RESUMO

Exposure to environmental lead (Pb) is a mild risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive degeneration of motor neurons. However, recent evidence has paradoxically linked higher Pb levels in ALS patients with longer survival. We investigated the effects of low-level Pb exposure on survival of mice expressing the ALS-linked superoxide dismutase-1 G93A mutation (SOD1(G93A)). SOD1(G93A) mice exposed to Pb showed longer survival and increased expression of VEGF in the ventral horn associated with reduced astrocytosis. Pretreatment of cultured SOD1(G93A) astrocytes with low, non toxic Pb concentrations upregulated VEGF expression and significantly abrogated motor neuron loss in coculture, an effect prevented by neutralizing antibodies to VEGF. The actions of Pb on astrocytes might explain its paradoxical slowing of disease progression in SOD1(G93A) mice and the improved survival of ALS patients. Understanding how Pb stimulates astrocytic VEGF production and reduces neuroinflammation may yield a new therapeutic approach for treating ALS.

Assuntos

Esclerose Lateral Amiotrófica/tratamento farmacológico , Astrócitos/efeitos dos fármacos , Chumbo/farmacologia , Medula Espinal/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Anticorpos Neutralizantes/farmacologia , Astrócitos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Gliose/tratamento farmacológico , Gliose/etiologia , Gliose/fisiopatologia , Chumbo/uso terapêutico , Camundongos , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Degeneração Neural/tratamento farmacológico , Degeneração Neural/etiologia , Degeneração Neural/fisiopatologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Medula Espinal/citologia , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Taxa de Sobrevida , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo

RESUMO

Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons. Mutations in the Cu/Zn superoxide dismutase (SOD1) are found in approximately 20% of patients with familial ALS. Mutant SOD1 causes motor neuron death through an acquired toxic property. Although the molecular mechanism underlying this toxic gain-of-function remains unknown, evidence support the role of mutant SOD1 expression in nonneuronal cells in shaping motor neuron degeneration. We have previously found that in contrast to nontransgenic cells, SOD1(G93A)-expressing astrocytes induced apoptosis of cocultured motor neurons. This prompted us to investigate whether the effect on motor neuron survival was related to a change in the gene expression profile. Through high-density oligonucleotide microarrays, we found changes in the expression of genes involved in transcription, signaling, cell proliferation, extracellular matrix synthesis, response to stress, and steroid and lipid metabolism. The most up-regulated gene was decorin (Dcn), a small multifunctional extracellular proteoglycan. Down-regulated genes included the insulin-like growth factor-1 receptor (Igf-1r) and the RNA binding protein ROD1. Rod1 was also found down-regulated in purified motor neurons expressing SOD1(G93A). Changes in the expression of Dcn, Igf-1r, and Rod1 were found in the spinal cord of asymptomatic animals, suggesting these changes occur before overt neuronal degeneration and potentially influence astrocyte-motor neuron interaction in the course of the disease. The astrocyte-specific gene expression profile might contribute to the identification of possible candidates for cell type-specific therapies in ALS.

Assuntos

Esclerose Lateral Amiotrófica/genética , Astrócitos/metabolismo , Comunicação Celular/genética , Regulação da Expressão Gênica/genética , Neurônios/metabolismo , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Animais Geneticamente Modificados , Astrócitos/citologia , Sobrevivência Celular/genética , Células Cultivadas , Decorina , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Perfilação da Expressão Gênica , Humanos , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Neurônios/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Proteoglicanas/genética , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Receptor IGF Tipo 1/genética , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Regulação para Cima/genética

RESUMO

Mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Recent reports indicate that astrocytes expressing the mutations of superoxide dismutase-1 (SOD1) may contribute to motor neuron injury in ALS. Here, we provide evidence that mitochondrial dysfunction in SOD1(G93A) rat astrocytes causes astrocytes to induce apoptosis of motor neurons. Mitochondria from SOD1(G93A) rat astrocytes displayed a defective respiratory function, including decreased oxygen consumption, lack of ADP-dependent respiratory control, and decreased membrane potential. Protein 3-nitrotyrosine was detected immunochemically in mitochondrial proteins from SOD1(G93A) astrocytes, suggesting that mitochondrial defects were associated with nitroxidative damage. Furthermore, superoxide radical formation in mitochondria was increased in SOD1(G93A) astrocytes. Similar defects were found in mitochondria isolated from the spinal cord of SOD1(G93A) rats, and pretreatment of animals with the spin trap 5,5-dimethyl-1-pyrroline N-oxide restored mitochondrial function, forming adducts with mitochondrial proteins in vivo. As shown previously, SOD1(G93A) astrocytes induced death of motor neurons in cocultures, compared with nontransgenic ones. This behavior was recapitulated when nontransgenic astrocytes were treated with mitochondrial inhibitors. Remarkably, motor neuron loss was prevented by preincubation of SOD1(G93A) astrocytes with antioxidants and nitric oxide synthase inhibitors. In particular, low concentrations (approximately 10 nm) of two mitochondrial-targeted antioxidants, ubiquinone and carboxy-proxyl nitroxide, each covalently coupled to a triphenylphosphonium cation (Mito-Q and Mito-CP, respectively), prevented mitochondrial dysfunction, reduced superoxide production in SOD1(G93A) astrocytes, and restored motor neuron survival. Together, our results indicate that mitochondrial dysfunction in astrocytes critically influences motor neuron survival and support the potential pharmacological utility of mitochondrial-targeted antioxidants in ALS treatment.

Assuntos

Antioxidantes/administração & dosagem , Astrócitos/enzimologia , Mitocôndrias/enzimologia , Neurônios Motores/enzimologia , Degeneração Neural/enzimologia , Superóxido Dismutase/genética , Substituição de Aminoácidos/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/prevenção & controle , Animais , Animais Geneticamente Modificados , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Células Cultivadas , Sistemas de Liberação de Medicamentos/métodos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Degeneração Neural/genética , Degeneração Neural/prevenção & controle , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/fisiologia

RESUMO

Nerve growth factor (NGF) can induce apoptosis by signaling through the p75 neurotrophin receptor (p75(NTR)) in several nerve cell populations. Cultured embryonic motor neurons expressing p75(NTR) are not vulnerable to NGF unless they are exposed to an exogenous flux of nitric oxide (*NO). In the present study, we show that p75(NTR)-mediated apoptosis in motor neurons involved neutral sphingomyelinase activation, increased mitochondrial superoxide production, and cytochrome c release to the cytosol. The mitochondria-targeted antioxidants mitoQ and mitoCP prevented neuronal loss, further evidencing the role of mitochondria in NGF-induced apoptosis. In motor neurons overexpressing the amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1(G93A) (SOD1(G93A)) mutation, NGF induced apoptosis even in the absence of an external source of *NO. The increased susceptibility of SOD1(G93A) motor neurons to NGF was associated to decreased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and downregulation of the enzymes involved in glutathione biosynthesis. In agreement, depletion of glutathione in nontransgenic motor neurons reproduced the effect of SOD1(G93A) expression, increasing their sensitivity to NGF. In contrast, rising antioxidant defenses by Nrf2 activation prevented NGF-induced apoptosis. Together, our data indicate that p75(NTR)-mediated motor neuron apoptosis involves ceramide-dependent increased mitochondrial superoxide production. This apoptotic pathway is facilitated by the expression of ALS-linked SOD1 mutations and critically modulated by Nrf2 activity.

Assuntos

Apoptose/fisiologia , Mitocôndrias/metabolismo , Neurônios Motores/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Medula Espinal/citologia , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Células Cultivadas , Citocromos c/metabolismo , Embrião de Mamíferos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Mitocôndrias/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Doadores de Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/farmacologia

RESUMO

Nerve growth factor (NGF) overexpression and increased production of peroxynitrite occur in several neurodegenerative diseases. We investigated whether NGF could undergo posttranslational oxidative or nitrative modifications that would modulate its biological activity. Compared to native NGF, peroxynitrite-treated NGF showed an exceptional ability to induce p75(NTR)-dependent motor neuron apoptosis at physiologically relevant concentrations. Whereas native NGF requires an external source of nitric oxide (NO) to induce motor neuron death, peroxynitrite-treated NGF induced motor neuron apoptosis in the absence of exogenous NO. Nevertheless, NO potentiated the apoptotic activity of peroxynitrite-modified NGF. Blocking antibodies to p75(NTR) or downregulation of p75(NTR) expression by antisense treatment prevented motor neuron apoptosis induced by peroxynitrite-treated NGF. We investigated what oxidative modifications were responsible for inducing a toxic gain of function and found that peroxynitrite induced tyrosine nitration in a dose-dependent manner. Moreover, peroxynitrite triggered the formation of stable high-molecular-weight oligomers of NGF. Preventing tyrosine nitration by urate abolished the effect of peroxynitrite on NGF apoptotic activity. These results indicate that the oxidation of NGF by peroxynitrite enhances NGF apoptotic activity through p75(NTR) 10,000-fold. To our knowledge, this is the first known posttranslational modification that transforms a neurotrophin into an apoptotic agent.

Assuntos

Apoptose/fisiologia , Neurônios Motores/metabolismo , Fator de Crescimento Neural/química , Ácido Peroxinitroso/farmacologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Espectrometria de Massas , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Fator de Crescimento Neural/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ratos , Receptor de Fator de Crescimento Neural/antagonistas & inibidores , Receptor de Fator de Crescimento Neural/efeitos dos fármacos , Receptor de Fator de Crescimento Neural/metabolismo , Tirosina/metabolismo

RESUMO

The p75 neurotrophin receptor (p75NTR) is expressed by degenerating spinal motor neurons in amyotrophic lateral sclerosis (ALS). The mature and pro-form of nerve growth factor (NGF) activate p75NTR to trigger motor neuron apoptosis. However, attempts to modulate p75NTR-mediated neuronal death in ALS models by downregulating or antagonizing p75NTR with synthetic peptides have led to only modest results. Recently, a novel ligand of p75NTR, compound LM11A-24, has been identified. It is a non-peptidyl mimetic of the neurotrophin loop 1 domain that promotes hippocampal neuron survival through p75NTR and exerts protection against p75NTR-mediated apoptosis of oligodendrocytes induced by proNGF. Thus, LM11A-24 appears to activate p75NTR-linked survival but not death mechanisms, and may interfere with the ability of neurotrophins to induce apoptosis. Given these findings, we hypothesized that LM11A-24 might be a particularly potent inhibitor of motor neuron degeneration. We examined the effects of LM11A-24 on apoptosis of cultured rat embryonic motor neurons. Interestingly, in contrast to the effects observed in hippocampal cultures, LM11A-24 was unable to prevent motor neuron apoptosis induced by trophic factor deprivation. However, picomolar concentrations of LM11A-24 prevented p75NTR-dependent motor neuron death induced by either exogenous addition of NGF or spinal cord extracts from symptomatic superoxide dismutase-1G93A mice, in the presence of low steady-state concentrations of nitric oxide. LM11A-24 also inhibited motor neuron death induced by NGF-producing reactive astrocytes in co-culture conditions. These studies suggest that modulation of p75NTR by small molecule ligands targeting this receptor might constitute a novel strategy for preventing motor neuron degeneration.

Assuntos

Neurônios Motores/efeitos dos fármacos , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/farmacologia , Receptor de Fator de Crescimento Neural/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Astrócitos , Cafeína/análogos & derivados , Cafeína/farmacologia , Contagem de Células/métodos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Lipopolissacarídeos/farmacologia , Neurônios Motores/fisiologia , Fator de Crescimento Neural/farmacologia , Estrutura Terciária de Proteína , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Medula Espinal/citologia

RESUMO

Reactive astrocytes surround amyloid depositions and degenerating neurons in Alzheimer's disease (AD). It has been previously shown that beta-amyloid peptide induces inflammatory-like responses in astrocytes, leading to neuronal pathology. Reactive astrocytes up-regulate nerve growth factor (NGF), which can modulate neuronal survival by signaling through TrkA or p75 neurotrophin receptor (p75NTR). Here, we analyzed whether soluble Abeta peptide 25-35 (Abeta) stimulated astrocytic NGF expression, modulating the survival of cultured embryonic hippocampal neurons. Hippocampal astrocytes incubated with Abeta up-regulated NGF expression and release to the culture medium. Abeta-stimulated astrocytes increased tau phosphorylation and reduced the survival of cocultured hippocampal neurons. Neuronal death and tau phosphorylation were reproduced by conditioned media from Abeta-stimulated astrocytes and prevented by caspase inhibitors or blocking antibodies to NGF or p75NTR. Moreover, exogenous NGF was sufficient to induce tau hyperphosphorylation and death of hippocampal neurons, a phenomenon that was potentiated by a low steady-state concentration of nitric oxide. Our findings show that Abeta-activated astrocytes potently stimulate NGF secretion, which in turn causes the death of p75-expressing hippocampal neurons, through a mechanism regulated by nitric oxide. These results suggest a potential role for astrocyte-derived NGF in the progression of AD.

Assuntos

Astrócitos/metabolismo , Hipocampo/citologia , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/química , Astrócitos/efeitos dos fármacos , Western Blotting/métodos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Tempo

RESUMO

Astrocytes may modulate the survival of motor neurons in amyotrophic lateral sclerosis (ALS). We have previously shown that fibroblast growth factor-1 (FGF-1) activates astrocytes to increase secretion of nerve growth factor (NGF). NGF in turn induces apoptosis in co-cultured motor neurons expressing the p75 neurotrophin receptor (p75NTR) by a mechanism involving nitric oxide (NO) and peroxynitrite formation. We show here that FGF-1 increased the expression of inducible nitric oxide synthase and NO production in astrocytes, making adjacent motor neurons vulnerable to NGF-induced apoptosis. Spinal cord astrocytes isolated from transgenic SOD1G93A rats displayed increased NO production and spontaneously induced apoptosis of co-cultured motor neurons. FGF-1 also activates the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in astrocytes. Because Nrf2 increases glutathione (GSH) biosynthesis, we investigated the role of GSH production by astrocytes on p75NTR-dependent motor neuron apoptosis. The combined treatment of astrocytes with FGF-1 and t-butylhydroquinone (tBHQ) increased GSH production and secretion, preventing motor neuron apoptosis. Moreover, Nrf2 activation in SOD1G93A astrocytes abolished their apoptotic activity. The protection exerted by increased Nrf2 activity was overcome by adding the NO donor DETA-NONOate to the co-cultures or by inhibiting GSH synthesis and release from astrocytes. These results suggest that activation of Nrf2 in astrocytes can reduce NO-dependent toxicity to motor neurons by increasing GSH biosynthesis.

Assuntos

Astrócitos/metabolismo , Glutationa/biossíntese , Neurônios Motores/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Antioxidantes/farmacologia , Astrócitos/efeitos dos fármacos , Western Blotting/métodos , Contagem de Células/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura/métodos , Interações Medicamentosas , Embrião de Mamíferos , Ativação Enzimática/efeitos dos fármacos , Fator 1 de Crescimento de Fibroblastos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa/metabolismo , Hidroquinonas/farmacologia , Metionina/análogos & derivados , Metionina/farmacologia , Fator de Crescimento Neural/farmacologia , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Propionatos/farmacologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Medula Espinal/citologia , Superóxido Dismutase , Transfecção/métodos , Ureia/análogos & derivados , Ureia/farmacologia

RESUMO

Fibroblast growth factor-1 (FGF-1) is highly expressed in motor neurons and can be released in response to sublethal cell injury. Because FGF-1 potently activates astroglia and exerts a direct neuroprotection after spinal cord injury or axotomy, we examined whether it regulated the expression of inducible and cytoprotective heme oxygenase-1 (HO-1) enzyme in astrocytes. FGF-1 induced the expression of HO-1 in cultured rat spinal cord astrocytes, which was dependent on FGF receptor activation and prevented by cycloheximide. FGF-1 also induced Nrf2 mRNA and protein levels and prompted its nuclear translocation. HO-1 induction was abolished by transfection of astrocytes with a dominant-negative mutant Nrf2, indicating that FGF-1 regulates HO-1 expression through Nrf2. FGF-1 also modified the expression of other antioxidant genes regulated by Nrf2. Both Nrf2 and HO-1 levels were increased and co-localized with reactive astrocytes in the degenerating lumbar spinal cord of rats expressing the amyotrophic lateral sclerosis-linked SOD1 G93A mutation. Overexpression of Nrf2 in astrocytes increased survival of co-cultured embryonic motor neurons and prevented motor neuron apoptosis mediated by nerve growth factor through p75 neurotrophin receptor. Taken together, these results emphasize the key role of astrocytes in determining motor neuron fate in amyotrophic lateral sclerosis.

Assuntos

Astrócitos/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Fator 1 de Crescimento de Fibroblastos/farmacologia , Heme Oxigenase (Desciclizante)/genética , Medula Espinal/citologia , Transativadores/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Animais , Astrócitos/citologia , Astrócitos/enzimologia , Comunicação Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1 , Humanos , Proteínas de Membrana , Neurônios Motores/citologia , Fator 2 Relacionado a NF-E2 , Degeneração Neural/metabolismo , Ratos , Ratos Mutantes , Ratos Sprague-Dawley , Superóxido Dismutase/genética , Transativadores/genética

RESUMO

Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75(NTR)-dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75(NTR) neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75(NTR)-dependent mechanism.

Assuntos

Esclerose Lateral Amiotrófica/patologia , Apoptose/fisiologia , Astrócitos/efeitos dos fármacos , Fator 1 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Neurônios Motores/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Medula Espinal/citologia , Fatores Etários , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Caspase 3 , Contagem de Células , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos , Fator 1 de Crescimento de Fibroblastos/metabolismo , Imunofluorescência , Sequestradores de Radicais Livres/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Metaloporfirinas/farmacologia , Camundongos , Camundongos Transgênicos , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/imunologia , Fator de Crescimento Neural/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fator de Crescimento Neural/imunologia , Superóxido Dismutase/genética , Tirosina/análogos & derivados , Ureia/análogos & derivados

RESUMO

Neurons and surrounding glial cells compose a highly specialized functional unit. In amyotrophic lateral sclerosis (ALS) astrocytes interact with motor neurons in a complex manner to modulate neuronal survival. Experiments using chimeric mice expressing ALS-linked mutations to Cu,Zn superoxide dismutase (SOD-1) suggest a critical modulation exerted by neighboring non-neuronal cell types on disease phenotype. When perturbed by primary neuronal damage, e.g. expression of SOD-1 mutations, neurons can signal astrocytes to proliferate and become reactive. Fibroblast growth factor-1 (FGF-1) can be released by motor neurons in response to damage to induce astrocyte activation by signaling through the receptor FGFR1. FGF-1 stimulates nerve growth factor (NGF) expression and secretion, as well as activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor. Nrf2 leads to the expression of antioxidant and cytoprotective enzymes such as heme oxygenase-1 and a group of enzymes involved in glutathione metabolism that prevent motor neuron degeneration. However, prolonged stimulation with FGF-1 or SOD-mediated oxidative stress in astrocytes may disrupt the normal neuron-glia interactions and lead to progressive neuronal degeneration. The re-expression of p75 neurotrophin receptor and neuronal NOS in motor neurons in parallel with increased NGF secretion by reactive astrocytes may be a mechanism to eliminate critically damaged neurons. Consequently, astrocyte activation in ALS may have a complex pathogenic role.

Assuntos

Esclerose Lateral Amiotrófica/fisiopatologia , Astrócitos/metabolismo , Comunicação Celular/fisiologia , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Astrócitos/patologia , Morte Celular/fisiologia , Fator 1 de Crescimento de Fibroblastos/metabolismo , Gliose/metabolismo , Humanos , Camundongos , Neurônios Motores/patologia , Fator de Crescimento Neural/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1

RESUMO

A strong glial reaction typically surrounds the affected upper and lower motor neurons and degenerating descending tracts of ALS patients. Reactive astrocytes in ALS contain protein inclusions, express inflammatory makers such as the inducible forms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2), display nitrotyrosine immunoreactivity and downregulate the glutamate transporter EAAT2. In this review, we discuss the evidence sustaining an active role for astrocytes in the induction and propagation of motor neuron loss in ALS. Available evidence supports the view that glial activation could be initiated by proinflammatory mediators secreted by motor neurons in response to injury, axotomy or muscular pathology. In turn, reactive astrocytes produce nitric oxide and peroxynitrite, which cause mitochondrial damage in cultured neurons and trigger apoptosis in motor neurons. Astrocytes may also contribute to the excitotoxic damage of motor neurons by decreasing glutamate transport or actively releasing the excitotoxic amino acid. In addition, reactive astrocytes secrete pro-apoptotic mediators, such as nerve growth factor (NGF) or Fas-ligand, a mechanism that may serve to eliminate vulnerable motor neurons. The comprehensive understanding of the interactions between motor neurons and glia in ALS may lead to a more accurate theory of the pathogenesis of the disease.

Assuntos

Esclerose Lateral Amiotrófica/metabolismo , Apoptose/fisiologia , Astrócitos/metabolismo , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Astrócitos/imunologia , Comunicação Celular/fisiologia , Gliose/metabolismo , Gliose/patologia , Gliose/fisiopatologia , Ácido Glutâmico/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Neurônios Motores/patologia

RESUMO

Production of nitric oxide (NO) by glial cells has been proposed to mediate cytotoxic effects on neighboring neurons. Although extensive genetic data implicate the beta amyloid peptide (Abeta) in the neurodegenerative cascade of Alzheimer's disease (AD), the molecular mechanisms underlying its effects on neurons and glia and the relationship between glial activation and neuronal death are not well understood. In AD, Abeta is sufficient to induce glial activation and promote the generation of inflammatory mediators including NO. We examined whether Abeta stimulated astrocytes to express nitric oxide synthase and produce NO. Also, we investigated whether astrocytic NO contributes to degenerative changes occurring in co-cocultured hippocampal neurons. We found that the treatment of rat hippocampal astrocyte cultures with Abeta(25-35) fragment up-regulated the mRNA and protein levels of both the inducible and neuronal forms of nitric oxide synthase (iNOS and nNOS, respectively) and increased the production of nitric oxide. Remarkably, hippocampal neurons co-cultured with astrocytes, previously stimulated with Abeta, displayed hyperphosphorylation of the microtubule-associated protein tau. This effect was attenuated by iNOS inhibitors, suggesting the role of overproduction of NO by reactive astrocytes in AD pathogenesis.

Assuntos

Astrócitos/fisiologia , Hipocampo/fisiologia , Neurônios/fisiologia , Óxido Nítrico/fisiologia , Proteínas tau/metabolismo , Doença de Alzheimer , Animais , Animais Recém-Nascidos , Células Cultivadas , Primers do DNA , Cinética , Modelos Animais , NG-Nitroarginina Metil Éster/farmacologia , Neurônios/efeitos dos fármacos , Fosforilação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas tau/genética

RESUMO

BACKGROUND: Overproduction of nitric oxide (NO) has been recognized as a major mechanism of neurotoxicity. NO reacts with superoxide to generate peroxynitrite, a strong oxidizing and nitrating species. Peroxynitrite is formed in glial cells and degenerating neurons in neuropathological conditions, including amyotrophic lateral sclerosis (ALS). In ALS, motor neurons re-express the p75 neurotrophin receptor (p75NTR) and might become vulnerable to NGF. In the present study, we investigated whether peroxynitrite stimulated nerve growth factor (NGF) expression in spinal cord astrocytes. MATERIALS AND METHODS: Astrocyte monolayers were exposed to peroxynitrite and nitrotyrosine formation was determined by immunofluorescence. mRNA levels for NGF, brain derived neutrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2) were measured by semi-quantitative RT-PCR and NGF release was determined by ELISA. RESULTS AND DISCUSSION: A single exposure to peroxynitrite specifically induced NGF expression and secretion in astrocytes coincident with reactive morphological changes and increased nitrotyrosine immunoreactivity. These results suggest that NGF expression in reactive astrocytes is under the control of oxidative stress.

Assuntos

Astrócitos/fisiologia , Fator Neurotrófico Derivado do Encéfalo/genética , Fator 2 de Crescimento de Fibroblastos/genética , Fator de Crescimento Neural/genética , Ácido Peroxinitroso/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Cinética , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo

RESUMO

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.

Assuntos

Esclerose Lateral Amiotrófica/metabolismo , Apoptose , Astrócitos/metabolismo , Neurônios Motores/metabolismo , Fator de Crescimento Neural/biossíntese , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Astrócitos/patologia , Extratos Celulares/farmacologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Fator de Crescimento Neural/antagonistas & inibidores , Fator de Crescimento Neural/metabolismo , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Ratos , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/antagonistas & inibidores , Receptores de Fator de Crescimento Neural/biossíntese , Medula Espinal/química , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/genética

RESUMO

The vulnerability of oligodendrocytes to excitatory amino acids may account for the pathology of white matter occurring following hypoxia/ischemia or autoimmune attack. Here, we examined the vulnerability of immature oligodendrocytes (positively labeled by galactocerobroside-C and not expressing myelin basic protein) from neonatal rat spinal cord to kainate, an agonist of excitatory amino acid receptors that induces long-lasting inward currents in immature oligodendrocytes. In particular, we studied whether kainate toxicity was linked to the endogenous production of nitric oxide. We found cultured oligodendrocytes to be highly sensitive to 24-48 h exposure to 0.5-1 mM kainate. The toxin induced striking morphological changes in oligodendrocytes, characterized by the disruption of the process network around the cell body and the growth of one or two long, thick and non-branched processes. A longer exposure to kainate resulted in massive death of oligodendrocytes, which was prevented by 6,7, dinitroquinoxaline-2,3-dione (DNQX) (30 micro M), the antagonist of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic/kainate receptors. Remarkably, we found that those oligodendrocytes displaying bipolar morphology following kainate exposure, also expressed the inducible form of nitric oxide synthase (iNOS) and nitrotyrosine immunoreactivity, suggesting that peroxynitrite could be formed by the reaction of nitric oxide with superoxide. Moreover, kainate toxicity was significantly prevented by addition of the NOS inhibitor nitro-L-arginine methyl ester (L-NAME), further suggesting that nitric oxide-derived oxidants contribute to excitotoxic mechanisms in immature oligodendrocytes.

Assuntos

Agonistas de Aminoácidos Excitatórios/toxicidade , Ácido Caínico/toxicidade , Óxido Nítrico/fisiologia , Oligodendroglia/efeitos dos fármacos , Tirosina/análogos & derivados , Animais , Animais Recém-Nascidos , Contagem de Células , Morte Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Inibidores Enzimáticos/farmacologia , Imunofluorescência , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ácido Peroxinitroso/metabolismo , Quinoxalinas/farmacologia , Ratos , Ratos Wistar , Medula Espinal/citologia , Células-Tronco/efeitos dos fármacos , Tirosina/metabolismo

RESUMO

Oxidative stress mediated by nitric oxide (NO) and its toxic metabolite peroxynitrite has previously been associated with motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Degenerating spinal motor neurons in familial and sporadic ALS are typically surrounded by reactive astrocytes expressing the inducible form of NO synthase (iNOS), suggesting that astroglia may have a pathogenic role in ALS. We report here that a brief exposure of spinal cord astrocyte monolayers to peroxynitrite (0.25-1 mM) provoked long-lasting reactive morphological changes characterized by process-bearing cells displaying intense glial fibrillary acidic protein and iNOS immunoreactivity. Furthermore, peroxynitrite caused astrocytes to promote apoptosis of embryonic motor neurons subsequently plated on the monolayers. Neuronal death occurred within 24 hr after plating, as evidenced by the presence of degenerating motor neurons positively stained for activated caspase-3 and nitrotyrosine. Motor neuron death was largely prevented by NOS inhibitors and peroxynitrite scavengers but not by trophic factors that otherwise will support motor neuron survival in the absence of astrocytes. The bacterial lipopolysaccharide, a well-known inflammatory stimulus that induces iNOS expression in astrocytes, provoked the same effects on astrocytes as peroxynitrite. Thus, spinal cord astrocytes respond to extracellular peroxynitrite by adopting a phenotype that is cytotoxic to motor neurons through peroxynitrite-dependent mechanisms.

Assuntos

Esclerose Lateral Amiotrófica/metabolismo , Apoptose/fisiologia , Astrócitos/metabolismo , Neurônios Motores/metabolismo , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Medula Espinal/metabolismo , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Sequestradores de Radicais Livres/farmacologia , Gliose/metabolismo , Gliose/patologia , Gliose/fisiopatologia , Lipopolissacarídeos/farmacologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Óxido Nítrico Sintase/metabolismo , Ácido Peroxinitroso/toxicidade , Fenótipo , Ratos , Receptor de Fator de Crescimento Neural/efeitos dos fármacos , Receptor de Fator de Crescimento Neural/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiopatologia

RESUMO

We have established a cell culture model of spinal cord astrocytes to study the cytotoxicity of peroxynitrite. Nitric oxide (NO) has been implicated as a key contributor to neurotoxicity. NO reacts with superoxide to generate peroxynitrite, a strong oxidant and nitrating agent with deleterious cytotoxic and pro-apoptotic effects. Peroxynitrite and nitrotyrosine are formed in damaged motor neurons in amyotrophic lateral sclerosis (ALS), which are surrounded by reactive astrocytes. To determine the effects of extracellular addition of peroxynitrite, purified astrocyte monolayers prepared from neonatal rat spinal cords were exposed to peroxynitrite (0.25-0.75 mM) for 5 min and further incubated in culture medium for 24-72h. Peroxynitrite exposure did not result in apparent cell loss or damage of the monolayer. However, a substantial number of cells adopted reactive features, with long processes displaying intense immunoreactivity to glial fibrillary acidic protein (GFAP). Western blot analysis performed 24h after peroxynitrite treatment showed that GFAP levels were not modified by the oxidant. There were no changes in cell viability parameters in astrocyte cultures after peroxyintrite, indicating that astrocytes are more resistant to the oxidant than other cell types. Peroxynitrite reacts with protein-bound tyrosine residues to form nitrotyrosine. We observed a modest to strong nitrotyrosine immunoreactivity in astrocytes 24h following peroxynitrite exposure. There was a remarkable association between nitrotyrosine and high-intensity GFAP immunoreactivity in astrocytes bearing long processes. These results suggest that peroxynitrite induces a characteristic long-lasting reactive astrocytic phenotype and provide new insight into understanding the origin of reactive astrocytes occurring in ALS.