RESUMO
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major components of plaque, beta-amyloid peptides (Abetas), are produced from amyloid precursor protein (APP) by the activity of beta- and gamma-secretases. beta-secretase activity cleaves APP to define the N-terminus of the Abeta1-x peptides and, therefore, has been a long- sought therapeutic target for treatment of AD. The gene encoding a beta-secretase for beta-site APP cleaving enzyme (BACE) was identified recently. However, it was not known whether BACE was the primary beta-secretase in mammalian brain nor whether inhibition of beta-secretase might have effects in mammals that would preclude its utility as a therapeutic target. In the work described herein, we generated two lines of BACE knockout mice and characterized them for pathology, beta-secretase activity and Abeta production. These mice appeared to develop normally and showed no consistent phenotypic differences from their wild-type littermates, including overall normal tissue morphology and brain histochemistry, normal blood and urine chemistries, normal blood-cell composition, and no overt behavioral and neuromuscular effects. Brain and primary cortical cultures from BACE knockout mice showed no detectable beta-secretase activity, and primary cortical cultures from BACE knockout mice produced much less Abeta from APP. The findings that BACE is the primary beta-secretase activity in brain and that loss of beta-secretase activity produces no profound phenotypic defects with a concomitant reduction in beta-amyloid peptide clearly indicate that BACE is an excellent therapeutic target for treatment of AD.
Assuntos
Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/enzimologia , Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Encéfalo/metabolismo , Linhagem Celular , Células Cultivadas , Técnicas de Cultura , Endopeptidases , Inibidores Enzimáticos/uso terapêutico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos KnockoutRESUMO
Amyloid plaques are a neuropathological hallmark of Alzheimer's disease (AD), but their relationship to neurodegeneration and dementia remains controversial. In contrast, there is a good correlation in AD between cognitive decline and loss of synaptophysin-immunoreactive (SYN-IR) presynaptic terminals in specific brain regions. We used expression-matched transgenic mouse lines to compare the effects of different human amyloid protein precursors (hAPP) and their products on plaque formation and SYN-IR presynaptic terminals. Four distinct minigenes were generated encoding wild-type hAPP or hAPP carrying mutations that alter the production of amyloidogenic Abeta peptides. The platelet-derived growth factor beta chain promoter was used to express these constructs in neurons. hAPP mutations associated with familial AD (FAD) increased cerebral Abeta(1-42) levels, whereas an experimental mutation of the beta-secretase cleavage site (671(M-->I)) eliminated production of human Abeta. High levels of Abeta(1-42) resulted in age-dependent formation of amyloid plaques in FAD-mutant hAPP mice but not in expression-matched wild-type hAPP mice. Yet, significant decreases in the density of SYN-IR presynaptic terminals were found in both groups of mice. Across mice from different transgenic lines, the density of SYN-IR presynaptic terminals correlated inversely with Abeta levels but not with hAPP levels or plaque load. We conclude that Abeta is synaptotoxic even in the absence of plaques and that high levels of Abeta(1-42) are insufficient to induce plaque formation in mice expressing wild-type hAPP. Our results support the emerging view that plaque-independent Abeta toxicity plays an important role in the development of synaptic deficits in AD and related conditions.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/genética , Fragmentos de Peptídeos/biossíntese , Placa Amiloide/genética , Placa Amiloide/metabolismo , Sinapses/genética , Sinapses/fisiologia , Envelhecimento/patologia , Doença de Alzheimer/genética , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Animais , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Degeneração Neural/genética , Fragmentos de Peptídeos/genética , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores Pré-Sinápticos/genética , Receptores Pré-Sinápticos/metabolismoRESUMO
Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.
Assuntos
Ácido Aspártico Endopeptidases/isolamento & purificação , Encéfalo/enzimologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Células CHO , Linhagem Celular , Membrana Celular/enzimologia , Clonagem Molecular , Cricetinae , Endopeptidases , Inibidores Enzimáticos/farmacologia , Escherichia coli , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , TransfecçãoRESUMO
Autosomal dominant forms of familial Alzheimer's disease (FAD) are associated with increased production of the amyloid beta peptide, Abeta42, which is derived from the amyloid protein precursor (APP). In FAD, as well as in sporadic forms of the illness, Abeta peptides accumulate abnormally in the brain in the form of amyloid plaques. Here, we show that overexpression of FAD(717V-->F)-mutant human APP in neurons of transgenic mice decreases the density of presynaptic terminals and neurons well before these mice develop amyloid plaques. Electrophysiological recordings from the hippocampus revealed prominent deficits in synaptic transmission, which also preceded amyloid deposition by several months. Although in young mice, functional and structural neuronal deficits were of similar magnitude, functional deficits became predominant with advancing age. Increased Abeta production in the context of decreased overall APP expression, achieved by addition of the Swedish FAD mutation to the APP transgene in a second line of mice, further increased synaptic transmission deficits in young APP mice without plaques. These results suggest a neurotoxic effect of Abeta that is independent of plaque formation.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/patologia , Rede Nervosa/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/biossíntese , Precursor de Proteína beta-Amiloide/genética , Animais , Modelos Animais de Doenças , Eletrofisiologia , Humanos , Camundongos , Camundongos Transgênicos , MutaçãoRESUMO
The interaction of insulin-like growth factor-I with glycosylated and nonglycosylated human recombinant IGFBP-3 was studied by competition binding and by realtime biospecific interaction analysis (BIA). No significant difference was found in the affinity of the two forms of recombinant IGFBP-3 for IGF-I. Combining the results from the two different methods of binding analysis, a dissociation constant of 50 +/- 14 pM was determined. This value compares favorably to that reported for IGF-I binding to natural human plasma-derived IGFBP-3. Subcutaneous injection into rats of IGF-I either bound to glycosylated or nonglycosylated IGFBP-3 yielded an area under the curve (AUC) that was essentially the same for either form of IGFBP-3 and was twice as large as the AUC obtained after injection of IGF-I in the free form. Circulating IGF-I peak levels were reached in less than 1 h post-injection for free IGF-I, 4 and 8 h post-injection for the nonglycosylated and glycosylated IGF-I/IGFBP-3 complexes respectively. Neutral gel filtration of rat serum samples 4 and 8 h post-injection revealed that the IGF-I delivered bound to the nonglycosylated IGFBP-3 circulated as a 40-50 kDa complex at the 4 h time point and as a 130-140 kD complex at the 8 h time point. IGF-I delivered as a complex with glycosylated IGFBP-3 was found to circulate as a 140 kD complex at 4 and 8 h post-injection.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas de Transporte/fisiologia , Somatomedinas , Animais , Células CHO , Proteínas de Transporte/genética , Proteínas de Transporte/farmacocinética , Cromatografia em Gel , Clonagem Molecular , Cricetinae , Escherichia coli , Glucose/metabolismo , Glicosilação , Humanos , Injeções Subcutâneas , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacocinética , Masculino , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinéticaRESUMO
Recombinant human insulin-like growth factor binding protein 3 (hIGFBP-3) stably expressed in chinese hamster ovary cells (CHO cells) has been purified to homogeneity from serum-free culture media. The purified protein migrates as a doublet (45/43 kDa) upon SDS-PAGE. The purified recombinant hIGFBP-3 is fully active and binds one mole of IGF-I per mole of recombinant binding protein. When the transfected CHO cells are treated with tunicamycin a single 29 kDa hIGFBP-3 protein is observed. This expressed hIGFBP-3 protein maintains its ability to bind IGF-I. N-Glycanase treatment of the purified hIGFBP-3 protein results in a protein that migrates similar to E. coli-derived IGFBP-3 upon SDS-PAGE under reducing conditions (30 kDa). Carboxymethylation of hIGFBP-3 suggests that all 18 cysteines are involved in disulfide linkages. These results represent the first purification and characterization of recombinant hIGFBP-3 expressed in CHO cells.
Assuntos
Proteínas de Transporte/isolamento & purificação , Fator de Crescimento Insulin-Like I/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Peso Molecular , Ovário , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
We report for the first time the isolation of a cDNA encoding the complete amino acid sequence for bovine growth hormone-dependent insulin-like growth factor binding protein-3 (bIGFBP-3). The deduced amino acid sequence from the cDNA revealed a mature polypeptide consisting of 264 amino acids and a 27 amino acid putative signal peptide. The amino acid sequence is over 80% homologous with human IGFBP-3 with complete conservation of the 18 cysteine residues and the 3 Asn-linked glycosylation sites. Between the two species there are 44 amino acid substitutions. Northern analysis of the bIGFBP-3 mRNA in bovine tissue revealed a single mRNA species of 1.65 kilobases.
Assuntos
Proteínas de Transporte/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Proteínas de Transporte/metabolismo , Bovinos , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Fígado/metabolismo , Dados de Sequência Molecular , Poli A/genética , Poli A/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Somatomedinas/metabolismoRESUMO
Insulin-like growth factors (IGFs) found in plasma and other body fluids circulate in association with specific binding proteins. We report here the cloning and the nucleotide sequence of cDNAs for the growth-hormone-dependent acid-stable IGF binding protein, hIGF-BP3. The derived protein begins with a putative 27-amino acid signal peptide followed by 264 residues of the mature polypeptide. The predicted sequence contains three potential N-linked glycosylation sites and shares two region of homology with the low-molecular-weight non-growth-hormone-dependent binding proteins BP-1 and BP-2. The protein contains 18 cysteine residues clustered in the amino and carboxy termini. Chinese Hamster ovary cells transfected with this cDNA secrete a 43-45 kD protein doublet, which bound IGF. The expressed IGF-binding protein is indistinguishable from the native BP-3 found in human plasma.