RESUMO
Diabetic neuropathy (DNP), afflicting sensory and motor nerve fibers, is a major complication in diabetes. The underlying cellular mechanisms of axon degeneration are poorly understood. IGFBP5, an inhibitory binding protein for insulin-like growth factor 1 (IGF1) is highly up-regulated in nerve biopsies of patients with DNP. We investigated the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP5 in motor axons and sensory nerve fibers. These mice develop motor axonopathy and sensory deficits similar to those seen in DNP. Motor axon degeneration was also observed in mice in which the IGF1 receptor (IGF1R) was conditionally depleted in motoneurons, indicating that reduced activity of IGF1 on IGF1R in motoneurons is responsible for the observed effect. These data provide evidence that elevated expression of IGFBP5 in diabetic nerves reduces the availability of IGF1 for IGF1R on motor axons, thus leading to progressive neurodegeneration. Inhibition of IGFBP5 could thus offer novel treatment strategies for DNP.
Assuntos
Axônios/fisiologia , Proteínas de Transporte/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Neuropatias Diabéticas/fisiopatologia , Neurônios Motores/fisiologia , Degeneração Neural/fisiopatologia , Animais , Axônios/patologia , Crescimento Celular , Sobrevivência Celular/fisiologia , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Neuropatias Diabéticas/patologia , Humanos , Camundongos Transgênicos , Atividade Motora/fisiologia , Neurônios Motores/patologia , Degeneração Neural/patologia , Nervo Frênico/patologia , Nervo Frênico/fisiopatologia , Receptor IGF Tipo 1/metabolismo , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia , Sensação/fisiologiaRESUMO
Insulin-like growth factor I (IGF-I) has been successfully tested in the SOD1-G93A mouse model of familial amyotrophic lateral sclerosis (ALS) and proposed for clinical treatment. However, beneficial effects required gene therapy or intrathecal application. Circumventing the dosing issues we recently found that polyethylene glycol (PEG) modified IGF-I (PEG-IGF-I) modulated neuromuscular function after systemic application, and protected against disease progression in a motor neuron disease model. Here we investigated its effects in two SOD1-G93A mouse lines, the G1L with a milder and the G1H with a more severe phenotype. Results showed that in G1L mice, PEG-IGF-I treatment significantly improved muscle force, motor coordination and animal survival. In contrast, treatment of G1H mice with PEG-IGF-I or IGF-I even at high doses did not beneficially affect survival or functional outcomes despite increased signalling in brain and spinal cord by both agents. In conclusion, the data point towards further investigation of the therapeutic potential of PEG-IGF-I in ALS patients with less severe clinical phenotypes.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Fator de Crescimento Insulin-Like I/uso terapêutico , Neurônios Motores/efeitos dos fármacos , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Índice de Gravidade de DoençaRESUMO
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). So far, immunological mechanisms responsible for demyelination have been the focus of interest. However, mechanisms regulating axon maintenance as well as glial precursor-cell proliferation and oligodendrocyte survival might also influence disease outcome. The cytokine ciliary neurotrophic factor (CNTF), which was originally identified as a survival factor for isolated neurons, promotes differentiation, maturation and survival of oligodendrocytes. To investigate the role of endogenous CNTF in inflammatory demyelinating disease, we studied myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in CNTF-deficient and wild-type C57BL/6 mice. Disease was more severe in CNTF-deficient mice and recovery was poor, with a 60% decrease in the number of proliferating oligodendrocyte precursor cells (OPCs) and a more than 50% increase in the rate of oligodendrocyte apoptosis. In addition, vacuolar dystrophy of myelin and axonal damage were more severe in CNTF-deficient mice. These specific pathological features could be prevented by treatment with an antiserum against tumor necrosis factor-alpha, suggesting that endogenous CNTF may counterbalance this effect of TNF-alpha (ref. 7). Here we identify a factor that modulates, in an inflammatory environment, glial cell survival and is an outcome determinant of EAE.
Assuntos
Fator Neurotrófico Ciliar/fisiologia , Doenças Desmielinizantes/prevenção & controle , Encefalomielite Autoimune Experimental/patologia , Esclerose Múltipla/prevenção & controle , Fatores de Crescimento Neural/fisiologia , Animais , Fator Neurotrófico Ciliar/deficiência , Fator Neurotrófico Ciliar/genética , Citocinas/fisiologia , Inflamação/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Mutations in Ribosomal s6 kinase 2 (Rsk2) are associated with severe neuronal dysfunction in Coffin-Lowry syndrome (CLS) patients, flies and mice. So far, the mechanisms of how Rsk2 regulates development, maintenance and activity of neurons are not understood. We have investigated the consequences of Rsk2 deficiency in mouse spinal motoneurons. Survival of isolated Rsk2 deficient motoneurons is not reduced, but these cells grow significantly longer neurites. Conversely, overexpression of a constitutively active form of Rsk2 leads to reduced axon growth. Increased axon growth in Rsk2 deficient neurons was accompanied by higher Erk 1/2 phosphorylation, and the knockout phenotype could be rescued by pharmacological inhibition of MAPK/Erk kinase (Mek). These data indicate that Rsk2 negatively regulates axon elongation via the MAPK pathway. Thus, the functional defects observed in the nervous system of CLS patients and animal models with Rsk2 deficiency might be caused by dysregulated neurite growth rather than primary neurodegeneration.
Assuntos
Axônios/fisiologia , Neurônios Motores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Sobrevivência Celular , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Medula Espinal/citologiaRESUMO
Members of the ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/cardiotrophin gene family are potent survival factors for embryonic and lesioned motoneurons. These factors act via receptor complexes involving gp130 and LIFR-beta and ligand binding leads to activation of various signaling pathways, including phosphorylation of Stat3. The role of Stat3 in neuronal survival was investigated in mice by Cre-mediated gene ablation in motoneurons. Cre is expressed under the neurofilament light chain (NF-L) promoter, starting around E12 when these neurons become dependent on neurotrophic support. Loss of motoneurons during the embryonic period of naturally occurring cell death is not enhanced in NF-L-Cre; Stat3(flox/KO) mice although motoneurons isolated from these mice need higher concentrations of CNTF for maximal survival in culture. In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult. These neurons, however, can be rescued by the addition of neurotrophic factors, including CNTF. Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons. Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Traumatismos do Nervo Facial/patologia , Neurônios Motores/citologia , Proteínas do Tecido Nervoso , Sistema Nervoso/embriologia , Sistema Nervoso/patologia , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Envelhecimento , Animais , Animais Recém-Nascidos , Axotomia , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Fator Neurotrófico Ciliar/farmacologia , Traumatismos do Nervo Facial/genética , Traumatismos do Nervo Facial/metabolismo , Deleção de Genes , Integrases/genética , Integrases/metabolismo , Litostatina , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Proteínas de Neurofilamentos/genética , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3 , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteína bcl-XRESUMO
Bag1 is a cochaperone for the heat-shock protein Hsp70 that interacts with C-Raf, B-Raf, Akt, Bcl-2, steroid hormone receptors and other proteins. Here we use targeted gene disruption in mice to show that Bag1 has an essential role in the survival of differentiating neurons and hematopoietic cells. Cells of the fetal liver and developing nervous system in Bag1-/- mice underwent massive apoptosis. Lack of Bag1 did not disturb the primary function of Akt or Raf, as phosphorylation of the forkhead transcription factor FKHR and activation of extracellular signal-regulated kinase (Erk)-1/2 were not affected. However, the defect was associated with the disturbance of a tripartite complex formed by Akt, B-Raf and Bag1, in addition to the absence of Bad phosphorylation at Ser136. We also observed reduced expression of members of the inhibitor of apoptosis (IAP) family. Our data show that Bag1 is a physiological mediator of extracellular survival signals linked to the cellular mechanisms that prevent apoptosis in hematopoietic and neuronal progenitor cells.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Membrana/fisiologia , Neurônios Motores/fisiologia , Medula Espinal/citologia , Fosfatase Alcalina/metabolismo , Análise de Variância , Animais , Southern Blotting/métodos , Western Blotting/métodos , Proteínas de Transporte/metabolismo , Contagem de Células/métodos , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Embrião de Mamíferos , Proteínas do Olho/metabolismo , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP70/deficiência , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Proteínas de Filamentos Intermediários/metabolismo , Isoenzimas/metabolismo , Fígado/citologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Mutação , Proteínas do Tecido Nervoso/metabolismo , Nestina , Proteínas de Neurofilamentos/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Propídio , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Fatores de Transcrição , Transfecção/métodos , Proteína de Morte Celular Associada a bclRESUMO
The Hey basic helix-loop-helix transcription factors are downstream effectors of Notch signaling in the cardiovascular system. Mice lacking Hey2 develop cardiac hypertrophy, often associated with congenital heart defects, whereas combined Hey1/Hey2 deficiency leads to severe vascular defects and embryonic lethality around embryonic day E9.5. The molecular basis of these disorders is poorly understood, however, since target genes of Hey transcription factors in the affected tissues remain elusive. To identify genes regulated by Hey factors we have generated a conditional Hey1 knockout mouse. This strain was used to generate paired Hey2- and Hey1/2-deficient embryonic stem cell lines. Comparison of these cell lines by microarray analysis identified GATA4 and GATA6 as differentially expressed genes. Loss of Hey1/2 leads to elevated GATA4/6 and ANF mRNA levels in embryoid bodies, while forced expression of Hey factors strongly represses expression of the GATA4 and GATA6 promoter in various cell lines. In addition, the promoter activity of the GATA4/6 target gene ANF was inhibited by Hey1, Hey2, and HeyL. Protein interaction and mutation analyses suggest that repression is due to direct binding of Hey proteins to GATA4 and GATA6, blocking their transcriptional activity. In Hey2-deficient fetal hearts we observed elevated mRNA levels of ANF and CARP. Expression of ANF and Hey2 is normally restricted to the trabecular and compact myocardial layer, respectively. Intriguingly, loss of Hey2 leads to ectopic ANF expression in the compact layer, suggesting a direct role for Hey2 in limiting ANF expression in this cardiac compartment.
Assuntos
Fator Natriurético Atrial/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Coração Fetal/metabolismo , Fator de Transcrição GATA4/genética , Proteínas Repressoras/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , DNA Complementar/genética , Regulação da Expressão Gênica , Marcação de Genes , Sequências Hélice-Alça-Hélice/genética , Histona Desacetilases/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Musculares , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genéticaRESUMO
Leukemia inhibitory factor (LIF) receptor beta (LIFRbeta) is a receptor for a variety of neurotrophic cytokines, including LIF, ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1). These cytokines play an essential role for the survival and maintenance of developing and postnatal somatic motoneurons. CNTF may also serve the maintenance of autonomic, preganglionic sympathetic neurons (PSNs) in the spinal cord, as suggested by its capacity to prevent their death after destruction of one of their major targets, the adrenal medulla. Although somatic motoneurons and PSNs share a common embryonic origin, they are distinct in several respects, including responses to lesions. We have studied PSNs in mice with targeted deletions of the LIFRbeta or CT-1 genes, respectively. We show that LIF, CNTF, and CT-1 are synthesized in embryonic adrenal gland and spinal cord and that PSNs express LIFRbeta. In embryonic day 18.5 LIFRbeta (-/-) and CT-1 (-/-) mice, PSNs were reduced by approximately 20%. PSNs projecting to the adrenal medulla were more severely affected (-55%). Although LIFRbeta (-/-) mice revealed normal numbers of adrenal chromaffin cells and axons terminating on chromaffin cells, levels of adrenaline and numbers of adrenaline-synthesizing cells were significantly reduced. We conclude that activation of LIFRbeta is required for normal development of PSNs and one of their prominent targets, the adrenal medulla. Thus, both somatic motoneurons and PSNs in the spinal cord depend on LIFRbeta signaling for their development and maintenance, although PSNs seem to be overall less affected than somatic motoneurons by LIFRbeta deprivation.
Assuntos
Medula Suprarrenal/fisiologia , Citocinas/fisiologia , Interleucina-6/fisiologia , Fibras Nervosas/fisiologia , Neurônios/fisiologia , Actinas/fisiologia , Medula Suprarrenal/efeitos dos fármacos , Medula Suprarrenal/inervação , Animais , Benzilaminas/farmacologia , Citocinas/deficiência , Citocinas/genética , Maleato de Dizocilpina/farmacologia , Hipocampo/fisiologia , Interleucina-6/deficiência , Interleucina-6/genética , Fator Inibidor de Leucemia , Camundongos , Fibras Nervosas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oligopeptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologia , Sistema Nervoso Simpático/embriologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Membranas Sinápticas/efeitos dos fármacos , Membranas Sinápticas/fisiologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/fisiologiaRESUMO
Members of the ciliary neurotrophic factor (CNTF)-leukemia inhibitory factor (LIF) gene family play an essential role for survival of developing and postnatal motoneurons. When subunits of the shared receptor complex are inactivated by homologous recombination, the mice die at approximately birth and exhibit reduced numbers of motoneurons in the spinal cord and brainstem nuclei. However, mice in which cntf, lif, or cardiotrophin-1 (ct-1) are inactivated can survive and show less motoneuron cell loss. This suggests cooperative and redundant roles of these ligands. However, their cooperative functions are not well understood. We generated cntf/lif/ct-1 triple-knock-out and combinations of double-knock-out mice to study the individual and combined roles of CNTF, LIF and CT-1 on postnatal motoneuron survival and function. Triple-knock-out mice exhibit increased motoneuron cell loss in the lumbar spinal cord that correlates with muscle weakness during early postnatal development. LIF deficiency leads to pronounced loss of distal axons and motor endplate alterations, whereas CNTF-and/or CT-1-deficient mice do not show significant changes in morphology of these structures. In cntf/lif/ct-1 triple-knock-out mice, various degrees of muscle fiber type grouping are found, indicating that denervation and reinnervation had occurred. We conclude from these findings that CNTF, LIF, and CT-1 have distinct functions for motoneuron survival and function and that LIF plays a more important role for postnatal maintenance of distal axons and motor endplates than CNTF or CT-1.
Assuntos
Fator Neurotrófico Ciliar/fisiologia , Citocinas/fisiologia , Interleucina-6/fisiologia , Neurônios Motores/fisiologia , Fibras Musculares Esqueléticas/patologia , Debilidade Muscular/fisiopatologia , Animais , Fator Neurotrófico Ciliar/deficiência , Fator Neurotrófico Ciliar/genética , Citocinas/deficiência , Citocinas/genética , Força da Mão , Interleucina-6/genética , Fator Inibidor de Leucemia , Camundongos , Camundongos Knockout , Placa Motora/ultraestrutura , Denervação Muscular , Debilidade Muscular/genética , Debilidade Muscular/patologia , Degeneração Neural , Nervos Periféricos/patologia , Medula Espinal/patologiaRESUMO
The microtubule associated tumor suppressor gene 1 (MTUS1) is a recently published tumor suppressor gene, which has also been shown to act as an early component in the growth inhibitory signaling cascade of the angiotensin II type 2 receptor (AT2R). In this study we report the generation of MTUS1 knock-out (KO) mice, which develop normally but reveal higher body weights and slightly decreased blood pressure levels. Twenty-eight percent of the studied MTUS1 KO mice also developed heart hypertrophy and 12% developed nephritis, independent of blood pressure levels. Forty-three percent of the MTUS1 KO mice revealed lymphoid hyperplasia affecting spleen (20%), kidney (37%), lung (23%), lymph nodes (17%), and liver (17%) accompanied with leukocytosis, lymphocytosis, and mild anemia. One animal (3%) developed a marginal zone B-cell lymphoma affecting submandibular salivary gland and regional lymph nodes. The symptoms of all mentioned animals are consistent with a B-cell lymphoproliferative disease with features of systemic lupus erythematosus. In addition, body weight of the MTUS1 KO mice was significantly increased and isolated skin fibroblasts showed increased cell proliferation and decreased cell size, compared to wild-type (WT) fibroblasts in response to depleted FCS concentration and lack of growth factors. In conclusion we herein report the first generation of a MTUS1 KO mouse, developing spontaneous heart hypertrophy and increased cell proliferation, confirming once more the anti-proliferative effect of MTUS1, and a SLE-like lymphoproliferative disease suggesting crucial role in regulation of inflammation. These MTUS1 KO mice can therefore serve as a model for further investigations in cardiovascular disease, autoimmune disease and carcinogenesis.
Assuntos
Cardiomegalia/patologia , Linfoma de Zona Marginal Tipo Células B/patologia , Transtornos Linfoproliferativos/patologia , Proteínas Supressoras de Tumor/deficiência , Animais , Cardiomegalia/sangue , Cardiomegalia/genética , Cardiomegalia/metabolismo , Proteínas de Transporte/genética , Processos de Crescimento Celular/fisiologia , Fibroblastos/patologia , Imuno-Histoquímica , Linfoma de Zona Marginal Tipo Células B/sangue , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma de Zona Marginal Tipo Células B/metabolismo , Transtornos Linfoproliferativos/sangue , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/metabolismo , Camundongos , Camundongos Knockout , Pele/patologia , Proteínas Supressoras de Tumor/genéticaRESUMO
Based on its potent neurotrophic and myotrophic activities, insulin-like growth factor I (IGF-I) has been proposed for treatment of neuromuscular disorders such as muscular dystrophies and amyotrophic lateral sclerosis (ALS). However, the short half life in the circulation limits its use in vivo. At least in mouse models, beneficial effects are generally only observed by dosing regimens such as minipumps or gene therapy that are difficult to translate to patients. We have developed a polyethylene glycol coupled IGF-I (PEG-IGF-I) that could circumvent these problems by longer half-life, showing all features of a therapeutic agent supporting muscular and neuronal function. Here we investigated its effects in the pmn mutant mouse, a model with typical dying-back motoneuron degeneration. In vitro, PEG-IGF-I and rhIGF-I profoundly promoted survival axonal growth of wild-type as well as pmn mutant embryonic motoneurons, suggesting that PEG-IGF-I had a fully conserved neurotrophic activity via its receptor. In vivo, treatment of pmn mutant mice with PEG-IGF-I prolonged survival, protected against late stage weight loss and significantly maintained muscle force and motor coordination. Consistently, PEG-IGF-I treatment rescued facial and lumbar motoneurons from cell death and partially preserved phrenic nerve myelinated axons. The data support that PEG-IGF-I could be used for treatment of neuromuscular diseases in a clinically feasible manner.
Assuntos
Desenho de Fármacos , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/farmacocinética , Doença dos Neurônios Motores/tratamento farmacológico , Neurônios Motores/efeitos dos fármacos , Polietilenoglicóis/farmacocinética , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Feto/citologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Neurônios Motores/citologia , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Nervo Frênico/citologia , Gravidez , Proteínas Recombinantes/farmacologia , Medula Espinal/citologiaRESUMO
The relative contribution of the two tryptophan hydroxylase (TPH) isoforms, TPH1 and TPH2, to brain serotonergic system function is controversial. To investigate the respective role of TPH2 in neuron serotonin (5-HT) synthesis and the role of 5-HT in brain development, mice with a targeted disruption of Tph2 were generated. The preliminary results indicate that in Tph2 knockout mice raphe neurons are completely devoid of 5-HT, whereas no obvious alteration in morphology and fiber distribution are observed. The findings confirm the exclusive specificity of Tph2 in brain 5-HT synthesis and suggest that Tph2-synthesized 5-HT is not required for serotonergic neuron formation.
Assuntos
Química Encefálica/genética , Encéfalo/citologia , Neurônios/fisiologia , Serotonina/deficiência , Triptofano Hidroxilase/fisiologia , Animais , Deleção de Genes , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Núcleos da Rafe/citologia , Núcleos da Rafe/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serotonina/biossíntese , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Triptofano Hidroxilase/genéticaRESUMO
The neurotrophic cytokines ciliary neurotrophic factor and leukemia inhibitory factor (LIF) play a key role in neuronal and oligodendrocyte survival and as protective factors in neuroinflammation. To further elucidate the potential of endogenous LIF in modulating neuroinflammation, we studied myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LIF knockout mice (LIF(-/-) mice). In the late phase of active myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, LIF(-/-) mice exhibited a markedly milder disease course. The inflammatory infiltrate in LIF(-/-) mice was characterized by an increase in neutrophilic granulocytes early and fewer infiltrating macrophages associated with less demyelination later in the disease. In good correlation with an effect of endogenous LIF on the immune response, we found an Ag-specific T cell-priming defect with impaired IFN-gamma production in LIF(-/-) mice. On the molecular level, the altered recruitment of inflammatory cells is associated with distinct patterns of chemokine production in LIF(-/-) mice with an increase of CXCL1 early and a decrease of CCL2, CCL3, and CXCL10 later in the disease. These data reveal that endogenous LIF is an immunologically active molecule in neuroinflammation. This establishes a link between LIF and the immune system which was not observed in the ciliary neurotrophic factor knockout mouse.
Assuntos
Quimiocinas/fisiologia , Quimiotaxia de Leucócito/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Fator Inibidor de Leucemia/deficiência , Fator Inibidor de Leucemia/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Predisposição Genética para Doença , Glicoproteínas/imunologia , Glicoproteínas/toxicidade , Fator Inibidor de Leucemia/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Receptores de OSM-LIF/biossíntese , Receptores de OSM-LIF/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Neurotrophins are potent survival factors for developing and injured neurons. However, they are not being used to treat neurodegenerative diseases because of difficulties in administration and numerous side effects that have been encountered in previous clinical trials. Their biological activities use Trk (tropomyosin-related kinase) transmembrane tyrosine kinases. Therefore, one alternative approach is to use transactivation pathways such as adenosine 2A receptor agonists, which can activate Trk receptor signaling independent of neurotrophin binding. However, the relevance in vivo and applicability of these transactivation events during neurodegenerative and injury conditions have never been extensively studied. Here we demonstrate that motoneuron survival after facial nerve lesioning is significantly enhanced by transactivation of Trk receptor tyrosine kinases by adenosine agonists. Moreover, survival of motoneurons directly required the activation of the BDNF receptor TrkB and an increase in Akt (AKT8 virus oncogene cellular homolog) activity. The ability of small molecules to activate a trophic response by using Trk signaling provides a unique mechanism to promote survival signals in motoneurons and suggests new strategies for using transactivation in neurodegenerative diseases.
Assuntos
Neurônios Motores/citologia , Neurônios Motores/enzimologia , Receptor trkB/genética , Receptores A2 de Adenosina/metabolismo , Ativação Transcricional/genética , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina , Animais , Axotomia , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/enzimologia , Ativação Enzimática/efeitos dos fármacos , Nervo Facial/efeitos dos fármacos , Nervo Facial/patologia , Camundongos , Neurônios Motores/efeitos dos fármacos , Fenetilaminas/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
UNLABELLED: We previously reported that exogenous cardiotrophin-1 (CT-1), a member of the IL-6 family of cytokines, exerts hepatoprotective effects. Because CT-1 is expressed in the normal liver, we hypothesized that this cytokine may constitute an endogenous defense of the liver against proapoptotic stimuli. Here, we found that CT-1-/- mice died faster than wild-type animals after challenge with a lethal dose of the Fas agonist Jo-2. At sublethal doses of Jo-2, all wild-type mice survived whereas CT-1-/- animals developed extensive hepatocyte apoptosis with 50% mortality at 24 hours. Pretreatment with CT-1 improved survival and reduced injury in both CT-1-/- and wild-type animals. Upon Fas ligation the activation of STAT-3, a molecule that defends the liver against apoptosis, was lower in CT-1-/- mice than in wild-type animals despite similar IL-6 up-regulation in the 2 groups. Analysis of liver transcriptome in CT-1-/- and wild-type mice showed that 9 genes reported to be associated with cell survival/death functions were differentially expressed in the 2 groups. Four of these genes [IGFBP1, peroxiredoxin3, TNFR1, and calpastatin (endogenous inhibitor of calpain)] could be validated by real-time PCR. All of them were down-regulated in CT-1-/- mice and were modulated by CT-1 administration. Treatment of CT-1-/- animals with the calpain inhibitor MDL28170 afforded significant protection against Fas-induced liver injury. CONCLUSION: CT-1-/- mice are highly sensitive to Fas-mediated apoptosis due in part to deficient STAT-3 activation and inadequate control of calpain activity during the apoptotic process. Our data show that CT-1 is a natural defense of the liver against apoptosis. This cytokine may have therapeutic potential.
Assuntos
Apoptose/fisiologia , Citocinas/fisiologia , Hepatócitos/fisiologia , Fígado/patologia , Animais , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/fisiologia , Citocinas/genética , Regulação da Expressão Gênica , Hepatócitos/patologia , Fígado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxidases/genética , Peroxidases/fisiologia , Peroxirredoxina III , Peroxirredoxinas , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/fisiologia , Receptor fas/imunologiaRESUMO
The transcription factor Sox10 regulates early neural crest development, specification of neural crest-derived lineages and terminal differentiation of oligodendrocytes in the central nervous system. Here, we generated two novel hypomorphic Sox10 alleles in the mouse. Mutant mice either expressed a Sox10 protein with a triple alanine substitution in the dimerization domain, or a Sox10 protein with a deletion in the central portion that we define as a cell-specific transactivation domain. Phenotypic analysis revealed important roles for a functional dimerization domain and the newly defined novel transactivation domain in melanocyte and enteric nervous system development, whereas early neural crest development and oligodendrocyte differentiation were surprisingly little disturbed in both mutants. Unique requirements were additionally detected for the novel transactivation domain in satellite glia differentiation and during Schwann cell myelination, whereas DNA-dependent dimerization was needed for immature Schwann cells to enter the promyelinating stage. These two hypomorphic alleles thus uncover novel functions of Sox10 in satellite glia and Schwann cells during late developmental stages and reveal important developmental differences between these two types of peripheral glia and oligodendrocytes regarding their reliance on Sox10.
Assuntos
Linhagem da Célula , Proteínas de Grupo de Alta Mobilidade/fisiologia , Neuroglia/fisiologia , Sistema Nervoso Periférico/embriologia , Fatores de Transcrição/fisiologia , Alelos , Animais , Linhagem da Célula/genética , Dimerização , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Melanócitos/fisiologia , Camundongos , Camundongos Mutantes , Mutação , Bainha de Mielina , Neuroglia/citologia , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Fatores de Transcrição SOXE , Células de Schwann , Deleção de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Recent evidence has shown that the activation of receptor tyrosine kinases is not only dependent on binding of their ligands but in addition requires adhesion molecules as coreceptors. We have identified CD44v6 as a coreceptor for c-Met in several tumor and primary cells. The CD44v6 ectodomain is required for c-Met activation, whereas the cytoplasmic tail recruits ERM proteins and the cytoskeleton into a signalosome complex. Here we demonstrate that c-Met (and hepatocyte growth factor and Gab1) is haploinsufficient in a cd44-/- background, as the cd44-/-; met+/- (and cd44-/-; hgf+/- and cd44-/-; gab1+/-) mice die at birth. They have impaired synaptic transmission in the respiratory rhythm-generating network and alterations in the phrenic nerve. These results are the first genetic data showing that CD44 and c-Met collaborate in vivo and that they are involved in synaptogenesis and axon myelination in the central and peripheral nervous systems.
Assuntos
Haploidia , Receptores de Hialuronatos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Fator de Crescimento de Hepatócito/metabolismo , Pulmão/anormalidades , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/patologia , Fibras Nervosas/patologia , Nervo Frênico/patologia , Nervo Frênico/ultraestrutura , Transmissão SinápticaRESUMO
Transgenic mice expressing the superoxide dismutase G93A mutation (SOD1(G93A)) were used to investigate the role of glial inwardly rectifying K(+) (Kir)4.1 channels, which buffer extracellular K(+) increases in response to neuronal excitation. A progressive decrease in Kir4.1 immunoreactivity was observed predominantly in the ventral horn of SOD1(G93A) mutants. Immunoblotting of spinal cord extracts mirrored these changes by showing a loss of Kir4.1 channels from presymptomatic stages onwards. Kir4.1 channels were found to be expressed in the spinal cord grey matter, targetting astrocytes and clustering around capillaries, supporting their role in clearance of extracellular K(+). To understand the functional implications of extracellular K(+) increases, we challenged the NSC34 motor neurone cell line with increasing extracellular K(+) concentrations. Exposure to high extracellular K(+) induced progressive motor neurone cell death. We suggest that loss of Kir4.1 impairs perineural K(+) homeostasis and may contribute to motor neurone degeneration in SOD1(G93A) mutants by K(+) excitotoxic mechanisms.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Neuroglia/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Medula Espinal/metabolismo , Superóxido Dismutase/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Aquaporina 4/metabolismo , Astrócitos/metabolismo , Western Blotting , Capilares/citologia , Capilares/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Espaço Extracelular/metabolismo , Imunofluorescência , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Potássio/metabolismo , Medula Espinal/citologia , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
Neuronal degeneration in spinal muscular atrophy is caused by reduced expression of the survival motor neuron (SMN) protein. SMN and the tightly interacting Gemin2 form part of a macromolecular complex (SMN complex) that mediates assembly of spliceosomal small nuclear ribonucleoproteins (U snRNPs). We used mouse genetics to investigate the function of this complex in motoneuron maintenance. Reduced Smn/Gemin2 protein levels lead to disturbed U snRNP assembly as indicated by reduced nuclear accumulation of Sm proteins. This finding correlates with enhanced motoneuron degeneration in Gemin2(+/-)/Smn(+/-) mice. Our data provide in vivo evidence that impaired production of U snRNPs contributes to motoneuron degeneration.
Assuntos
Neurônios Motores/patologia , Proteínas do Tecido Nervoso/genética , Animais , Morte Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Modelos Animais de Doenças , Marcação de Genes , Camundongos , Camundongos Knockout , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Proteínas de Ligação a RNA , Mapeamento por Restrição , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas do Complexo SMNRESUMO
Selenoprotein P (SePP), the major selenoprotein in plasma, has been implicated in selenium transport, selenium detoxification or antioxidant defence. We generated SePP-knockout mice that were viable, but exhibited reduced growth and developed ataxia. Selenium content was elevated in liver, but low in plasma and other tissues, and selenoenzyme activities changed accordingly. Our data reveal that SePP plays a pivotal role in delivering hepatic selenium to target tissues.