RESUMO
AIM: Previous studies have shown that tenuigenin, a crude extract of Polygala tenuifolia Willd. that is commonly used in traditional Chinese herbal medicine for memory loss, can reduce the secretion of Abeta from cultured cells. However, the mechanism underlying this effect and the active compound derived from tenuigenin is unknown. In this study, a purified component of tenuigenin, tenuifolin, was examined and revealed to be an effective compound in vitro. METHODS: Abeta secretion from three sets of COS-7 cells, each carrying a plasmid expressing a different form of APP was examined following the treatment with tenuifolin. Initially, tenuifolin was determined to have no inherent toxicity to either the transfected or wild type cells at the effective concentrations. Cells were then treated with 0.5-2.0 microg mL(-1) tenuifolin for 12 h and their media were examined via an ELISA for Abeta1-40 and Abeta-42. RESULTS: We found that treatment with 2.0 microg mL(-1) tenuifolin significantly decreased Abeta secretion from COS-7 cells without altering the ratio of Abeta1-40 and Abeta-42. This effect is most probably due to inhibition of the beta-site APP cleaving enzyme as Abeta secretion was not inhibited from cells expressing the C99 fragment. CONCLUSION: Tenuifolin is an effective compound from tenuigenin. We believe that this finding should lead the way for future experiments to determine the exact mechanism for tenuifolin's effect on Abeta secretion.
Assuntos
Peptídeos beta-Amiloides , Diterpenos do Tipo Caurano , Medicamentos de Ervas Chinesas , Animais , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Chlorocebus aethiops , Células COS , Diterpenos do Tipo Caurano/administração & dosagem , Diterpenos do Tipo Caurano/efeitos adversos , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/efeitos adversos , Ensaio de Imunoadsorção Enzimática , Fitoterapia , Polygala , Fatores de TempoRESUMO
OBJECTIVE: Update of the Hohenheim consensus on monosodium glutamate from 1997: Summary and evaluation of recent knowledge with respect to physiology and safety of monosodium glutamate. DESIGN: Experts from a range of relevant disciplines received and considered a series of questions related to aspects of the topic. SETTING: University of Hohenheim, Stuttgart, Germany. METHOD: The experts met and discussed the questions and arrived at a consensus. CONCLUSION: Total intake of glutamate from food in European countries is generally stable and ranged from 5 to 12 g/day (free: ca. 1 g, protein-bound: ca. 10 g, added as flavor: ca. 0.4 g). L-Glutamate (GLU) from all sources is mainly used as energy fuel in enterocytes. A maximum intake of 6.000 [corrected] mg/kg body weight is regarded as safe. The general use of glutamate salts (monosodium-L-glutamate and others) as food additive can, thus, be regarded as harmless for the whole population. Even in unphysiologically high doses GLU will not trespass into fetal circulation. Further research work should, however, be done concerning the effects of high doses of a bolus supply at presence of an impaired blood brain barrier function. In situations with decreased appetite (e.g., elderly persons) palatability can be improved by low dose use of monosodium-L-glutamate.
Assuntos
Qualidade de Produtos para o Consumidor , Aditivos Alimentares/administração & dosagem , Aditivos Alimentares/efeitos adversos , Glutamato de Sódio/administração & dosagem , Glutamato de Sódio/efeitos adversos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Regulação do Apetite/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Feminino , Aromatizantes/administração & dosagem , Aromatizantes/efeitos adversos , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Gravidez , Efeitos Tardios da Exposição Pré-NatalRESUMO
Although amyloid beta (Abeta) deposition has been a hallmark of Alzheimer's disease (AD), the absence of a phenotype in the beta amyloid precursor protein (APP) knockout mouse, tends to detract our attention away from the physiological functions of APP. Although much attention has been focused on the neurotoxicity of Abeta, many studies suggest the involvement of APP in neuroplasticity. We found that secreted amyloid precursor protein (sAPP) increased the differentiation of human neural stem cells (hNSCs) in vitro, while an antibody-recognizing APP dose-dependently inhibited these activities. With a high dose of sAPP treatment or wild-type APP gene transfection, hNSCs were differentiated into astrocytes rather than neurons. In vivo, hNSCs transplanted into APP-transgenic mouse brain exhibited glial differentiation rather than neural differentiation. Our results suggest that APP regulates neural stem cell biology in the adult brain, and that altered APP metabolism in Down syndrome or AD may have implications for the pathophysiology of these diseases.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/farmacologia , Diferenciação Celular/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Animais , Apoptose , Movimento Celular/efeitos dos fármacos , Transplante de Células , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroglia/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Plaques containing amyloid beta-peptides (Abeta) are a major feature in Alzheimer's disease (AD), and GM1 ganglioside is an important component of cellular plasma membranes and especially enriched in lipid raft. GM1-bound Abeta (GM1/Abeta), found in brains exhibiting early pathological changes of AD including diffuse plaques, has been suggested to be involved in the initiation of amyloid fibril formation in vivo by acting as a seed. However, the role of GM1 in amyloid beta-protein precursor (APP) processing is not yet defined. In this study, we report that exogenous GM1 ganglioside promotes Abeta biogenesis and decreases sAPPalpha secretion in SH-SY5Y and COS7 cells stably transfected with human APP695 cDNA without affecting full-length APP and the sAPPbeta levels. We also observe that GM1 increases extracellular levels of Abeta in primary cultures of mixed rat cortical neurons transiently transfected with human APP695 cDNA. These findings suggest a regulatory role for GM1 in APP processing pathways.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Gangliosídeo G(M1)/farmacologia , Gangliosídeo G(M1)/fisiologia , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases , Células COS , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Córtex Cerebral/citologia , Chlorocebus aethiops , DNA Complementar/genética , Endopeptidases/metabolismo , Líquido Extracelular/metabolismo , Humanos , Neuroblastoma/patologia , Neurônios/citologia , Neurônios/metabolismo , Fragmentos de Peptídeos/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção , Regulação para Cima/efeitos dos fármacosRESUMO
To rapidly respond to invading microorganisms, humans call on their innate immune system. This occurs by microbe-detecting receptors, such as CD14, that activate immune cells to eliminate the pathogens. Here, we link the lipopolysaccharide receptor CD14 with Alzheimer's disease, a severe neurodegenerative disease resulting in dementia. We demonstrate that this key innate immunity receptor interacts with fibrils of Alzheimer amyloid peptide. Neutralization with antibodies against CD14 and genetic deficiency for this receptor significantly reduced amyloid peptide induced microglial activation and microglial toxicity. The observation of strongly enhanced microglial expression of the LPS receptor in brains of animal models of Alzheimer's disease indicates a clinical relevance of these findings. These data suggest that CD14 may significantly contribute to the overall neuroinflammatory response to amyloid peptide, highlighting the possibility that the enormous progress currently being made in the field of innate immunity could be extended to research on Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Receptores de Lipopolissacarídeos/fisiologia , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Anticorpos Monoclonais/farmacologia , Imunidade Inata , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/imunologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidadeRESUMO
Programmed cell death, or apoptosis, has been implicated in Alzheimer's disease. Mutations in the presenilin (PS) genes, PS1 and PS2, are a major cause of early-onset familial Alzheimer's disease (FAD). Previous studies have suggested that the PS play a role in apoptosis. However, the mechanisms whereby presenilins affect apoptosis and the relationship of FAD-associated presenilin mutants to the apoptotic effect have not been elucidated. In the present study, in an attempt to further explore the effect of PS2 on apoptosis we examined whether overexpression of wild-type or mutant PS2 can directly induce apoptosis or increase cell susceptibility to apoptosis in various cell lines, such as N2a, CHO, and HEK 293T. Wild-type or mutant PS2 was transiently transfected into these cell lines and the viability of the transfected cells was evaluated by their morphology, DNA fragmentation and condensation, appearance of sub-G(1/0) cells, and caspase activation. We also examined the susceptibility of the PS2-transfected cells to apoptosis induced by the apoptotic inducers staurosporine and H(2)O(2). Our results showed that overexpression of either wild type or mutant PS2 in these cell lines did not directly induce apoptosis or increase the susceptibility to apoptosis induced by staurosporine or H(2)O(2). Taken together, these results suggest that overexpression of PS2 does not cause pro-apoptotic effects, at least not in the cellular systems and conditions employed in this study, and therefore it seems unlikely that apoptosis plays a prominent role in the neuropathological effects of PS2 in Alzheimer's disease.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Apoptose/fisiologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Mutação/fisiologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Regulação da Expressão Gênica/fisiologia , Predisposição Genética para Doença , Humanos , Camundongos , Presenilina-2 , Células Tumorais CultivadasRESUMO
There is circumstantial evidence that the reelin signaling pathway may contribute to neurodegeneration in the adult brain and could be linked to Alzheimer's disease (AD). In the present immunohistochemical report we studied the reelin expression profile in double-transgenic mice that express both human mutant beta-amyloid precursor protein (APP) and human mutant presenilin-1. We were able to demonstrate that reelin immunostaining was found together with human APP in the neuritic component of many AD-typical plaques in both hippocampus and neocortex. This observation gives the first evidence for the association of reelin with amyloid deposits.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Placa Amiloide/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Moléculas de Adesão Celular Neuronais/genética , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Proteínas da Matriz Extracelular/genética , Feminino , Hipocampo/patologia , Hipocampo/fisiopatologia , Imuno-Histoquímica , Interneurônios/metabolismo , Interneurônios/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso , Placa Amiloide/genética , Placa Amiloide/patologia , Presenilina-1 , Células Piramidais/metabolismo , Células Piramidais/patologia , Proteína Reelina , Serina EndopeptidasesRESUMO
The beta-amyloid protein (Abeta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APP-CTFbeta), which is subsequently cleaved by gamma-secretase to produce Abeta. The aim of this study was to examine the cleavage of APP-CTFbeta by gamma-secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTFbeta sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [35S]methionine and [35S]cysteine. Labelled APP-CTFbeta and Abeta were then immunoprecipitated with a monoclonal antibody (WO2) specific for the transgene sequences. Approximately 30% of the human APP-CTFbeta (hAPP-CTFbeta) was converted to human Abeta (hAbeta), which was rapidly secreted. The remaining 70% of the hAPP-CTFbeta was degraded by an alternative pathway. The cleavage of hAPP-CTFbeta to produce hAbeta was inhibited by specific gamma-secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTFbeta and hAbeta levels, suggesting that the alternative pathway was proteasome-dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APPcyt) directly. The study suggests that in primary cortical neurons, APP-CTFbeta is degraded by two distinct pathways, one involving gamma-secretase, which produces Abeta, and a second major pathway involving direct cleavage of APP-CTFbeta within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Abeta production in the AD brain.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Western Blotting , Células Cultivadas , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Complexos Multienzimáticos/antagonistas & inibidores , Neurônios/metabolismo , Inibidores de Proteases/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
The beta amyloid peptide is the major component of the neuritic plaques, the characteristic lesions in Alzheimer's disease. Mutations in three genes (APP, PS-1, and PS-2) cause familial Alzheimer's disease by alteration of the rate of generation of amyloid peptide or the length of this peptide. However, in the 90% non-familial cases, other factors play a major pathogenetic role. These include the apolipoprotein E genotype, the "plaque-associated" proteins promoting the formation of toxic fibrillar aggregates or the chronic inflammatory responses. The aim of this review is to explain the steps in the complex cascade leading to Alzheimer's disease and, based on this, to report the current efforts to intervene in these different pathophysiological events in order to prevent progression of Alzheimer's disease. Whereas acetylcholine substitution is currently used in clinical practice, future therapeutical strategies to combat Alzheimer's disease may include anti-inflammatory treatments, vaccination against beta amyloid peptide, or treatment with cholesterol-lowering drugs.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Humanos , Proteínas de Membrana/genética , Mutação , Presenilina-1 , Presenilina-2RESUMO
Transmissible spongiform encephalopathies are characterised by the transformation of the normal cellular prion protein (PrP(C)) into an abnormal isoform (PrP(TSE)). Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor antagonists can inhibit glutathione depletion and neurotoxicity induced by PrP(TSE) and a toxic prion protein peptide, PrP106-126, in vitro. NMDA receptor activation is known to increase intracellular accumulation of Ca(2+), resulting in up-regulation of arachidonic acid (AA) metabolism. This can stimulate the lipoxygenase pathways that may generate a number of potentially neurotoxic metabolites. Because of the putative relationship between AA breakdown and PrP106-126 neurotoxicity, we investigated AA metabolism in primary cerebellar granule neuron cultures treated with PrP106-126. Our studies revealed that PrP106-126 exposure for 30 min significantly up-regulated AA release from cerebellar granule neurons. PrP106-126 neurotoxicity was mediated through the 5-lipoxygenase (5-LOX) pathway, as shown by abrogation of neuronal death with the 5-LOX inhibitors quinacrine, nordihydroguaiaretic acid, and caffeic acid. These inhibitors also prevented PrP106-126-induced caspase 3 activation and annexin V binding, indicating a central role for the 5-LOX pathway in PrP106-126-mediated proapoptosis. Interestingly, inhibitors of the 12-lipoxygenase pathway had no effect on PrP106-126 neurotoxicity or proapoptosis. These studies clearly demonstrate that AA metabolism through the 5-LOX pathway is an important early event in PrP106-126 neurotoxicity and consequently may have a critical role in PrP(TSE)-mediated cell loss in vivo. If this is so, therapeutic intervention with 5-LOX inhibitors may prove beneficial in the treatment of prion disorders.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Fragmentos de Peptídeos/toxicidade , Doenças Priônicas/enzimologia , Príons/metabolismo , Príons/toxicidade , Animais , Anexina A5/efeitos dos fármacos , Anexina A5/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Caspase 3 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Córtex Cerebelar/efeitos dos fármacos , Córtex Cerebelar/enzimologia , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Memantina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/enzimologia , Doenças Priônicas/fisiopatologia , Quinacrina/farmacologiaRESUMO
Mutations in the presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease by altering gamma-secretase cleavage of the amyloid precursor protein, the last step in the generation of Abeta peptide. Ablation of presenilin (PS) genes, or mutation of two critical aspartates, abolishes gamma-secretase cleavage, suggesting that PS may be the gamma-secretases. Independently, inhibition experiments indicate that gamma-secretase is an aspartyl protease. To characterize the putative gamma-secretase activity associated with presenilins, lysates from human neuroblastoma SH-SY5Y and human brain homogenates were incubated with biotin derivatives of pepstatin, followed by immunoprecipitation of PS and associated proteins, and biotin detection by Western blotting. Precipitation with PS1 antibodies, directed to either N-terminal or loop regions, yielded the same 43 kDa band, of apparent molecular mass consistent with that of full-length PS1, although it may represent an aspartyl protease complexed with PS1. Incubation of cell lysates with pepstatin-biotin, followed by streptavidin precipitation and PS1 Western blotting, revealed PS1 fragments and full-length protein, indicating that pepstatin-biotin bound to both cleaved and uncleaved PS1. Binding could be competed by gamma-secretase inhibitor L-685,458 and could not be achieved with a PS1 mutant lacking the two transmembrane aspartates. Pepstatin-biotin was also shown to bind to PS2. PS1 was specifically absorbed to pepstatin-agarose, with an optimal pH of 6. Binding of pepstatin-biotin to PS1 from lymphocytes of a heterozygous carrier of pathologic exon 9 deletion was markedly decreased as compared to control lymphocytes, suggesting that this PS1 mutation altered the pepstatin binding site.
Assuntos
Doença de Alzheimer/enzimologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Pepstatinas/metabolismo , Inibidores de Proteases/metabolismo , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide , Animais , Ligação Competitiva , Biotina/metabolismo , Células COS , Células Cultivadas , Ácidos Cólicos , Detergentes , Éxons/genética , Humanos , Hidrólise , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Peso Molecular , Testes de Precipitina , Presenilina-1 , Presenilina-2 , Ligação Proteica/genética , Deleção de Sequência , Células Tumorais CultivadasRESUMO
We reported previously that the carbohydrate domain of the amyloid precursor protein is involved in amyloid precursor protein (APP)-APP interactions. Functional in vitro studies suggested that this interaction occurs through the collagen binding site of APP. The physiological significance remained unknown, because it is not understood whether and how APP dimerization occurs in vivo. Here we report that cellular APP exists as homodimers matching best with a two-site model. Consistent with our published crystallographic data, we show that a deletion of the entire sequence after the kunitz protease inhibitor domain did not abolish APP homodimerization, suggesting that two domains are critically involved but that neither is essential for homodimerization. Finally, we generated stabilized dimers by expressing mutant APP with a single cysteine in the ectodomain juxtamembrane region. Mutation of Lys(624) to cysteine produced approximately 6-8-fold more A beta than cells expressing normal APP. Our results suggest that amyloid A beta production can in principle be positively regulated by dimerization in vivo. We suggest that dimerization could be a physiologically important mechanism for regulating the proposed signal activity of APP.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/química , Animais , Sítios de Ligação , Western Blotting , Células COS , Membrana Celular/metabolismo , Cromatografia em Gel , Colágeno/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Cisteína/química , Dimerização , Dissulfetos , Humanos , Immunoblotting , Lisina/química , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transfecção , Células Tumorais CultivadasRESUMO
Inhibition of neocortical beta-amyloid (Abeta) accumulation may be essential in an effective therapeutic intervention for Alzheimer's disease (AD). Cu and Zn are enriched in Abeta deposits in AD, which are solubilized by Cu/Zn-selective chelators in vitro. Here we report a 49% decrease in brain Abeta deposition (-375 microg/g wet weight, p = 0.0001) in a blinded study of APP2576 transgenic mice treated orally for 9 weeks with clioquinol, an antibiotic and bioavailable Cu/Zn chelator. This was accompanied by a modest increase in soluble Abeta (1.45% of total cerebral Abeta); APP, synaptophysin, and GFAP levels were unaffected. General health and body weight parameters were significantly more stable in the treated animals. These results support targeting the interactions of Cu and Zn with Abeta as a novel therapy for the prevention and treatment of AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Quelantes/farmacologia , Clioquinol/farmacologia , Cobre/metabolismo , Zinco/metabolismo , Fatores Etários , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Sinaptofisina/metabolismoRESUMO
beta-Amyloid peptides are key molecules that are involved in the pathology of Alzheimer's disease (AD). The source and place of the neurotoxic action of Abeta, however, is still a matter of controversial debates. In the present report, we studied the neuropathological events in a transgenic mouse model expressing human mutant beta-amyloid precursor protein and human mutant presenilin-1 in neurons. Western blot and immunohistochemical analysis revealed that intracellular Abeta staining preceded plaque deposition, which started in the hippocampal formation. At later stages, many neuritic Abeta positive plaques were found in all cortical, hippocampal and many other brain areas. Interestingly, intraneuronal Abeta staining was no longer detected in the brain of aged double-transgenic mice, which correlates with the typical neuropathology in the brain of chronic AD patients.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Placa Amiloide/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Gliose/genética , Gliose/patologia , Gliose/fisiopatologia , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Mutação/genética , Neurônios/patologia , Placa Amiloide/genética , Placa Amiloide/patologia , Presenilina-1RESUMO
The accumulation of amyloid plaques and amyloid congophilic angiopathy (ACA) in the brains of affected individuals is one of the main pathological features of Alzheimer's disease. Within these deposits, the beta A4 (Ass) polypeptide represents a major component with the C-terminal 39-43 amino acid variants being most abundant. Using a mouse IgG1 MoAb produced by hybridoma beta A4[35-43]-95.2 3B9, which reacts with the epitope is defined by the amino acid residues beta A438[GVV]40, this study has identified a unique conformation within the carboxyl terminus of human beta A4[1-42]. Although the beta A438[GVV]40 sequence is present within the C-termini of human beta A4[1-40] and beta A4[1-43] and the beta A4-containing region of human APP, the beta A4[35-43]-95.2 3B9 MoAb (designated MoAb 3B9) does not bind these polypeptides, demonstrating a high degree of specificity for the beta A438[GVV]40 epitope as presented within the beta A4[1-42] sequence. The beta A4[1-42] epitope bound by MoAb 3B9 is sensitive to heating (100 degrees C for 5 min) and is denatured by SDS but not by oxidative radio-iodination of beta A4 or by adsorption to plastic surfaces or nitrocellulose. The recognition of beta A4 plaque deposits and ACA by MoAb 3B9 within formalin-fixed sections of human AD brain demonstrates the potential of these antibodies for investigating the role of the unique beta A4[1-42] conformation in the development of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Especificidade de Anticorpos , Mapeamento de Epitopos , Epitopos , Humanos , Hibridomas , Dados de Sequência MolecularAssuntos
Demência/diagnóstico , Demência/genética , Paraparesia Espástica/diagnóstico , Paraparesia Espástica/genética , Proteínas Adaptadoras de Transdução de Sinal , Doença de Alzheimer/complicações , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Amiloide/genética , Demência/complicações , Diagnóstico Diferencial , Doença de Gerstmann-Straussler-Scheinker/diagnóstico , Doença de Gerstmann-Straussler-Scheinker/genética , Humanos , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Mutação , Paraparesia Espástica/complicações , Presenilina-1 , Príons/genética , SíndromeRESUMO
Recent epidemiological studies show a strong reduction in the incidence of Alzheimer's disease in patients treated with cholesterol-lowering statins. Moreover, elevated Abeta42 levels and the varepsilon4 allele of the lipid-carrier apolipoprotein E are regarded as risk factors for sporadic and familial Alzheimer's disease. Here we demonstrate that the widely used cholesterol-lowering drugs simvastatin and lovastatin reduce intracellular and extracellular levels of Abeta42 and Abeta40 peptides in primary cultures of hippocampal neurons and mixed cortical neurons. Likewise, guinea pigs treated with high doses of simvastatin showed a strong and reversible reduction of cerebral Abeta42 and Abeta40 levels in the cerebrospinal fluid and brain homogenate. These results suggest that lipids are playing an important role in the development of Alzheimer's disease. Lowered levels of Abeta42 may provide the mechanism for the observed reduced incidence of dementia in statin-treated patients and may open up avenues for therapeutic interventions.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Anticolesterolemiantes/farmacologia , Isoformas de Proteínas/metabolismo , Sinvastatina/farmacologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/química , Animais , Colesterol/sangue , Predisposição Genética para Doença , Cobaias , Técnicas In Vitro , Mutação , Fragmentos de Peptídeos/metabolismo , Isoformas de Proteínas/química , Padrões de ReferênciaRESUMO
Neurodegenerative disorders such as prion diseases and Alzheimer's disease (AD) are characterized by neuronal dysfunction and accumulation of amyloidogenic protein. In vitro studies have demonstrated that these amyloidogenic proteins can induce cellular oxidative stress and therefore may contribute to the neuronal dysfunction observed in these illnesses. Although the neurotoxic pathways are not fully elucidated, recent studies in AD have demonstrated up-regulation of caspases in neurons treated with amyloid beta (Abeta) peptide, suggesting involvement of apoptotic processes. To examine the role of proapoptotic pathways in prion diseases we treated primary mouse cortical neurons with the toxic prion protein peptide PrP106-126 and measured caspase activation and annexin V binding. We found that PrP106-126 induced a rapid and marked elevation in caspase 3, 6, and 8-like activity in neuronal cultures. Increased annexin V binding was observed predominantly on cortical cell neurites in peptide-treated cultures. Interestingly, these effects were induced by sublethal (5-50 microM) or lethal (100-200 microM) concentrations of PrP106-126. Sublethal concentrations of PrP106-126 maintained elevated caspase activation for at least 10 days with no loss of cell viability. Abeta1-40 also up-regulated caspase 3 activity and annexin V binding at both sublethal (5 microM) and lethal (25 microM) concentrations. There were no changes to proapoptotic marker expression in cultures treated with scrambled PrP106-126 (200 microM) or Abeta1-28 (25 microM) peptides. These studies demonstrate that amyloidogenic peptides can induce prolonged activation of proapoptotic marker expression in cultured neurons even at sublethal concentrations. These effects could contribute to chronic neuronal dysfunction and increase susceptibility to additional metabolic insults in neurodegenerative disorders. If so, targeting of therapeutic strategies against neuronal caspase activation early in the disease course could be beneficial in AD and prion diseases.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Doenças Priônicas/metabolismo , Príons/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Animais , Anexina A5/efeitos dos fármacos , Anexina A5/metabolismo , Apoptose/fisiologia , Biomarcadores/análise , Caspase 3 , Caspase 6 , Caspase 8 , Caspase 9 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas/metabolismo , Células Cultivadas/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Relação Dose-Resposta a Droga , Feto , Camundongos , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Doenças Priônicas/fisiopatologia , Príons/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Oxidative stress may have an important role in the progression of neurodegenerative disorders such as Alzheimer's disease (AD) and prion diseases. Oxidative damage could result from interactions between highly reactive transition metals such as copper (Cu) and endogenous reducing and/or oxidizing molecules in the brain. One such molecule, homocysteine, a thiol-containing amino acid, has previously been shown to modulate Cu toxicity in HeLa and endothelial cells in vitro. Due to a possible link between hyperhomocysteinemia and AD, we examined whether interaction between homocysteine and Cu could potentiate Cu neurotoxicity. Primary mouse neuronal cultures were treated with homocysteine and either Cu (II), Fe (II or III) or Zn (II). Homocysteine was shown to selectively potentiate toxicity from low micromolar concentrations of Cu. The toxicity of homocysteine/Cu coincubation was dependent on the ability of homocysteine to reduce Cu (II) as reflected by the inhibition of toxicity with the Cu (I)-specific chelator, bathocuproine disulphonate. This was supported by data showing that homocysteine reduced Cu (II) more effectively than cysteine or methionine but did not reduce Fe (III) to Fe (II). Homocysteine also generated high levels of hydrogen peroxide in the presence of Cu (II) and promoted Abeta/Cu-mediated hydrogen peroxide production and neurotoxicity. The potentiation of metal toxicity did not involve excitotoxicity as ionotropic glutamate receptor antagonists had no effect on neurotoxicity. Homocysteine alone also had no effect on neuronal glutathione levels. These studies suggest that increased copper and/or homocysteine levels in the elderly could promote significant oxidant damage to neurons and may represent additional risk factor pathways which conspire to produce AD or related neurodegenerative conditions.