RESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) constitute the most common known cause of Parkinson's disease (PD), accounting for both familial and sporadic forms of the disease. We analyzed the tempo-spatial activity of leucine-rich repeat kinase 1 (LRRK1) and LRRK2 at the cellular level in human and rat tissues including development and aging. Lrrk2 mRNA is expressed in adult rat striatum, hippocampus, cerebral cortex, sensory and sympathetic ganglia, lung, spleen and kidney. In the developing rat striatum, Lrrk2 transcription is first observed at postnatal day (P) 8 followed by increasing mRNA levels during the following 3 weeks, as revealed by quantitative in situ hybridization, after which levels remain up to 24 months of age. The time-course of postnatal development of Lrrk2 activity in striatum thus closely mirrors the postnatal development of the dopamine innervation of striatum. Lrrk2 mRNA is seen in P1 rat lung, heart, and kidney, whereas Lrrk1 is found in many areas of the P1 rat. Lrrk1 is present in adult rat brain, adrenal gland, liver, lung, spleen and kidney and also in embryonic brain, with declining gene activity after birth. LRRK1 and LRRK2 are active in the adult human cortex cerebri, hippocampus and LRRK2, but not LRRK1, in striatum. Transcription of both genes is also seen in the young human thymus and LRRK2 is active in tubular parts of the adult human kidney. Our findings suggest that the two paralogous genes have partly complementary expression patterns in the brain, as well as in certain peripheral organs including lymphatic tissues. While the strong presence of Lrrk2 message in striatum is intriguing in relation to PD, the many other neuronal and non-neuronal sites of Lrrk2 activity also needs to be taken into account in deciphering possible pathogenic pathways.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Adulto , Fatores Etários , Idoso , Estruturas Animais/embriologia , Estruturas Animais/crescimento & desenvolvimento , Estruturas Animais/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/anatomia & histologia , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Embrião de Mamíferos , Feminino , Feto , Humanos , Rim/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Timo/metabolismoRESUMO
Transforming growth factor-betas (TGF-betas) constitute an expanding family of multifunctional cytokines with prominent roles in development, cell proliferation, differentiation, and repair. We have cloned, expressed, and raised antibodies against a distant member of the TGF-betas, growth/differentiation factor-15 (GDF-15). GDF-15 is identical to macrophage inhibitory cytokine-1 (MIC-1). GDF-15/MIC-1 mRNA and protein are widely distributed in the developing and adult CNS and peripheral nervous systems, including choroid plexus and CSF. GDF-15/MIC-1 is a potent survival promoting and protective factor for cultured and iron-intoxicated dopaminergic (DAergic) neurons cultured from the embryonic rat midbrain floor. The trophic effect of GDF-15/MIC-1 was not accompanied by an increase in cell proliferation and astroglial maturation, suggesting that GDF-15/MIC-1 probably acts directly on neurons. GDF-15/MIC-1 also protects 6-hydroxydopamine (6-OHDA)-lesioned nigrostriatal DAergic neurons in vivo. Unilateral injections of GDF-15/MIC-1 into the medial forebrain bundle just above the substantia nigra (SN) and into the left ventricle (20 microgram each) immediately before a 6-OHDA injection (8 microgram) prevented 6-OHDA-induced rotational behavior and significantly reduced losses of DAergic neurons in the SN. This protection was evident for at least 1 month. Administration of 5 microgram of GDF-15/MIC-1 in the same paradigm also provided significant neuroprotection. GDF-15/MIC-1 also promoted the serotonergic phenotype of cultured raphe neurons but did not support survival of rat motoneurons. Thus, GDF-15/MIC-1 is a novel neurotrophic factor with prominent effects on DAergic and serotonergic neurons. GDF-15/MIC-1 may therefore have a potential for the treatment of Parkinson's disease and disorders of the serotonergic system.
Assuntos
Citocinas/metabolismo , Dopamina/metabolismo , Mesencéfalo/metabolismo , Neurônios/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Clonagem Molecular , Corpo Estriado/efeitos dos fármacos , Citocinas/genética , Citocinas/farmacologia , Feminino , Fator 15 de Diferenciação de Crescimento , Humanos , Ferro/farmacologia , Cinese/efeitos dos fármacos , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/embriologia , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Especificidade de Órgãos , Oxidopamina/antagonistas & inibidores , Núcleos da Rafe/citologia , Núcleos da Rafe/efeitos dos fármacos , Núcleos da Rafe/embriologia , Núcleos da Rafe/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Serotonina/metabolismo , Medula Espinal , Substância Negra/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Dopamine (DA) replacement therapy continues to be the gold standard treatment for Parkinson's disease (PD), as it improves key motor symptoms including bradykinesia and gait disturbances. With time, treatment induces side effects in the majority of patients, known as L-DOPA-induced dyskinesia (LID), which are often studied in animals by the use of unilateral, toxin-induced rodent models. In this study, we used the progressive, genetic PD model MitoPark to specifically evaluate bilateral changes in motor behavior following long-term L-DOPA treatment at three different stages of striatal DA depletion. Besides locomotor activity, we assessed changes in gait with two automated gait analysis systems and the development of dyskinetic behavior. Long-term treatment with a moderate, clinically relevant dose of L-DOPA (8 mg/kg) gradually produced age-dependent hyperactivity in MitoPark mice. In voluntary and forced gait analyses, we show that MitoPark mice with severe DA depletion have distinct gait characteristics, which are normalized to control levels following long-term L-DOPA treatment. The cylinder test showed an age-dependent and gradual development of bilateral LID. Significant increase in striatal FosB and prodynorphin expression was found to accompany the behavior changes. Taken together, we report that MitoPark mice model both behavioral and biochemical characteristics of long-term L-DOPA treatment in PD patients and provide a novel, consistent and progressive animal model of dyskinesia to aid in the discovery and evaluation of better treatment options to counteract LID.
Assuntos
Discinesia Induzida por Medicamentos/etiologia , Marcha/efeitos dos fármacos , Levodopa/efeitos adversos , Atividade Motora/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Animais , Comportamento Animal/efeitos dos fármacos , Benserazida/administração & dosagem , Benserazida/efeitos adversos , Modelos Animais de Doenças , Esquema de Medicação , Discinesia Induzida por Medicamentos/fisiopatologia , Feminino , Levodopa/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/fisiopatologia , Distribuição AleatóriaRESUMO
We have studied the regulation of survival and serotonergic markers by neurotrophins and several trophically active cytokines in neurons cultured from the embryonic rat raphe region under defined conditions. At embryonic day 14, saturating concentrations of brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4 and basic fibroblast growth factor elicited a two- to 2.5-fold increase in numbers of tryptophan hydroxylase- and serotonin-immunoreactive neurons over a four-day culture period. Transforming growth factor beta-1 and glial cell line-derived neurotrophic factor were less potent, while fibroblast growth factor-5 was only marginally effective. Distinct responses to different factors were noted depending on embryonic age and regional origin of serotonergic neurons. Thus, brain-derived neurotrophic factor augmented numbers of tryptophan hydroxylase-positive neurons at embryonic day 16 by a factor of seven, but only 1.5- to two-fold when cultures were established from day 13 or 14 embryos. In cultures of rostral serotonergic groups (B4-B9), numbers of tryptophan hydroxylase-positive neurons decreased in the absence of factors, whereas numbers of tryptophan hydroxylase-immunoreactive neurons in cultures from caudal serotonergic groups (B1-B3) increased during a 12-day culture period. There was no evidence that serotonergic neurons undergo apoptosis (as visualized by terminal deoxynucleotidyl transferase dUTP nick end labeling) or proliferate (as visualized by 5-bromodeoxyuridine incorporation) in culture. Numbers of serotonergic neurons also increased when cultures were treated with a brief 24-h pulse of brain-derived neurotrophic factor, supporting the notion that changes in numbers of serotonergic neurons reflected alterations of phenotype rather than cell death or proliferation. The ability of cells to specifically take up the serotonin analog 5,7-dihydroxytryptamine was also up-regulated by brain-derived neurotrophic factor in both rostral and caudal raphe cultures. Lability of the serotonergic phenotype was further suggested by the observation that ciliary neurotrophic factor fully prevented the brain-derived neurotrophic factor-mediated increase in tryptophan hydroxylase-positive neurons. The effect of ciliary neurotrophic factor was dependent on the presence of astrocytes. We conclude that serotonergic neurons show spatially and temporally distinct responses to neurotrophic factors, which seem to have a profound influence of the transmitter phenotype rather than on survival.
Assuntos
Proteínas do Tecido Nervoso/farmacologia , Neurônios/citologia , Núcleos da Rafe/química , Serotonina/genética , 5,7-Di-Hidroxitriptamina/farmacocinética , Animais , Anticorpos , Astrócitos/citologia , Astrócitos/fisiologia , Química Encefálica/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Fator Neurotrófico Ciliar , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Fatores de Crescimento Neural/farmacologia , Neurônios/química , Neurônios/enzimologia , Neurotrofina 3 , Fenótipo , Gravidez , Núcleos da Rafe/citologia , Ratos , Ratos Wistar , Serotoninérgicos/farmacocinética , Triptofano Hidroxilase/análise , Triptofano Hidroxilase/imunologiaRESUMO
Corpora amylacea (CA) are spherical bodies routinely observed throughout the aged human brain, normally found at high frequencies under the ependymal lining of the ventricles. We identified clusters of CA under the ependyma of the lateral and fourth ventricles in post-mortem brain material from Parkinson patients as well as age-matched controls. Using a monoclonal antibody we found CA to be immunoreactive for nestin, a marker of neural stem cells, while no other structures in the investigated brain areas were labeled by this antibody. Nestin filaments are therefore possible structural components of CA, a finding which may trigger new hypotheses regarding their biogenesis and function.
Assuntos
Envelhecimento/patologia , Encéfalo/patologia , Corpos de Inclusão/patologia , Proteínas de Filamentos Intermediários/análise , Proteínas do Tecido Nervoso , Idoso , Idoso de 80 Anos ou mais , Anticorpos , Anticorpos Monoclonais , Feminino , Humanos , Imuno-Histoquímica , Corpos de Inclusão/química , Proteínas de Filamentos Intermediários/imunologia , Masculino , Nestina , Doença de Parkinson/patologiaRESUMO
We have cloned, expressed, and raised antibodies against a novel member of the TGF-beta superfamily, growth/differentiation factor-15 (GDF-15). The predicted protein is identical to macrophage inhibitory cytokine-1 (MIC-1), which was discovered simultaneously. GDF-15 is a more distant member of the TGF-beta superfamily and does not belong to one of the known TGF-beta subfamilies. In the CNS, GDF-15/MIC-1 mRNA is abundantly expressed by the choroid plexus. In addition we have preliminary evidence that GDF-15/MIC-1 is a potent trophic factor for selected classes of neurons in vitro and in vivo. Thus, GDF-15 is a novel neurotrophic factor with prospects for the treatment of disorders of the CNS.
Assuntos
Citocinas , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Lesões Encefálicas/metabolismo , Lesões Encefálicas/fisiopatologia , Líquido Cefalorraquidiano/metabolismo , Fator 15 de Diferenciação de Crescimento , Humanos , Estrutura Terciária de Proteína/fisiologia , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Abnormally low plasma cystine levels have been found in the late asymptomatic stage of HIV infection and several other diseases associated with progressive loss of skeletal muscle mass. The phenomenon is commonly associated with a low NK cell activity, skeletal muscle wasting or muscle fatigue and increased rates of urea production. In its extreme form, the negative nitrogen balance leads to overt cachexia and is associated with severe debilitation and psychological stress. The low NK cell activity is in most cases not life-threatening but may be disasterous in HIV infection, because it may compromise the initially stable balance between immune system and virus and trigger disease progression. This review summarizes briefly (i) the role of cysteine in the physiological regulation of body cell mass and the development of skeletal muscle wasting, and (ii) the role of glutathione in the immune system.
Assuntos
Caquexia/fisiopatologia , Cisteína/fisiologia , Glutationa/fisiologia , Infecções por HIV/fisiopatologia , Doenças Musculares/fisiopatologia , Transdução de Sinais/fisiologia , Animais , Cistina/sangue , Cistina/deficiência , Progressão da Doença , Infecções por HIV/sangue , Infecções por HIV/imunologia , Humanos , Células Matadoras Naturais/imunologia , Músculo Esquelético/fisiopatologia , Doenças Musculares/sangue , Doenças Musculares/imunologiaRESUMO
The MitoPark mouse, in which the mitochondrial transcription factor Tfam is selectively removed in midbrain dopamine (DA) neurons, is a genetic model for Parkinson's disease (PD) that replicates the slow and progressive development of key symptoms. To further validate this model, we have extended both behavioral and biochemical analyses in these animals. We found that vertical movements decline earlier and faster than horizontal movements, possibly modeling the early occurrence of axial, postural instability in PD. L-DOPA induces different locomotor responses depending on the age: in young MitoPark mice the L-DOPA-induced motor activation is small; middle-aged MitoPark mice respond in a dose-dependent manner to L-DOPA, whereas aged MitoPark mice display a double-peaked locomotor response to a high dose of L-DOPA that includes an intermittent period of very low motor activity, similar to the 'on-off' phenomenon in PD. To correlate behavior with biochemical data, we analyzed monoamine levels in three different brain areas that are highly innervated by the DA system: striatum, anterior cortex and olfactory bulb. DA levels declined earlier and faster in striatum than in cortex; only at the latest time-point analyzed, DA levels were found to be significantly lower than control levels in the olfactory bulb. Interestingly, the ratio between homovanillic acid (HVA) and DA differed between regions over time. In striatum and olfactory bulb, the ratio increased steeply indicating increased DA turnover. In contrast, the ratio decreased over time in cortex, revealing important differences between DA cells in substantia nigra and the ventral tegmental area.
Assuntos
Proteínas de Ligação a DNA/deficiência , Proteínas de Grupo de Alta Mobilidade/deficiência , Levodopa/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Animais , Pareamento de Bases , Primers do DNA , Proteínas de Ligação a DNA/genética , Progressão da Doença , Relação Dose-Resposta a Droga , Feminino , Proteínas de Grupo de Alta Mobilidade/genética , Masculino , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Camundongos , Camundongos Knockout , Modelos Genéticos , Atividade Motora , Doença de Parkinson/fisiopatologia , Reação em Cadeia da Polimerase , PosturaAssuntos
Acetilcisteína/farmacologia , Ativação Linfocitária/efeitos dos fármacos , NF-kappa B/metabolismo , Compostos de Sulfidrila/farmacologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Células Cultivadas , Técnicas de Cultura/métodos , Cisteína/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Glutationa/análogos & derivados , Glutationa/metabolismo , Glutationa/farmacologia , Dissulfeto de Glutationa , Glutationa Redutase/metabolismo , Humanos , Hidrocortisona/análogos & derivados , Hidrocortisona/farmacologia , Imunoterapia , Leucemia L5178/imunologia , Leucemia L5178/terapia , Mercaptoetanol/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos DBA , Receptores de Interleucina-2/biossíntese , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaAssuntos
Acetilcisteína/uso terapêutico , Caquexia/metabolismo , Cisteína/metabolismo , Glutationa/metabolismo , Infecções por HIV/complicações , Neoplasias/complicações , Aminoácidos/metabolismo , Caquexia/tratamento farmacológico , Caquexia/etiologia , Caquexia/imunologia , Infecções por HIV/metabolismo , Humanos , Músculo Esquelético/metabolismoRESUMO
Serotonergic neurons in the central nervous system are crucial in the control of autonomic functions and behavior. Mechanisms by which development and maintenance of the serotonergic transmitter phenotype is regulated include activation of protein kinase A (PKA). Using cultures established from the E14 rat raphe we show here that forskolin (10 microM) increases numbers of neurons expressing tryptophan hydroxylase (TpOH), the key enzyme of serotonin synthesis, and uptake of the false serotonergic transmitter 5, 7-dihydroxytryptamine (5,7-DHT). As shown by short-term treatments the effect is due to phenotype induction rather than survival. To begin to understand downstream or parallel signaling pathways required for the PKA-mediated induction of serotonergic markers, we have studied the putative implication of brain-derived neurotrophic factor (BDNF) and its receptor trkB. Treatment of raphe neurons with forskolin induced BDNF mRNA assayed by competitive RT-PCR. Moreover, trkB-IgG receptor bodies fully prevented the forskolin-induced numerical increase in TpOH- and 5,7-DHT-positive cells suggesting an implication of a TrkB-activated pathway. TrkC-IgG had no effect. K252b, a specific inhibitor of trk kinase activity likewise abolished the induction of serotonergic markers by forskolin. In turn, the inductive effect of BDNF on serotonergic markers was blocked by KT5720, a specific inhibitor of PKA. Taken together, these data suggest that co-activation of cAMP- and trkB-dependent signaling pathways plays a crucial role in the regulation of the serotonergic neuronal phenotype.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , AMP Cíclico/fisiologia , Neurônios/metabolismo , Receptor trkB/fisiologia , Serotonina/metabolismo , 5,7-Di-Hidroxitriptamina/metabolismo , Animais , Carbazóis/farmacologia , Células Cultivadas , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Alcaloides Indólicos , RNA Mensageiro/metabolismo , Núcleos da Rafe/citologia , Ratos , Receptor trkB/antagonistas & inibidores , Receptores de Serotonina/fisiologia , Receptores 5-HT1 de Serotonina , Triptofano Hidroxilase/metabolismoRESUMO
Serotonin (5-HT) is an important factor controlling survival, differentiation, and plasticity of neurons in serotonergic target regions of the brain and has been implicated in major psychiatric and autonomic disorders. Relatively little is known, however, of factors controlling differentiation and plasticity of developing and adult 5-HT neurons. We show now that 5-HT, the 5-HT1(A) receptor, brain-derived neurotrophic factor (BDNF), and its receptor, trkB, form an auto/paracrine loop for the regulation of the serotonergic phenotype. Serotonin applied to cultures from E14 rat raphe increased numbers of neurons expressing serotonergic markers in a dose-dependent manner. Agonists of the 5-HT1(A) receptor, BP-554 and 8-OH-DPAT, but not agonists of the 5-HT1(B) and 5-HT1(D) receptors, mimicked this effect, while the specific 5-HT1(A) antagonist, WAY-100635, inhibited it. Serotonin also increased BDNF mRNA and protein in embryonic raphe cultures. Induction of serotonergic markers by serotonin was suppressed by a trkB-IgG fusion protein but not by trkC-IgG. Taken together, our data indicate that serotonin acts on 5-HT1(A) autoreceptors, causing up-regulation of BDNF, which activates trkB to promote serotonergic phenotype-specific markers.
Assuntos
Neurônios/fisiologia , Receptor trkB/fisiologia , Receptores de Serotonina/fisiologia , Serotonina/fisiologia , Animais , Biomarcadores , Fator Neurotrófico Derivado do Encéfalo/genética , Carbazóis/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Alcaloides Indólicos , Fenótipo , Fosfotransferases/antagonistas & inibidores , RNA Mensageiro/metabolismo , Núcleos da Rafe/citologia , Núcleos da Rafe/efeitos dos fármacos , Núcleos da Rafe/metabolismo , Ratos , Ratos Wistar , Receptor trkC/fisiologia , Receptores 5-HT1 de Serotonina , Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologiaRESUMO
Oxidative conditions potentiate the activation of the nuclear transcription factor kappa B (NF kappa B) and the activator protein-1 (AP-1) in intact cells, but inhibit their DNA binding activity in vitro. We now show that both the activation of NF kappa B and the inhibition of its DNA binding activity is modulated in intact cells by the physiological oxidant glutathione disulphide (GSSG). NF kappa B activation in human T lineage cells (Molt-4) by 12-O-tetradecanoyl-phorbol 13-acetate was inhibited by dithiothreitol, and this was partly reversed by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or by hydrogen peroxide, indicating that GSSG may be required for NF kappa B activation. These effects of BCNU and hydrogen peroxide were not seen in glutathione-depleted cells. However, NF kappa B and AP-1 activation were potentiated by dithiothreitol if added to cell cultures 1 h after the phorbol ester, indicating that a shift of redox conditions may support optimal oxidative activation with minimal inhibition of DNA binding. The elevation of intracellular GSSG levels by BCNU before stimulation suppressed the chloramphenicol acetyltransferase expression dependent on NF kappa B but increased that dependent on AP-1. This selective suppression of NF kappa B was also demonstrable by electrophoretic mobility shift assays. In vitro, GSSG inhibited the DNA binding activity of NF kappa B more effectively than that of AP-1, while AP-1 was inhibited more effectively by oxidized thioredoxin.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Glutationa/análogos & derivados , Proteínas de Homeodomínio , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Linfócitos T/metabolismo , Transcrição Gênica , Animais , Antioxidantes/farmacologia , Sequência de Bases , Carmustina/farmacologia , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Glutationa/metabolismo , Dissulfeto de Glutationa , Peróxido de Hidrogênio/farmacologia , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Oxirredução , Pirrolidinas/farmacologia , Proteína de Replicação C , Linfócitos T/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Tiocarbamatos/farmacologia , Tiorredoxinas/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
HIV-infected individuals and SIV-infected rhesus macaques have, on the average, decreased plasma cysteine and cystine concentrations and decreased intracellular glutathione levels. We now show that a depletion of intracellular glutathione in a human T cell line (Molt-4) inhibits the activation and nuclear translocation of the transcription factor NF kappa B, whereas incubation with increasing extracellular concentrations of cysteine inhibits the DNA-binding and transactivating activity of NF kappa B. Because inhibition of DNA-binding activity is associated with increasing intracellular glutathione disulfide levels and GSSG can be shown to inhibit the DNA-binding activity directly in cell-free systems, our studies suggest that GSSG is a physiologically relevant inhibitor in intact cells also. NF kappa B controls many immunologically important genes, so our studies suggest that the immune system may be sensitive not only against a cysteine and glutathione deficiency but also against an excess of cysteine.
Assuntos
Cisteína/farmacologia , Glutationa/metabolismo , NF-kappa B/metabolismo , Acetilcisteína/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Glutationa/análogos & derivados , Dissulfeto de Glutationa , HIV , Infecções por HIV/metabolismo , Humanos , Cinética , Macaca mulatta , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Vírus da Imunodeficiência Símia , Compostos de Sulfidrila/metabolismo , Linfócitos T , Ativação Transcricional/efeitos dos fármacosRESUMO
Serotonergic (5-HT) neurons of the CNS develop as two separate clusters, a rostral and a caudal group, within the brain stem raphe. We show here that the transforming growth factors -beta2 and -beta3 (TGF-beta) and the TGF-beta type II receptor are expressed in the embryonic rat raphe, when 5-HT neurons develop and differentiate. To investigate putative roles of TGF-betas in the regulation of 5-HT neuron development we have generated serum-free cultures isolated either from the rostral or the caudal embryonic rat raphe, respectively. In cultures from the caudal E14 raphe saturating concentrations (5 ng/ml) of TGF-beta2 and -beta3 augmented numbers of tryptophan hydroxylase (TpOH) -immunoreactive neurons and cells specifically taking up 5,7-dihydroxytryptamine (5,7-DHT) by about 1.7-fold over a period of 4 days. Treatment with TGF-betas also increased uptake of 3H-5HT uptake about 1.7-fold. Alterations in 5-HT neuron numbers were due to the induction of serotonergic markers rather than increased survival, as shown by the efficacy of delayed short-term treatments. Comparing rostral and caudal raphe cultures from different embryonic ages suggests that distinct effects of TGF-betas reflect the responsiveness of 5-HT neurons at different ages rather than of different origins.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neurônios/fisiologia , Núcleos da Rafe/embriologia , Receptores de Fatores de Crescimento Transformadores beta/genética , Serotonina/metabolismo , Fator de Crescimento Transformador beta/farmacologia , 5,7-Di-Hidroxitriptamina/farmacocinética , Receptores de Ativinas , Animais , Transporte Biológico , Células Cultivadas , Meios de Cultura Livres de Soro , Desenvolvimento Embrionário e Fetal , Feminino , Idade Gestacional , Neurônios/citologia , Neurônios/efeitos dos fármacos , Gravidez , Proteínas Serina-Treonina Quinases/genética , Núcleos da Rafe/metabolismo , Ratos , Ratos Wistar , Receptores Proteína Tirosina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triptofano Hidroxilase/metabolismoRESUMO
Procaine has opposite effects on the active transport of Na+ when applied on the mucosal side of the frog skin [where it produces a stimulation of the short-circuit current (Isc)] or when added on the serosal side (where it produces an inhibition of Isc). In an attempt to reveal and localize the primary effect of procaine on either the apical or latero-basal membranes of the epithelial cells, we have tried to "chemically dissect" both membrane functions with inhibitors and ionophores. When applied on the apical side of the latero-basally depolarized epithelium, 25 mmol/l procaine increases Isc and Voc (transepithelial open-circuit potential), while decreasing the transepithelial resistance. The E1-E2 linearity domain of the I-V curves is narrowed. On the serosal side of the depolarized epithelium, the same concentration of procaine does not affect Isc and Voc (which are already inhibited) but it produces an increase in the transepithelial resistance (Rt). Procaine influence on the passive K+ permeability was studied by using the ionophore nystatin, which is assumed to form channels permeable to K+, when applied on the amiloride blocked apical membrane. In nystatin-treated epithelia, 25 mmol/l procaine on the apical side decreased Isc, Voc and Rt. In parallel experiments during Cl- substitution by SO2-(4), the procaine effects on Isc and Voc are no longer maintained, but transient.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Potássio/metabolismo , Procaína/farmacologia , Absorção Cutânea/efeitos dos fármacos , Sódio/metabolismo , Amilorida/farmacologia , Animais , Feminino , Técnicas In Vitro , Nistatina/farmacologia , Rana ridibundaRESUMO
Persephin (PSP) is the most recently discovered member of the GDNF family of neurotrophic factors. We have used an RT-PCR approach to start addressing the putative functional significance of PSP by determining sites of its synthesis in the neonatal rat brain. Generally, two transcripts were found. Sequence analysis of the transcripts identifies an 88 bp intronic sequence. Neural tissues analysed included cortex, hippocampus, striatum, diencephalon, mesencephalon, cerebellum, hindbrain and spinal cord as well as superior cervical, dorsal root ganglia, adrenal gland, and PC12 pheochromocytoma cells. As non-neuronal tissues, sciatic nerve, optic nerve, primary astroglial, oligodendroglial, O2A progenitor, and glioma cells (C6, B49) were also included. All tissues/cells except oligodendrocytes and O2A progenitor cells were strongly positive for PSP mRNA. To test the hypothesis of whether PSP might act as a target-derived factor, as suggested for GDNF, the motoneuron-muscle axis has been analysed. PSP is synthesized in skeletal muscle and, to a higher extent, in the spinal cord. Moreover, PSP is synthesized in purified embryonic motoneurons. Together, these data do not support a role for PSP as a typical target-derived neurotrophic factor for motoneurons. We conclude that PSP is synthesized throughout the nervous system and that it is presumably of both astroglial and neuronal origin, in contrast to GDNF and neurturin, which seem to be predominantly of neuronal origin.
Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/genética , Medula Espinal/metabolismo , Neoplasias das Glândulas Suprarrenais , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Complementar , Gânglios Espinais/metabolismo , Íntrons , Rim/metabolismo , Dados de Sequência Molecular , Fatores de Crescimento Neural/análise , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Oligodendroglia/metabolismo , Nervo Óptico/metabolismo , Especificidade de Órgãos , Células PC12 , Feocromocitoma , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nervo Isquiático/metabolismo , Análise de Sequência de DNA , Gânglio Cervical Superior/metabolismoRESUMO
Induction of tyrosine kinase activity of the insulin receptor (IR) beta-chain is believed to require its autophosphorylation at Tyr1162, Tyr1163, and Tyr1158. However, the mechanism of the initial phosphorylation is poorly understood. We show that treatment of IR-transfected Chinese hamster ovary cells with antioxidants inhibits insulin responsiveness. Conversely, partial inhibition of glutathione biosynthesis by buthionine sulfoximine (BSO) and glutathione reductase by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), i.e., procedures that intracellularly induce mildly oxidative conditions, caused a decrease in IR beta-chain sulfhydryl groups and enhanced synergistically the induction of IR tyrosine phosphorylation by insulin. The IR beta-chain from cells treated with BSO/BCNU in the absence of insulin was not detectably tyrosine phosphorylated, but nevertheless was functionally altered, as demonstrated in vitro by a moderate kinase activity at lowATP concentrations (5 nM) and a strong kinase activity at 25 microM ATP. This activity was found to be specific for tyrosine (not for serine or threonine), and tryptic peptide maps indicated that it is more selective than that induced by insulin. Moreover, the kinase activity from BSO/BCNU-treated cells showed a spontaneous decay that was not prevented by the phosphatase inhibitor vanadate. Together, these results suggest that optimal insulin responsiveness may require a process of 'redox priming' of the IR beta-chain that involves structural and functional changes in the absence of detectable tyrosine phosphorylation of the beta-chain.
Assuntos
Antimetabólitos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Butionina Sulfoximina/farmacologia , Carmustina/farmacologia , Insulina/fisiologia , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/efeitos dos fármacos , Receptor de Insulina/metabolismo , Tirosina/metabolismo , Animais , Antioxidantes/farmacologia , Células CHO/efeitos dos fármacos , Cricetinae , Indução Enzimática , Oxirredução , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/biossíntese , Receptor de Insulina/genética , TransfecçãoRESUMO
Bone morphogenetic proteins (BMPs), growth and differentiation factor 5 (GDF5) and glial cell line-derived neurotrophic factor (GDNF) are members of the transforming growth factor-beta superfamily that have been implicated in tissue growth and differentiation. Several BMPs are expressed in embryonic and adult brain. We show now that BMP-2, -6 and -7 and GDF5 are expressed in the embryonic rat hindbrain raphe. To start to define roles for BMPs in the regulation of serotonergic (5-HT) neuron development, we have generated serum-free cultures of 5-HT neurons isolated from the embryonic (E14) rat raphe. Addition of saturating concentrations (10 ng/mL) of BMP-6 and GDF5 augmented numbers of tryptophan hydroxylase (TpOH) -immunoreactive neurons and cells specifically taking up 5, 7-dihydroxytryptamine (5,7-DHT) by about two-fold. Alterations in 5-HT neuron numbers were due to the induction of serotonergic markers rather than increased survival, as shown by the efficacy of short-term treatments. Importantly, BMP-7 selectively induced 5, 7-DHT uptake without affecting TpOH immunoreactivity. BMP-6 and -7 also promoted DNA synthesis and increased numbers of cells immunoreactive for vimentin and glial fibrillary acidic protein (GFAP). Pharmacological suppression of cell proliferation or glial development abolished the induction of serotonergic markers by BMP-6 and -7, suggesting that BMPs act indirectly by stimulating synthesis or release of glial-derived serotonergic differentiation factors. Receptor bodies for the neurotrophin receptor trkB, but not trkC, abolished the BMP-mediated effects on serotonergic development, suggesting that the glia-derived factor is probably brain-derived neurotrophic factor (BDNF) or neurotrophin-4. In support of this notion, we detected increased levels of BDNF mRNA in BMP-treated cultures. Together, these data suggest both distinct and overlapping roles of several BMPs in regulating 5-HT neuron development.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Serotonina/metabolismo , 5,7-Di-Hidroxitriptamina/farmacocinética , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Neuroglia/fisiologia , Neurônios/metabolismo , Fenótipo , Núcleos da Rafe/embriologia , Ratos , Ratos Wistar , Rombencéfalo/embriologia , Células-Tronco/fisiologia , Triptofano Hidroxilase/farmacocinéticaRESUMO
Numerous studies have suggested that glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic molecule. We show now on a variety of cultured neurons including peripheral autonomic, sensory, and CNS dopaminergic neurons that GDNF is not trophically active unless supplemented with TGF-beta. Immunoneutralization of endogenous TGF-beta provided by serum or TGF-beta-secreting cells, as e.g., neurons, in culture abolishes the neurotrophic effect of GDNF. The dose-response relationship required for the synergistic effect of GDNF and TGF-beta identifies 60 pg/ml of either factor combined with 2 ng/ml of the other factor as the EC50. GDNF/TGF-beta signaling employs activation of phosphatidylinositol-3 (PI-3) kinase as an intermediate step as shown by the effect of the specific PI-3 kinase inhibitor wortmannin. The synergistic action of GDNF and TGF-beta involves protection of glycosylphosphatidylinositol (GPI)-linked receptors as shown by the restoration of their trophic effects after phosphatidylinositol-specific phospholipase C-mediated hydrolysis of GPI-anchored GDNF family receptor alpha. The biological significance of the trophic synergism of GDNF and TGF-beta is underscored by colocalization of the receptors for TGF-beta and GDNF on all investigated GDNF-responsive neuron populations in vivo. Moreover, the in vivo relevance of the TGF-beta/GDNF synergism is highlighted by the co-storage of TGF-beta and GDNF in secretory vesicles of a model neuron, the chromaffin cell, and their activity-dependent release. Our results broaden the definition of a neurotrophic factor by incorporating the possibility that two factors that lack a neurotrophic activity when acting separately become neurotrophic when acting in concert. Moreover, our data may have a substantial impact on the treatment of neurodegenerative diseases.