RESUMO
Mitochondrial dysfunction and oxidative stress play an important role in ageing and have been implicated in several age-related neurodegenerative conditions including Alzheimer's disease (AD) and other tauopathies characterized by the presence of intracellular accumulations of the hyperphosphorylated microtubule-associated protein tau. To study the interaction between mitochondrial dysfunction and tau pathology in vivo, we generated a novel mouse model by crossbreeding two existing lines: the Harlequin (Hq) mutant mice which suffer from mitochondrial dysfunction and oxidative stress due to a lack of the mitochondrial apoptosis-inducing factor (AIF), and the P301L tau transgenic mice, a mouse model of human tau pathology. Combined expression of the Hq mouse mutation and the tau transgene in the Tau/Hq double mutant mice led to an increase in tau pathology and apoptotic neurodegeneration when compared to single expression of the two mutations. Neurodegeneration was most prominent in the dentate gyrus and was significantly increased in the cerebellum leading to aggravated motor deficits. Functional activity measurements of the mitochondrial respiratory chain (MRC) in the Tau/Hq mice revealed early decreased activities of multiple MRC complexes and depleted ATP levels which preceded neurodegeneration and elevated oxidative stress markers. These results suggest an age-dependent mutual reinforcement of the tau pathology and mitochondrial dysfunction in vivo, which may contribute to neurodegeneration in patients suffering from AD and other age-related tauopathies.
Assuntos
Complexo I de Transporte de Elétrons/genética , Doenças Mitocondriais/genética , Mutação/genética , Degeneração Neural/genética , Tauopatias/genética , Proteínas tau/genética , Trifosfato de Adenosina/metabolismo , Fatores Etários , Animais , Apoptose/genética , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Caspase 3/metabolismo , Complexo I de Transporte de Elétrons/deficiência , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Predisposição Genética para Doença , Humanos , Marcação In Situ das Extremidades Cortadas , Leucina/genética , Peroxidação de Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Transtornos dos Movimentos/genética , Transtornos dos Movimentos/fisiopatologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo/genética , Prolina/genética , Bombas de Próton/metabolismo , Coloração pela Prata/métodos , Estatísticas não ParamétricasRESUMO
PURPOSE: This study aims at a first evaluation of the outcome of the Swiss national MD-PhD program during the last 16 years. METHOD: One hundred and twenty six former and current students in the Swiss national MD-PhD program were surveyed via a Web-based questionnaire in September 2007. Twenty-four questions assessed information regarding participant demographics, information on the PhD thesis and publication activity, current positions and research activity, as well as participant's opinions, attitudes and career goals. RESULTS: Eighty questionnaires were received from 126 MD-PhD students and graduates (63.5% response rate). The responders consisted of present students (36%), former graduates (56%), and dropouts (8%). The percentage of women in the program was 23%, and the average duration of the program was 4.2 +/- 1.4 years. Research interests were predominantly in the fields of neuroscience, immunology, molecular biology and cancer research. A considerable portion of the MD-PhD graduates had an excellent publication record stemming from their PhD research work, and 89% were planning to continue a research-orientated career. Over 50% of those MD-PhD graduates completing their thesis before 2002 had already reached an assistant or full professor position at the time of the survey. Nearly all participants considered the MD-PhD training helpful to their career and high quality standards were assigned to the acquired practical and intellectual skills. However, criticism was expressed concerning the general mentoring and the career related mentoring. Moreover, general mentoring and career related mentoring were significantly less well perceived in research groups employing more than seven PhD students at the same time. CONCLUSIONS: The MD-PhD students and graduates surveyed were satisfied with their education and most of them continued a research-orientated career. Regarding the overall positive evaluation, this study supports the view that MD-PhD graduates are well qualified for a successful career in academic medicine.
Assuntos
Atitude do Pessoal de Saúde , Pesquisa Biomédica/educação , Escolha da Profissão , Educação de Pós-Graduação em Medicina/organização & administração , Estudantes de Medicina/psicologia , Adulto , Autoria , Pesquisa Biomédica/organização & administração , Feminino , Humanos , Satisfação no Emprego , Masculino , Mentores , Pessoa de Meia-Idade , Avaliação de Programas e Projetos de Saúde , Inquéritos e Questionários , SuíçaRESUMO
Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24(-/-) mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased beta-secretase activity and diminished Abeta generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24(-/-) mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24(-/-) mouse.
Assuntos
Envelhecimento/metabolismo , Desmosterol/metabolismo , Microdomínios da Membrana/fisiologia , Proteínas do Tecido Nervoso/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Córtex Cerebral/citologia , Colesterol/deficiência , Fibrinolisina/metabolismo , Gangliosídeo G(M1)/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Plasminogênio/metabolismo , Ligação Proteica , Proteolipídeos/metabolismoRESUMO
DHCR24/seladin-1, a crucial enzyme in sterol synthesis, is of lower abundance in brain areas affected by Alzheimer's disease. While high levels of DHCR24/seladin-1 exert antiapoptotic function by conferring resistance against oxidative stress, the molecular mechanism for this protective effect is not fully understood. Here we show that DHCR24/seladin-1 expression is up-regulated in an acute response and down-regulated in a chronic response to oxidative stress. High levels of DHCR24/seladin-1 were associated with elevated cholesterol concentrations and a general increase in cholesterol biosynthesis upon oxidative stress exposure in neuroblastoma SH-SY5Y cells. DHCR24/seladin-1 overexpression conferred resistance to oxidative stress in a cholesterol-dependent manner. Mutating the reductase activity within DHCR24/seladin-1 abolished this protective effect. Conversely, DHCR24/seladin-1 levels diminished upon chronic exposure to oxidative stress. Low levels of DHCR24/seladin-1 were associated with reduced p53 levels, independent of DHCR24 activity and cholesterol concentrations. Additionally, ablation of DHCR24/seladin-1 prevented apoptosis of primary neurons in a p53-dependent manner and reduced the response of critical p53 targets due to deficient stabilization of p53 and therefore elevated p53 ubiquitination and degradation. Our findings reveal a dual capacity of DHCR24/seladin-1, which appears to be involved in two mechanistically independent prosurvival effects, exerting an acute response and a chronic response to oxidative stress.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Colesterol/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Camundongos , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , RNA Interferente Pequeno/genética , Fatores de Tempo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , UbiquitinaçãoRESUMO
The cholesterol-synthesizing enzyme seladin-1, encoded by the Dhcr24 gene, is a flavin adenine dinucleotide-dependent oxidoreductase and regulates responses to oncogenic and oxidative stimuli. It has a role in neuroprotection and is downregulated in affected neurons in Alzheimer's disease (AD). Here we show that seladin-1-deficient mouse brains had reduced levels of cholesterol and disorganized cholesterol-rich detergent-resistant membrane domains (DRMs). This was associated with inefficient plasminogen binding and plasmin activation, the displacement of beta-secretase (BACE) from DRMs to APP-containing membrane fractions, increased beta-cleavage of APP and high levels of Abeta peptides. In contrast, overexpression of seladin-1 increased both cholesterol and the recruitment of DRM components into DRM fractions, induced plasmin activation and reduced both BACE processing of APP and Abeta formation. These results establish a role of seladin-1 in the formation of DRMs and suggest that seladin-1-dependent cholesterol synthesis is involved in lowering Abeta levels. Pharmacological enhancement of seladin-1 activity may be a novel Abeta-lowering approach for the treatment of AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Colesterol/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Colesterol/metabolismo , Primers do DNA , Endopeptidases/metabolismo , Fibrinolisina/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Plasminogênio/metabolismo , Nexinas de Proteases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não ParamétricasRESUMO
Host cell reactions are a crucial determinant for tumor invasion. We analyzed on a genomewide scale gene expression differences between microdissected tissues taken from unaffected liver tissue of a human colorectal tumor (LS174) growing in the livers of nude mice and tissue from the host part of the invasive front. Due to the low degree of interspecies cross-hybridization of 15% as determined on Affymetrix microarrays, our xenograft model allowed for the distinction of genes of murine versus human origin even if the respective tissues could not be isolated separately. Using the gene ontology (GO) classification, we were able to determine patterns of up- and downregulated genes in the liver part of the invasive front. We observed a pronounced overrepresentation, e.g., of the GO terms "extracellular matrix," "cell communication," "response to biotic stimulus," "structural molecule activity" and "cell growth," indicating a very pronounced host cell response to tumor invasion. On the single gene level, hepatic stellate cell (HSC) activation markers were overrepresented in the liver part of the invasion front. Immunohistochemistry and qPCR confirmed an activation of HSC as well as an increased number of HSC in the invasive front as compared to the noninvaded liver tissue. In summary, our data demonstrate the feasibility of an interspecies differential gene expression approach on a genomewide scale.
Assuntos
Adenocarcinoma/patologia , Adenocarcinoma/secundário , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Invasividade Neoplásica/genética , Animais , Regulação para Baixo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase , Transplante Heterólogo , Regulação para CimaRESUMO
Numerous nuclear gene products are required for the correct expression of organellar genes. One such gene in the green alga Chlamydomonas reinhardtii is MCD1, whose product is required for stability of the chloroplast-encoded petD mRNA. In mcd1 mutants, which are non-photosynthetic, petD mRNA is degraded by a 5'-3' exonuclease activity, resulting in a failure to synthesize its product, subunit IV of the cytochrome b (6)/f complex. Here, we report the sequence of the wild-type MCD1 gene, which encodes a large and novel putative protein. Analysis of three mutant alleles showed that two harbored large deletions, but that one allele, mcd1-2, had a single base change resulting in a nonsense codon near the N-terminus. This same mutant allele can be suppressed by a second-site mutation in the nuclear MCD2 gene, whereas mcd2-1 cannot suppress the deletion in mcd1-1 (Esposito,D. Higgs,D.C. Drager,R.G. Stern, D.B. and Girard-Bascou,J. (2001) Curr. Genet., 39, 40-48). We report the cloning of mcd2-1, and show that the mutation lies in a tRNA(Ser)(CGA), which has been modified to translate the nonsense codon in mcd1-2. We discuss how the existence of a large tRNA(Ser) gene family may permit this suppression without pleiotropic consequences.
Assuntos
Chlamydomonas reinhardtii/genética , Complexo Citocromos b6f/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA de Transferência de Serina/genética , Supressão Genética , Proteínas de Algas/genética , Alelos , Animais , Sequência de Bases , Núcleo Celular/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Clonagem Molecular , Complexo Citocromos b6f/metabolismo , Genes Supressores , Teste de Complementação Genética , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA de Transferência de Serina/metabolismoRESUMO
PURPOSE: The use of desmopressin in the treatment of primary nocturnal enuresis (PNE) is accepted and based on the fact that this drug leads to renal water reabsorption. However, recent findings have also implicated that desmopressin regulates other molecules, such as sodium and potassium. We investigate if desmopressin influences renal Ca2+ handling. MATERIALS AND METHODS: A total of 32 children with PNE were enrolled in a prospective study. Patients received a standard 30 microg desmopressin intranasally before going to bed. All patients were treated for at least 4 weeks. Desmopressin was then withdrawn and reintroduced after 2 weeks. Urine samples were collected during all 3 phases of the study. Ca2+ measurement was performed in single morning spot urines as well as in 24-hour collections. Additionally, blood was sampled for analysis of Ca2+. The Wilcoxon signed rank test was used for statistical analysis. RESULTS: Wet nights decreased an average of 4.75 to 1.0 per week with desmopressin treatment. While blood concentrations did not change with or without medication, urinary Ca2+ excretion was significantly higher while patients were treated with desmopressin. This significant result was the same in single spot as well as in 24-hour samples. CONCLUSIONS: This study demonstrated the increased excretion of Ca2+ by desmopressin treatment in children with PNE. Since Ca2+ is a crucial molecule in growth and development, this finding indicates the necessity of larger followup studies concerning Ca2+ handling and growth in children on long-term desmopressin treatment.
Assuntos
Cálcio/urina , Desamino Arginina Vasopressina/uso terapêutico , Enurese/tratamento farmacológico , Enurese/urina , Fármacos Renais/uso terapêutico , Adolescente , Criança , Feminino , Humanos , Masculino , Estudos ProspectivosRESUMO
Abnormally high concentrations of beta-amyloid peptide (Abeta) and amyloid plaque formation in Alzheimer's disease (AD) may be caused either by increased generation or by decreased degradation of Abeta. Therefore, activation of mechanisms that lower brain Abeta levels is considered valuable for AD therapy. Neuronal upregulation of neprilysin (NEP) in young transgenic mice expressing the AD-causing amyloid precursor protein mutations (SwAPP) led to reduction of brain Abeta levels and delayed Abeta plaque deposition. In contrast, a comparable increase of brain NEP levels in aged SwAPP mice with pre-existing plaque pathology did not result in a significant reduction of plaque pathology. Therefore, we suggest that the potential of NEP for AD therapy is age-dependent and most effective early in the course of AD pathophysiology.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Neprilisina/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Fatores Etários , Peptídeos beta-Amiloides/administração & dosagem , Peptídeos beta-Amiloides/genética , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Camundongos , Neuroglia/citologia , Neuroglia/metabolismoRESUMO
The accumulation of the beta-amyloid peptide (Abeta) is a central event in the pathogenesis of Alzheimer's disease (AD). Abeta removal from the brain by immune therapy shows promising potential for the treatment of patients with AD, although the mechanisms of the antibody action are incompletely understood. In this study we compared the biological activities of antibodies raised against various Abeta fragments for Abeta reduction in vitro and in vivo. Antibodies against Abeta enhanced the uptake of Abeta42 aggregates up to 6-fold by primary microglial cells in vitro. The kinetics of Abeta42 uptake varied considerably among antibodies. Based on the activity to mediate Abeta42 uptake by microglial cells, we identified a bioactive antibody that significantly reduced Abeta42 levels in the brains of transgenic mice with neuronal expression of an AD-related mutated amyloid precursor protein. This effect depended on the epitopes recognized by the antibody. Our data suggest that the ability to facilitate Abeta42 uptake by primary microglia cells in vitro can be used to predict the biological activity of the antibody by passive immunization in vivo. This protocol may prove useful for the rapid validation of the activity of antibodies designed to be used in immune therapy of AD.