RESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, degenerative disorder of motor neurons. The causes of most cases of ALS are as yet undefined. In a previous study, it was shown that N-methyl-D-aspartate (NMDA) and H(2)O(2) stimuli reduce neuronal survival in cortical neurons in culture (Boutahar et al., 2008). To identify variations in gene expression in response to these neurotoxins in transgenic vs. control cortical neurons cultures, both microarray and RT-PCR analysis were performed. High-density oligonucleotide microarrays showed changes in the expression of about 600 genes involved in protein degradation, neurotrophic factors pathway, cell cycle, inflammation, cytoskeleton, cell adhesion, transcription, or signalling. The most up-regulated genes following H(2)O(2) treatment were involved in cytoskeletal organization and axonal transport, such as ARAP2, KIF17, and DKK2, or in trophic factors pathways, such as insulin-like growth factor-binding protein 4 (IGFBP4), FGF17, and serpin2. The most down-regulated genes were involved in ion transport, such as TRPV1. After NMDA treatment, the most up-regulated genes were involved in protein degradation, such as ubiquitin-conjugating enzyme E2I and cathepsin H, and the most down-regulated genes were involved in ion transport, such as SCN7A. We conclude that these neurotoxins act through different transcriptional inductions, and these changes may reflect an adaptative cellular response to the cellular stress induced by the neurotoxins involved in ALS in the presence of mutant human SOD1.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Neurônios/metabolismo , Superóxido Dismutase/metabolismo , Análise de Variância , Animais , Sobrevivência Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Perfilação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , N-Metilaspartato/farmacologia , Neurotoxinas/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Oxidantes/farmacologia , Estresse Oxidativo , Estatísticas não Paramétricas , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
INTRODUCTION: The objective of this study was to determine the ability of various parameters commonly used for the diagnosis of acute meningitis to differentiate between bacterial and viral meningitis, in adult patients with a negative direct cerebrospinal fluid (CSF) examination. METHODS: This was a prospective study, started in 1997, including all patients admitted to the emergency unit with acute meningitis and a negative direct CSF examination. Serum and CSF samples were taken immediately on admission. The patients were divided into two groups according to the type of meningitis: bacterial (BM; group I) or viral (VM; group II). The CSF parameters investigated were cytology, protein, glucose, and lactate; the serum parameters evaluated were C-reactive protein and procalcitonin. CSF/serum glucose and lactate ratios were also assessed. RESULTS: Of the 254 patients with meningitis with a negative direct CSF examination, 35 had BM and 181, VM. The most highly discriminative parameters for the differential diagnosis of BM proved to be CSF lactate, with a sensitivity of 94%, a specificity of 92%, a negative predictive value of 99%, a positive predictive value of 82% at a diagnostic cut-off level of 3.8 mmol/L (area under the curve (AUC), 0.96; 95% confidence interval (CI), 0.95 to 1), and serum procalcitonin, with a sensitivity of 95%, a specificity of 100%, a negative predictive value of 100%, and a positive predictive value of 97% at a diagnostic cut-off level of 0.28 ng/ml (AUC, 0.99; 95% CI, 0.99 to 1). CONCLUSIONS: Serum procalcitonin and CSF lactate concentrations appear to be the most highly discriminative parameters for the differential diagnosis of BM and VM.
Assuntos
Calcitonina/sangue , Ácido Láctico/líquido cefalorraquidiano , Meningites Bacterianas/diagnóstico , Meningite Viral/diagnóstico , Precursores de Proteínas/sangue , Doença Aguda , Adolescente , Adulto , Idoso , Área Sob a Curva , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Proteína C-Reativa/análise , Peptídeo Relacionado com Gene de Calcitonina , Líquido Cefalorraquidiano/química , Líquido Cefalorraquidiano/citologia , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Diagnóstico Diferencial , Feminino , Glucose/líquido cefalorraquidiano , Humanos , Masculino , Meningites Bacterianas/sangue , Meningites Bacterianas/líquido cefalorraquidiano , Meningite Viral/sangue , Meningite Viral/líquido cefalorraquidiano , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Adulto JovemRESUMO
Neurotrophins protect neurons against glutamate and oxidative stress, but the underlying mechanism remains unclear. We investigated the neuroprotective role of the neurotrophin brain-derived neurotrophic factor (BDNF) in neuronal cultures subjected to NMDA or H(2)O(2) toxicity and analyzed the molecular mechanisms involved, particularly those related to regulation of cell cycle or endoplasmic reticulum (ER) stress. Preincubation with BDNF of cortical neuron cultures prevented NMDA- or H(2)O(2)-induced neuronal death as well as MAPK-ERK1/2 activation. Inhibition of phosphatidylinositol 3-kinase (PI3-K) abolished the protective effect of BDNF. NMDA and H(2)O(2) induced activation of cell cycle reentry regulators such as retinoblastoma (Rb) protein and E2F1 transcription factor. However, BDNF abolished the activation of both factors. NMDA-induced expression of chaperone encoding gene BIP was slightly inhibited by BDNF, but it did not affect expression of ER stress protein CHOP. Our results suggest that BDNF neuroprotection may be mediated through inhibition of Ras-MAPK pathway and cell cycle reentry during oxidative or excitotoxic stress responses. However, BDNF did not modify expression of ER stress signal induced by NMDA.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Ciclo Celular/fisiologia , Retículo Endoplasmático/metabolismo , Neurônios/fisiologia , Estresse Oxidativo/fisiologia , Estresse Fisiológico/fisiologia , Animais , Morte Celular/fisiologia , Córtex Cerebral/fisiologia , Peróxido de Hidrogênio/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , N-Metilaspartato/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de TempoRESUMO
Clinical magnetic resonance imaging (MRI) offers a noninvasive diagnostic tool for neurodegenerative diseases. MRI was performed on mice to investigate a relationship between brain atrophy and overexpression of two genes involved in such diseases, SOD1 (superoxide dismutase) and APP (amyloid precursor protein), which have been associated with pathogenesis of Alzheimer's disease or Down syndrome. Additionaly, we investigated how life span and growth rate were affected by genetic background. T2-weighted MRI made possible the measurement of the volume of brain regions of interest in living transgenic mice that overexpress normal APP, SOD1, or both. The most pronounced alterations in gray matter volume were observed in 1-year-old double APP/SOD1 transgenic mice. Hippocampus, entorhinal, and cingulate cortex volumes were decreased by 8% to 25%. In contrast, mice homozygous for SOD1 exhibited atrophy specifically in cortex regions (cingulate, retrosplenial, and temporoparietal cortex), but no significant modification was found in the hippocampus region. None of these alterations was seen in single APP transgenics. However, the life span of these mice was significantly shortened. SOD1 overexpression prevented APP toxicity with regard to premature death, especially in double APP/SOD1 transgenic animals homozygous for SOD1, and increase in life span was significantly correlated to SOD1 activity. In conclusion, overexpression of both APP and SOD1, in contrast to single APP transgenics, produced a robust effect on brain anatomy but did not impair growth or life span. Consequences of genotype alterations on brain atrophy may be dissociated from their effect on life span.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide/genética , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Imageamento por Ressonância Magnética , Superóxido Dismutase/genética , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Atrofia , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Transgênicos , RadiografiaRESUMO
Oxidative stress and excitotoxicity are both involved in the pathogenesis of neuronal degenerative diseases like ALS. In order to compare their action, some key proteins involved in their respective signaling pathways, particularly ERK and p53, were analyzed in primary cultures of cortical neurons subjected to NMDA or H(2)O(2) treatment. Early ERK activation was detected after NMDA treatment and was maintained during 24 h, but not after H(2)O(2) treatment. Early p53 expression was also found after NMDA treatment but diminished later. On the other hand, it progressively increased from 6 h to 24 h after H(2)O(2) treatment. Blocking ERK1/2 activation with the upstream inhibitor U0126 inhibited NMDA-mediated p53 expression, suggesting that ERK1/2 signals drive the cells to apoptosis under these conditions. In order to identify the initial membrane target of these neurotoxins, PAK1 was analyzed. Early increase of PAK1 expression was measured after NMDA treatment and was still present after 24 h. Conversely increased PAK1 expression was only detected 24 h after H(2)O(2) treatment. In order to define the components through which NMDA or H(2)O(2) induce the final elements of these pathways, p21 and c-jun, we have performed a detailed functional analysis of c-jun and p21 promoters following plasmid transfection. Both p21 and c-jun were activated after NMDA treatment, but this activation was abolished after H(2)O(2) treatment. We conclude that NMDA induces an early effect that involves activation of p53, ERK, PAK1, p21 and c-jun. On the other hand, H(2)O(2) induces long-term p53 expression, late expression of PAK1 without activation of p21 promoter. The timing differences of the action of these neurotoxins may explain why the presence of both compounds is needed to induce neuronal death.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Agonistas de Aminoácidos Excitatórios/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Radicais Livres/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Peróxido de Hidrogênio/farmacologia , Camundongos , N-Metilaspartato/metabolismo , Neurônios/citologia , Neurotoxinas/farmacologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Tempo , Proteína Supressora de Tumor p53/efeitos dos fármacosRESUMO
BACKGROUND: In cerebrospinal fluid (CSF), which is a rich source of biomarkers for neurological diseases, identification of biomarkers requires methods that allow reproducible detection of low abundance proteins. It is therefore crucial to decrease dynamic range and improve assessment of protein abundance. RESULTS: We applied LC-MS/MS to compare the performance of two CSF enrichment techniques that immunodeplete either albumin alone (IgYHSA) or 14 high-abundance proteins (IgY14). In order to estimate dynamic range of proteins identified, we measured protein abundance with APEX spectral counting method.Both immunodepletion methods improved the number of low-abundance proteins detected (3-fold for IgYHSA, 4-fold for IgY14). The 10 most abundant proteins following immunodepletion accounted for 41% (IgY14) and 46% (IgYHSA) of CSF protein content, whereas they accounted for 64% in non-depleted samples, thus demonstrating significant enrichment of low-abundance proteins. Defined proteomics experiment metrics showed overall good reproducibility of the two immunodepletion methods and MS analysis. Moreover, offline peptide fractionation in IgYHSA sample allowed a 4-fold increase of proteins identified (520 vs. 131 without fractionation), without hindering reproducibility. CONCLUSIONS: The novelty of this study was to show the advantages and drawbacks of these methods side-to-side. Taking into account the improved detection and potential loss of non-target proteins following extensive immunodepletion, it is concluded that both depletion methods combined with spectral counting may be of interest before further fractionation, when searching for CSF biomarkers. According to the reliable identification and quantitation obtained with APEX algorithm, it may be considered as a cheap and quick alternative to study sample proteomic content.
Assuntos
Acetilcisteína/efeitos adversos , Bloqueadores dos Canais de Cálcio/efeitos adversos , Ponte de Artéria Coronária sem Circulação Extracorpórea , Diltiazem/efeitos adversos , Sequestradores de Radicais Livres/efeitos adversos , Coração/efeitos dos fármacos , Cuidados Intraoperatórios , Estresse Oxidativo/efeitos dos fármacos , Acetilcisteína/administração & dosagem , Bloqueadores dos Canais de Cálcio/administração & dosagem , Diltiazem/administração & dosagem , Quimioterapia Combinada , Sequestradores de Radicais Livres/administração & dosagem , Humanos , Traumatismo por Reperfusão/prevenção & controleRESUMO
Oxidative stress and excitotoxicity have been implicated as triggering factors in various neurodegenerative diseases or acute neurological insults. Cu/Zn superoxide dismutase (SOD1), a potent free radical scavenging factor, may prevent the progression of such diseases. In the present study, we show that SOD1 overexpression promoted the survival of cortical neuronal cultures originating from mice carrying the human SOD1 transgene. SOD1 overexpression significantly protected against the deleterious effect of reactive oxygen species, ceramide, or N-methyl-D-aspartate (NMDA). It also preserved cortical neurons against apoptosis induced by NMDA or ceramide, as revealed by a smaller increase in caspase 3 activity. SOD1 overexpression was correlated with higher SOD1 activity, and neurotoxins induced an increase in SOD1 activity in cultures from both mice. Moreover, the ratio of increase of SOD1 in cultures from nontransgenic vs. transgenic mice was similar in control cultures or following neurotoxins administration. The highest amount of neurotoxin-induced SOD1 activity was generated by NMDA. Moreover, following exposure to hydrogen peroxide, the cytoskeletal organization was altered, as evidenced by modifications of beta-tubulin or MAP2 labelling. The fact that increased superoxide dismutase activity protected neurons suggests that appropriate control of SOD1 activity is required for neuronal survival under stressful conditions.