabditis elegans , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Sistema Nervoso Central/metabolismo , Contactina 2 , Drosophila , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Medula Espinal/crescimento & desenvolvimento , Vertebrados , Proteínas RoundaboutRESUMO
A variety of molecules expressed at the midline of the developing central nervous system (CNS) control multiple aspects of pattern formation and axon guidance. We recently identified monoclonal antibody (mAb) CARO 2 as a novel marker of the ventral midline in the developing rat CNS, and the corresponding antigen as a membrane-associated 28-kDa protein. We report here the isolation of cDNA clones encoding the mAb CARO 2 antigen, which we rename VEMA, for ventral midline antigen. The deduced amino acid sequence of VEMA contains a single transmembrane domain near its N-terminus and several tyrosine-based internalization motifs. These structural features are consistent with the association of VEMA to intracellular membranes. In situ hybridization analyses demonstrate that VEMA mRNA is predominantly expressed at the ventral midline. The restricted distribution of VEMA, as well as several characteristics of its primary structure, suggest a role for this protein in regulating axon guidance in the mammalian CNS.
Assuntos
Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Medula Espinal/embriologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sequência de Bases , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular , Embrião de Mamíferos , Biblioteca Gênica , Humanos , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , RNA Mensageiro/genética , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Medula Espinal/citologia , Medula Espinal/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
Regionally expressed cell surface molecules are thought to mediate contact-dependent interactions that regulate pattern formation and axon pathfinding in the developing vertebrate central nervous system (CNS). We recently isolated monoclonal antibody (mAb) CARO 2 through a screen for positional markers in the developing rat CNS. Between embryonic day (E)11.5 and E13, mAb CARO 2 specifically labels both the floor plate and notochord in the developing spinal cord. In contrast to the distribution of several well-characterized ventral midline markers, mAb CARO 2 labeling is restricted to the apical portion of the floor plate and the outer surface of the notochord. The anterior limit of mAb CARO 2 immunoreactivity corresponds to the midbrain/hindbrain border. Floor plate labeling persists throughout embryogenesis, whereas notochord labeling is not detectable after E13. During later stages of embryonic development (E16-E20) apically restricted floor plate labeling is present only in the rostral spinal cord. In postnatal rats, mAb CARO immunoreactivity is not present in any region of the CNS. Immunoblot analyses show that mAb CARO 2 recognizes an epitope on a 28-kD protein that is enriched in the floor plate, transiently expressed during embryogenesis, and membrane-associated. Consistent with the latter result, mAb CARO 2 labels the surfaces of floor plate cells. These findings suggest that the CARO 2 antigen is a new cell surface marker of the floor plate and notochord which may participate in neural cell patterning and/or axon guidance in the developing rat spinal cord.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Notocorda/imunologia , Medula Espinal/embriologia , Medula Espinal/imunologia , Animais , Anticorpos Monoclonais/análise , Biomarcadores , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/imunologia , Immunoblotting , Imuno-Histoquímica , Proteínas de Membrana/química , Proteínas do Tecido Nervoso/química , Ratos , Fatores de Tempo , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Extensive necrotic death of MSN neuroblastoma cells could be induced after incubation with the calcium ionophore, A23187. The reaction was concentration-dependent and time course-dependent. Levels of the 66 kd/alpha-internexin neurofilament protein (NF-66) and the cognate heat shock protein 70 (Hsc 70) decreased during the Ca2+-activated cell death. Addition of the calcium chelator, ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) restored the normal level of NF-66 and partially that of the Hsc 70. Use of either calpain I or calpain II inhibitor could alleviate the reduction of 66 kd protein during the ionophore treatment whereas only calpain I inhibitor treatment was effective in restoring the normal level of the Hsc 70. Neither of these calpain inhibitors could block the ionophore triggered cell death. EGTA was toxic to cells in a wide range of concentration suggesting a calcium-independent activation of cell death mechanism.
Assuntos
Calcimicina/farmacologia , Proteínas de Choque Térmico HSP70 , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurofibrilas/metabolismo , Neurofibrilas/patologia , Calpaína/antagonistas & inibidores , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Morte Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSC70 , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Leupeptinas/farmacologia , Proteínas do Tecido Nervoso , Neurofibrilas/efeitos dos fármacos , Oligopeptídeos/farmacologia , Células Tumorais CultivadasRESUMO
Cytoplasmic sequestration of wild-type p53 protein occurs in a subset of primary human tumors including breast cancer, colon cancer, and neuroblastoma (NB). The sequestered p53 localizes to punctate cytoplasmic structures that represent large protein aggregates. One functional consequence of this blocked nuclear access is impairment of the p53-mediated G1 checkpoint after DNA damage. Here we show that cytoplasmic p53 from NB cells is incompetent for specific DNA binding, probably due to its sequestration. Importantly, the C-terminal domain of sequestered p53 is masked, as indicated by the failure of a C-terminally directed antibody to detect p53 in these structures. To determine (i) which domain of p53 is involved in the aggregation and (ii) whether this phenotype is potentially reversible, we generated stable NB sublines that coexpress the soluble C-terminal mouse p53 peptide DD1 (amino acids 302-390). A dramatic phenotypic reversion occurred in five of five lines. The presence of DD1 blocked the sequestration of wild-type p53 and relocated it to the nucleus, where it accumulated. The nuclear translocation is due to shuttling of wild-type p53 by heteroligomerization to DD1, as shown by coimmunoprecipitation. As expected, the nuclear heterocomplexes were functionally inactive, since DD1 is a dominant negative inhibitor of wild-type p53. In summary, we show that nuclear access of p53 can be restored in NB cells.