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1.
J Cell Biol ; 155(2): 217-26, 2001 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11604419

RESUMO

Peripherin, a neuronal intermediate filament protein associated with axonal spheroids in amyotrophic lateral sclerosis (ALS), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. To further clarify the selectivity and mechanism of peripherin-induced neuronal death, we analyzed the effects of peripherin overexpression in primary neuronal cultures. Peripherin overexpression led to the formation of cytoplasmic protein aggregates and caused the death not only of motor neurons, but also of dorsal root ganglion (DRG) neurons that were cultured from dissociated spinal cords of peripherin transgenic embryos. Apoptosis of DRG neurons containing peripherin aggregates was dependent on the proinflammatory central nervous system environment of spinal cultures, rich in activated microglia, and required TNF-alpha. This synergistic proapoptotic effect may contribute to neuronal selectivity in ALS.


Assuntos
Apoptose , Proteínas de Filamentos Intermediários/ultraestrutura , Glicoproteínas de Membrana , Neurônios Motores/ultraestrutura , Proteínas do Tecido Nervoso/ultraestrutura , Fator de Necrose Tumoral alfa/fisiologia , Esclerose Lateral Amiotrófica/patologia , Animais , Anticorpos/farmacologia , Células Cultivadas , Gânglios Espinais/ultraestrutura , Proteínas de Filamentos Intermediários/genética , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Microinjeções , Proteínas do Tecido Nervoso/genética , Periferinas , Medula Espinal/fisiologia , Fator de Necrose Tumoral alfa/imunologia
2.
Neuroreport ; 12(6): 1239-43, 2001 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-11338199

RESUMO

Enhanced production of nitrotyrosine and subsequent protein nitration has been proposed as the mechanism by which mutant SOD1 causes death of motor neurons in a familial form of amyotrophic lateral sclerosis (FALS-1). We have tested this hypothesis in a primary culture model in which mutant human SOD1 was expressed in motor neurons of dissociated spinal cord cultures. Preventing formation of nitrotyrosine by inhibiting nitric oxide synthase rescued cultured motor neurons from excitotoxic death induced by adding glutamate to the culture medium, but failed to significantly delay death of motor neurons expressing the G93A mutant SOD1. The results do not support generation of nitrotyrosine being the predominant lethal gain of function conferred by mutations in SOD1.


Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Neurônios Motores/enzimologia , Superóxido Dismutase/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico/farmacologia , Humanos , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/metabolismo
3.
J Biol Chem ; 276(16): 12791-6, 2001 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-11278741

RESUMO

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis. In humans and experimental models, death of motor neurons is preceded by formation of cytoplasmic aggregates containing mutant SOD-1 protein. In our previous studies, heat shock protein 70 (HSP70) prolonged viability of cultured motor neurons expressing mutant human SOD-1 and reduced formation of aggregates. In this paper, we report that mutant SOD-1 proteins have altered solubility in cells relative to wild-type SOD-1 and can form a direct association with HSP70 and other stress proteins. Whereas wild-type human and endogenous mouse SOD-1 were detergent-soluble, a portion of mutant SOD-1 was detergent-insoluble in protein extracts of NIH3T3 transfected with SOD-1 gene constructs, spinal cord cultures established from G93A SOD-1 transgenic mouse embryos, and lumbar spinal cord from adult G93A transgenic mice. A direct association of HSP70, HSP40, and alphaB-crystallin with mutant SOD-1 (G93A or G41S), but not wild-type or endogenous mouse SOD-1, was demonstrated by coimmunoprecipitation. Mutant SOD-1.HSP70 complexes were predominantly in the detergent-insoluble fraction. However, only a small percentage of total cellular mutant SOD-1 was detergent-insoluble, suggesting that mutation-induced alteration of protein conformation may not in itself be sufficient for direct interaction with heat shock proteins.


Assuntos
Proteínas de Choque Térmico/metabolismo , Doença dos Neurônios Motores/enzimologia , Doença dos Neurônios Motores/genética , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Células 3T3 , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Proteínas de Choque Térmico/química , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Superóxido Dismutase/genética , Transfecção
4.
Toxicol Sci ; 51(2): 178-83, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10543019

RESUMO

In vitro systems are widely used for investigation of neurotoxicant-induced perturbations of cellular functions. A variety of systems exist that demonstrate certain similarities to neurotoxicant-induced events in the intact animal are discussed, including single-cell types, systems that consider endpoints relevant in toxicology, and systems that consider heterogeneous cell interactions. Relationships between the in vitro and in vivo systems are examined in which ethanol, lead, polychlorinated biphenyl compounds, and organophosphate insecticides are examples. Situations in which the in vitro systems have been used to advantage are provided, along with cautions associated with their use.


Assuntos
Síndromes Neurotóxicas/diagnóstico , Testes de Toxicidade/métodos , Animais , Modelos Animais de Doenças , Humanos , Técnicas In Vitro
5.
J Neurochem ; 72(3): 1081-7, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10037479

RESUMO

PC12 cells are well characterized for their ability to differentiate into neuronal-like cells when challenged with nerve growth factor. It has been reported that the calpain and proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (CI) is also able to induce neurite outgrowth in PC12 cells. In this study, we report that the inhibitor of proteasomal chymotrypsin-like activity, carbobenzoxy-Ile-Glu-(O-tert-butyl)-Ala-Leu-aldehyde (PSI), can also induce differentiation of PC12 cells. Induction of neurite outgrowth with PSI, CI, or its close analogue, carbobenzoxy-Leu-Leu-leucinal (MG132), was associated with stress-activated protein kinase (SAPK) activation. Neurite formation induced by protease inhibition was independent of mitogen-activated protein kinase/extracellular signal-regulated kinase, p38/reactivating kinase, or phosphatidylinositol 3-kinase activities. The exact mechanism by which protease inhibition activates SAPKs remains to be elucidated; however, our results suggest that the SAPK signal transduction cascade may be an alternative and/or parallel pathway in the regulation of neuronal differentiation.


Assuntos
Proteínas Quinases Ativadas por Mitógeno , Neuritos/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Proteínas Quinases/metabolismo , Animais , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/fisiologia , Leupeptinas/farmacologia , Proteína Quinase 12 Ativada por Mitógeno , Neuritos/enzimologia , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , Testes de Precipitina , Inibidores de Proteínas Quinases , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno
6.
J Neurochem ; 72(2): 693-9, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9930742

RESUMO

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene underlie some familial cases of amyotrophic lateral sclerosis, a neurodegenerative disorder characterized by loss of cortical, brainstem, and spinal motor neurons. We present evidence that SOD-1 mutants alter the activity of molecular chaperones that aid in proper protein folding and targeting of abnormal proteins for degradation. In a cultured cell line (NIH 3T3), resistance to mutant SOD-1 toxicity correlated with increased overall chaperoning activity (measured by the ability of cytosolic extracts to prevent heat denaturation of catalase) as well as with up-regulation of individual chaperones/stress proteins. In transgenic mice expressing human SOD-1 with the G93A mutation, chaperoning activity was decreased in lumbar spinal cord but increased or unchanged in clinically unaffected tissues. Increasing the level of the stress-inducible chaperone 70-kDa heat shock protein by gene transfer reduced formation of mutant SOD-containing proteinaceous aggregates in cultured primary motor neurons expressing G93A SOD-1 and prolonged their survival. We propose that insufficiency of molecular chaperones may be directly involved in loss of motor neurons in this disease.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Chaperoninas/metabolismo , Superóxido Dismutase/genética , Células 3T3 , Animais , Sobrevivência Celular/fisiologia , Regulação Enzimológica da Expressão Gênica , Técnicas de Transferência de Genes , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/citologia , Neurônios Motores/enzimologia , Mutação/fisiologia , Fármacos Neuroprotetores/metabolismo , Medula Espinal/citologia , Superóxido Dismutase/metabolismo , Transfecção , Regulação para Cima/fisiologia
7.
J Neurosci ; 18(23): 9673-84, 1998 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-9822728

RESUMO

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a subset of familial cases of amyotrophic lateral sclerosis. Using a primary culture model, we have demonstrated that normally nontoxic glutamatergic input, particularly via calcium-permeable AMPA/kainate receptors, is a major factor in the vulnerability of motor neurons to the toxicity of SOD-1 mutants. Wild-type and mutant (G41R, G93A, or N139K) human SOD-1 were expressed in motor neurons of dissociated cultures of murine spinal cord by intranuclear microinjection of plasmid expression vector. Both a general antagonist of AMPA/kainate receptors (CNQX) and a specific antagonist of calcium-permeable AMPA receptors (joro spider toxin) reduced formation of SOD-1 proteinaceous aggregates and prevented death of motor neurons expressing SOD-1 mutants. Partial protection was obtained by treatment with nifedipine, implicating Ca2+ entry through voltage-gated calcium channels as well as glutamate receptors in potentiating the toxicity of mutant SOD-1 in motor neurons. Dramatic neuroprotection was obtained by coexpressing the calcium-binding protein calbindin-D28k but not by increasing intracellular glutathione levels or treatment with the free radical spin trap agent, N-tert-butyl-alpha-phenylnitrone. Thus, generalized oxidative stress could have contributed in only a minor way to death of motor neurons expressing the mutant SOD-1. These studies demonstrated that the toxicity of these mutants is calcium-dependent and provide direct evidence that calcium entry during neurotransmission, coupled with deficiency of cytosolic calcium-binding proteins, is a major factor in the preferential vulnerability of motor neurons to disease.


Assuntos
Cálcio/fisiologia , Ácido Glutâmico/farmacologia , Neurônios Motores/enzimologia , Superóxido Dismutase/genética , Sinapses/enzimologia , 2-Amino-5-fosfonovalerato/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Calbindina 1 , Calbindinas , Canais de Cálcio/fisiologia , Canais de Cálcio Tipo L , Células Cultivadas , Óxidos N-Cíclicos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Sequestradores de Radicais Livres/farmacologia , Gânglios Espinais/citologia , Glutationa/metabolismo , Ácido Cinurênico/farmacologia , Camundongos , Neurônios Motores/química , Mutagênese/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurotoxinas/farmacologia , Óxidos de Nitrogênio/farmacologia , Estresse Oxidativo/fisiologia , Receptores de AMPA/fisiologia , Proteína G de Ligação ao Cálcio S100/metabolismo , Venenos de Aranha/farmacologia , Medula Espinal/citologia , Superóxido Dismutase/metabolismo , Sinapses/química
8.
Nucleic Acids Res ; 26(2): 486-9, 1998 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-9421504

RESUMO

The immediate/early promoter/enhancer of cytomegalovirus (CMV promoter) is one of the most commonly used promoters for expression of transgenes in eukaryotic cells. In practice, the CMV promoter is often thought of as a constitutively active unregulated promoter. However, we have observed that transcription from the CMV promoter can be up-regulated by a variety of environmental stresses. Many forms of cellular stress stimulate MAP kinase signalling pathways, resulting in activation of stress-activated protein kinases [SAPKs, also called Jun N-terminal kinases (JNKs)] and p38 kinases. We have found that the same conditions that lead to activation of SAPK/JNKs and p38 kinases can also dramatically increase expression from the CMV promoter. Inhibitors of p38 kinases abolished basal transcription from the CMV promoter and completely blocked stress-induced up-regulation of the CMV promoter. Overexpression of a dominant negative JNK kinase had no effect on basal transcription, but significantly reduced up-regulation caused by stress. These results have grave implications for use of the CMV promoter. If the CMV promoter can be up-regulated by cellular stresses, inadvertent activation of the stress kinase pathways may complicate, if not invalidate, the interpretation of a wide range of experiments.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Citomegalovirus/genética , Regulação Viral da Expressão Gênica , Genes Precoces , MAP Quinase Quinase Quinase 1 , Proteínas Quinases Ativadas por Mitógeno , Regiões Promotoras Genéticas , Células 3T3 , Animais , Linhagem Celular , Ativação Enzimática , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/análise , Transcrição Gênica , Transfecção , beta-Galactosidase/genética
9.
J Neurosci Res ; 50(4): 514-21, 1997 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-9404713

RESUMO

Aberrant phosphorylation of neurofilaments, similar to that occurring in various motor neuron diseases, is produced in cultured motor neurons by activation of protein kinase C (PKC). Following exposure to synthetic diacylglycerol, persistent change in the phosphorylation state of C-terminal domains of neurofilament proteins was detected by increased perikaryal immunoreactivity with the antibody SMI34; this antibody recognizes NF-M/NF-H when C-terminal KSP repeat domains are highly phosphorylated. SMI34 labeling of perikarya and dendrites was prevented by pretreatment with either the NMDA receptor antagonist APV or by the Ca2+/calmodulin-dependent protein kinase (CaMK) inhibitor KN-62, but not by antagonists of AMPA/kainate or metabotropic glutamate receptors or by inhibitors of arachidonic acid metabolic pathways. Thus, activation of PKC may induce neurofilament phosphorylation in motor neurons by acting in cooperation with stimulation of NMDA receptors and activation of CaMK. These mechanisms may be relevant to motor neuron disease and other neuronal injuries in which increased PKC activity has been measured.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Proteínas de Neurofilamentos/metabolismo , Proteína Quinase C/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Ácido Araquidônico/metabolismo , Células Cultivadas , Ativação Enzimática , Antagonistas de Aminoácidos Excitatórios/farmacologia , Camundongos , Proteínas de Neurofilamentos/biossíntese , Fosforilação
10.
Neuroreport ; 8(9-10): 2111-5, 1997 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-9243594

RESUMO

First generation, replication-defective adenoviral vectors are highly effective for gene transfer into the central nervous system, but the host's immune response limits the utility of this vector for possible therapy of neurological disease or long-term gene transfer studies in experimental animals. We have demonstrated the effectiveness of FK506 (tacrolimus), a powerful immunosuppressant that readily crosses the blood-brain barrier, in maintaining adenovirus-mediated reporter gene transfer following stereotaxic injection of the recombinant (AdCMVlacZ) into mouse striatum. After 28 days, beta-galactosidase expression was reduced by 75% relative to day 10 in immunocompetent animals, accompanied by an inflammatory reaction in the region of transduced cells; however, in mice receiving daily s.c. injections of FK506, beta-galactosidase activity was maintained at the 10 days post-injection level.


Assuntos
Corpo Estriado/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Imunossupressores/farmacologia , Tacrolimo/farmacologia , Transgenes/genética , Adenoviridae , Animais , Corpo Estriado/metabolismo , Técnicas de Transferência de Genes , Masculino , Camundongos , Fatores de Tempo , beta-Galactosidase/metabolismo
11.
J Neuropathol Exp Neurol ; 56(5): 523-30, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9143265

RESUMO

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene underlie some familial cases of amyotrophic lateral sclerosis (FALS), a neurodegenerative disorder characterized by loss of cortical, brainstem, and spinal motor neurons. To investigate the mechanisms responsible for the toxicity of mutant enzyme, SOD-1 cDNAs bearing mutations found in FALS patients (mSOD) were expressed in cultured spinal motor neurons, dorsal root ganglion (DRG) and hippocampal neurons. Many features of motor neuron disease seen in humans with FALS and in transgenic mouse models were reproduced, including preferential susceptibility of motor neurons to toxicity of mSOD. Abnormal cytoplasmic aggregation of mSOD protein was observed in mSOD-expressing motor neurons, but never in neurons expressing SODwt enzyme, and was followed by evidence of apoptotic cell death. Such aggregates were not observed in nonvulnerable neuronal populations expressing mSOD (DRG or hippocampal neurons). Aggregation of SOD-1 may contribute significantly to the death of motor neurons expressing mutations associated with FALS-1 and the mechanisms leading to aggregation may pertain to the specific vulnerability of motor neurons in this disease.


Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Mutação , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Apoptose/fisiologia , Sobrevivência Celular , Células Cultivadas , Humanos , Camundongos/embriologia , Camundongos Endogâmicos , Doença dos Neurônios Motores/metabolismo , Neurônios Motores/enzimologia , Neurônios Motores/fisiologia , Proteínas de Neurofilamentos/metabolismo , Fosforilação , Medula Espinal/citologia , Medula Espinal/enzimologia , Frações Subcelulares/metabolismo , Distribuição Tecidual
12.
Exp Neurol ; 140(1): 14-20, 1996 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8682175

RESUMO

Replication-defective human type 5 adenoviral recombinants (AVR) are very efficient means of introducing foreign genes into neurons in vitro and in vivo; however, a significant reduction in the number of cells expressing reporter genes has been reported to occur over time. In vitro, this may be due to direct toxicity of the protein product of the transgene or adenoviral molecules. In vivo, in addition, an immune attack by the host could eliminate the transduced cells. To assess the direct toxicity of AVR or reporter gene products, a quantitative study of survival of transduced neurons over a period of 4 weeks was conducted in primary neural cultures. Cultures of dissociated murine spinal cord-dorsal root ganglia were exposed to AVR containing the Escherichia coli lacZ (E. coli lacZ) gene under control of either the very efficient cytomegalovirus enhancer/promoter or the fast muscle troponin I promoter, which is not active in these cells. Two factors contributed to loss of neuronal and nonneuronal cells: (i) direct toxicity of (E1 + E3)-deleted replication-incompetent AVR at high titers [> or = 5 x 10(8) viral particles/ml or multiplicity of infection (m.o.i.) 1000] and (ii) high levels of expression of the reporter gene product, beta-galactosidase, at titers that result in 55-75% transduction efficiency (5 x 10(7)-5 x 10(8) viral particles/ml or m.o.i. 100-1000). Despite the efficacy of adenoviral vectors in introducing foreign genes into primary, postmitotic cells, specific precautions must be taken in their use because of the narrow margin between concentrations of recombinants that transduce a sufficient percentage of cells and those that are cytotoxic.


Assuntos
Adenoviridae , Contagem de Células/efeitos dos fármacos , Galactosidases/efeitos dos fármacos , Sistema Nervoso/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos , Sistema Nervoso/metabolismo
13.
J Toxicol Environ Health ; 48(1): 57-69, 1996 May.
Artigo em Inglês | MEDLINE | ID: mdl-8637058

RESUMO

With the aim of identifying a surrogate marker for the neurotoxic effects of methylmercury using a peripheral blood sample, the sensitivity of microtubules in circulating blood cells to depolymerization by methylmercury was compared. Methylmercuric chloride was added to samples of human venous blood or to isolated platelets and lymphocytes (human or rabbit) suspended in RPMI medium plus 10% fetal calf serum. After 1 h, microtubular networks were visualized by immunolabeling with antibody specific for tubulin. The percentage of platelets without visible, intact microtubules and the percentage of viable, unactivated lymphocytes without microtubules visibly radiating from the centriolar region through the cytoplasm were counted. A concentration-dependent loss of microtubules was observed in both cell types. Loss of microtubules was more easily quantitated and was observed at significantly lower concentrations in platelets compared to lymphocytes. The IC50 (concentration of methylmercuric chloride resulting in dissolution of microtubules in 50% of the cells) was 3.1 microM in platelets and 7.4 microM in lymphocytes in samples exposed in culture medium without erythrocytes. When methylmercury was added to whole blood for 1 h, the IC50 increased to 182 microM in platelets and >700 microM in lymphocytes, consistent with the known sequestration of methylmercury in erythrocytes. With longer durations of exposure, much lower concentrations of methylmercury were effective in both cell types. When rabbit lymphocytes and platelets were exposed to methylmercury under culture conditions, IC50s in platelets/lymphocytes were 2.5 microM/4.8 microM after 1 h of exposure, 0.77 microM/1.12 microM after 20 h, and 0.51 microM/0.63 microM after 70 h. The results of this study suggest that platelets may be more suitable than lymphocytes as a cell type in which to monitor in vivo effects of methylmercury on microtubules.


Assuntos
Plaquetas/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Microtúbulos/efeitos dos fármacos , Adulto , Animais , Plaquetas/citologia , Plaquetas/ultraestrutura , Células Cultivadas , Meios de Cultura , Relação Dose-Resposta a Droga , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Dose Letal Mediana , Linfócitos/citologia , Linfócitos/ultraestrutura , Masculino , Compostos de Metilmercúrio/sangue , Microtúbulos/metabolismo , Coelhos
14.
J Neuropathol Exp Neurol ; 55(2): 246-56, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8786383

RESUMO

Characteristic responses of motor neurons to injury include an apparent increase in the phosphorylation of C-terminal domains of neurofilament proteins in the perikaryal and dendritic compartments. This change was induced in dissociated cultures of embryonic spinal cord by activation of protein kinase C (PKC). PKC was activated by: (a) exposure of cultures to 10 nM 12-o-tetradecanoyl phorbol 13-acetate (TPA); (b) microinjection of 1 mM dioctanoylglycerol (diC8) directly into perikaryal of motor neurons;(c) addition of 10 microM diC8 to culture medium. Activation of PKC led to different immediate and long term effects on neurofilaments of motor neurons. After 30 minutes (min), fragmentation of the neurofilament network was observed by labeling with antibodies to low and high molecular weight neurofilaments proteins; glial filaments were disassembled after 10 min and reassembled by 1 hour (h). From 4 to 24 h, motor neuron were observed with extensions of perikaryal cytoplasm or massive enlargements of proximal dendritic processes, both containing intact neurofilament networks. Over 1 to 12 days, there was a gradual increase in the number of motor neuronal perikarya immunoreactive with antibodies to neurofilament proteins phosphorylated at KSP sites on the C-terminal domains (SMI31, SMI34). It is proposed that activation of PKc secondary to other injurious events may contribute to the changes in neurofilaments observed in motor neuron diseases.


Assuntos
Doença dos Neurônios Motores/metabolismo , Neurônios Motores/metabolismo , Proteínas de Neurofilamentos/metabolismo , Proteína Quinase C/metabolismo , Animais , Células Cultivadas , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos , Fosforilação , Medula Espinal/metabolismo , Fatores de Tempo
15.
Clin Exp Pharmacol Physiol ; 22(5): 366-7, 1995 May.
Artigo em Inglês | MEDLINE | ID: mdl-7554432

RESUMO

1. Clonal cell lines, primary cultured neurones and transgenic animals expressing mutant genes linked to familial forms of neurodegenerative diseases provide models in which to examine the interaction between expression of a predisposing gene and exposure to neurotoxic chemicals. Methods of establishing these models are reviewed. 2. Mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD-1) have been identified in cases of familial amyotrophic lateral sclerosis linked to chromosome 21. We report that in clonal lines of PC12 cells, the cytotoxicity of a glutathione-depleting epoxide, styrene oxide, varied with SOD activity in a manner similar to that previously demonstrated for redox cycling chemicals. These preliminary data suggest that either low or high SOD-1 activities may be associated with greater toxicity of a variety of neurotoxic chemicals and their metabolites.


Assuntos
Esclerose Lateral Amiotrófica/genética , Compostos de Epóxi/toxicidade , Neurônios/citologia , Neurotoxinas/toxicidade , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/enzimologia , Animais , Linhagem Celular , Células Cultivadas , Cromossomos Humanos Par 21/efeitos dos fármacos , Células Clonais , Técnicas de Cultura , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Camundongos , Mutação/efeitos dos fármacos , Mutação/genética , Oxirredução , Células PC12/efeitos dos fármacos , Ratos , Superóxido Dismutase/metabolismo , Transfecção
16.
J Neurosci Res ; 38(6): 629-39, 1994 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-7807580

RESUMO

It was previously reported that a monoclonal antibody to beta-amyloid precursor protein (mab22C11; Boehringer Mannheim, Indianapolis, IN) labels an intermediate filament-associated protein (beta APP-IFAP) in cultured human skin fibroblasts (Dooley et al.: J Neurosci Res 33:60-67, 1992). The time course of its expression and association with different classes of intermediate filaments has been assessed in neurons, Schwann cells, and astrocytes in dissociated cultures of murine brain and spinal cord-dorsal root ganglia; in primary cultures of human muscle; and in the epithelial cell line PtK1. beta APP-IFAP was expressed in all non-neuronal cell types examined. Mab22C11 immunoreactivity was minimal or absent following dissociation or subculture, but gradually increased with time. In fibroblasts, myoblasts, and epithelial cells, the distribution eventually resembled that of vimentin. With the exception of glial fibrillary acidic protein (GFAP), beta APP-IFAP was not associated with the intermediate filament proteins characteristically found in differentiated cells, i.e., desmin, the cytokeratins, and neurofilament proteins. No labeling of neurons by mab22C11 was observed at any stage of in vitro maturation. In sections of Alzheimer's brain, the antibody labeled a subpopulation of reactive astrocytes. It is suggested that beta APP-IFAP may be the product of a member of the beta APP multigene family expressed developmentally in non-neuronal cells.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Sistema Nervoso Central/embriologia , Embrião de Mamíferos/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário e Fetal , Células Epiteliais , Epitélio/metabolismo , Fibroblastos/metabolismo , Humanos , Rim/citologia , Rim/metabolismo , Marsupiais , Camundongos/embriologia , Músculos/citologia , Músculos/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Vimentina/metabolismo
17.
Neurotoxicology ; 14(4): 381-6, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8164884

RESUMO

Methods to evaluate the neurotoxicity of chemicals in vitro must take into account that many xenobiotics exert their toxic effects through metabolites. S9 fraction of liver homogenate has been used in cultures of bacterial and mammalian cells as a system for metabolic activation. The suitability of the S9 activation system for long-term neurotoxicity studies in vitro was investigated in dissociated cultures of murine spinal cord-dorsal root ganglia. Exposure to amounts of S9 greater than 0.07 mg S9 protein/ml of culture medium for 4 days or longer was cytotoxic to all types of neurons in the cultures. Non-neuronal cells tolerated higher exposures, but contained numerous cytoplasmic inclusions when 0.35 mg S9 protein was included in the medium. It was demonstrated that cytotoxicity was caused by the particulate, microsomal fraction of S9. It is concluded that direct incorporation of S9 fraction in the growth medium (0.07 mg S9 protein/ml or greater) is not a suitable method of generating metabolites in dissociated cultures of central nervous system when several days are required to elicit a biological response. Cytotoxicity can be prevented by using tissue culture inserts to separate cultured cells from S9 particulate fraction by a microporous membrane.


Assuntos
Biotransformação/fisiologia , Fígado/metabolismo , Neurônios/fisiologia , Frações Subcelulares/metabolismo , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/citologia , Masculino , Ratos , Ratos Sprague-Dawley
18.
Neurotoxicology ; 14(4): 387-95, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-7909362

RESUMO

NSC-34 is a hybrid cell line produced by fusion of motor neuron enriched, embryonic mouse spinal cord cells with mouse neuroblastoma. Cultures contain two populations of cells: small, undifferentiated cells that have the capacity to undergo cell division and larger, multi-nucleate cells that express many properties of motor neurons. The utility of NSC-34 cells as a model for investigation of neurotoxicity was evaluated following exposure of cultures to a selection of chemicals known to be neurotoxic to motor neurons. NSC-34 responded to agents that affect voltage-gated ion channels, cytoskeletal organization and axonal transport. The sensitivity of action potential production to various ion channel blockers was similar to that in primary motor neurons in culture. 2,5-hexanedione induced focal aggregation of neurofilaments in perikarya and processes of NSC-34. Sodium pyridinethione induced swelling and retraction of processes. In contrast, NSC-34 was not a good model in which to investigate agents that affect synaptic transmission. No electrophysiological evidence of synaptic connections between NSC-34 cells was obtained. Exposure to 1 mM glutamate had no effect on cell morphology or action potential production. Difficulties in using this line to investigate chemical neurotoxicity were poor substrate adhesion, requirement for routine subculture and change in expression of the neuronal phenotype with repeated subculture.


Assuntos
Células Híbridas/fisiologia , Modelos Neurológicos , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neoplasias da Medula Espinal/patologia , Medula Espinal/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Linhagem Celular , Meios de Cultura , Estudos de Avaliação como Assunto , Glutamatos/farmacologia , Ácido Glutâmico , Hexanonas/farmacologia , Camundongos , Neurotransmissores/farmacologia , Piridinas/farmacologia , Medula Espinal/citologia , Tetrodotoxina/farmacologia , Tionas
19.
J Neurosci Res ; 33(1): 60-7, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1453484

RESUMO

The expression of beta-amyloid precursor protein (BAPP) and its mRNAs was studied in fibroblasts obtained from patients afflicted with Alzheimer's disease (AD) and age-matched controls. Using reverse transcriptase-polymerase chain reaction (RT-PCR), transcripts corresponding to 770, 751, 714, and 695 amino acids were detected in both AD and control fibroblasts. Antibody 22C11 against BAPP (Boehringer Mannheim) labeled an intracellular protein, specifically localized to the intermediate filament network. In addition to bands of the predicted molecular weights for BAPP (120-135 kDa), Western blotting revealed a 57 kDa band which was not evident in samples of human brain. As cytoskeletal elements are vital in maintaining cellular architecture and various cell interactions, localization of BAPP or a related molecule to the cytoskeleton suggests a possible structural role for this protein within the cell.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/imunologia , Fibroblastos/metabolismo , Proteínas de Filamentos Intermediários/imunologia , Doença de Alzheimer/imunologia , Especificidade de Anticorpos , Western Blotting , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Fibroblastos/imunologia , Humanos , Imuno-Histoquímica , Microinjeções , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Pele/imunologia , Pele/metabolismo , Vimentina/metabolismo
20.
Dev Dyn ; 194(3): 209-21, 1992 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1467557

RESUMO

We have developed a series of mouse-mouse neural hybrid cell lines by fusing the aminopterin-sensitive neuroblastoma N18TG2 with motor neuron-enriched embryonic day 12-14 spinal cord cells. Of 30 neuroblastoma-spinal cord (NSC) hybrids displaying a multipolar neuron-like phenotype, 10 express choline acetyltransferase, and 4 induce twitching in cocultured mouse myotubules. NSC-19, NSC-34, and their subclones express additional properties expected of motor neurons, including generation of action potentials, expression of neurofilament triplet proteins, and acetylcholine synthesis, storage, and release. In addition, NSC-34 cells induce acetylcholine receptor clusters on cocultured myotubes, and undergo a vimentin-neurofilament switch with maturation in culture, similar to that occurring in neuronal development. NSC cell lines appear to model selected aspects of motor neuron development in an immortalized clonal system.


Assuntos
Hibridização Genética , Neurônios Motores/fisiologia , Neuroblastoma/patologia , Medula Espinal/patologia , Acetilcolinesterase/metabolismo , Animais , Fusão Celular , Linhagem Celular , Técnicas Citológicas , Proteínas do Citoesqueleto/metabolismo , Eletrofisiologia , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/fisiopatologia , Sistema Nervoso Parassimpático/citologia , Medula Espinal/metabolismo , Medula Espinal/fisiologia , Sinapses/fisiologia
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