RESUMO
The tau protein is a microtubule-associated protein that promotes microtubule stabilization. The phosphorylation of the tau protein has been linked to its dissociation from microtubules. Here, we examined the relationship between neuronal depolarization activity and tau protein phosphorylation by employing model systems in culture as well as in vivo. The KCl-evoked depolarization of cultured neurons has often been used to investigate the effects of neuronal activity. We found dephosphorylation at AT8 sites (S202, T205), T212, AT180 sites (T231, S235), and S396 in KCl-simulated cultured neurons. We also found that the KCl-induced tau protein dephosphorylation increases the level of the tau protein fractionated with stable microtubules. In an in vivo experiment, we demonstrated that the exposure of mice to a new environment activates protein phosphatase 1 in the mouse hippocampus and induces tau protein dephosphorylation. We also found an increased amount of the tau protein in a stable microtubule fraction, suggesting that the dephosphorylation of the tau protein may lead to its increased microtubule association in vivo. These results suggest that the association of microtubules with tau proteins may be regulated by the tau protein phosphorylation status affected by neuronal electrical activity.
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Sleep apnea is regarded as an important risk factor in the pathogenesis of Alzheimer disease (AD). Chronic intermittent hypoxia treatment (IHT) given during the sleep period of the circadian cycle in experimental animals is a well-established sleep apnea model. Here we report that transient IHT for 4 days on AD model mice causes Aß overproduction 2 months after IHT presumably via upregulation of synaptic BACE1, side-by-side with tau hyperphosphorylation. These results suggest that even transient IHT may be sufficient to cause long-lasting changes in the molecules measured as AD biomarkers in the brain.
Assuntos
Peptídeos beta-Amiloides , Modelos Animais de Doenças , Síndromes da Apneia do Sono , Proteínas tau , Animais , Proteínas tau/metabolismo , Fosforilação , Peptídeos beta-Amiloides/metabolismo , Síndromes da Apneia do Sono/metabolismo , Síndromes da Apneia do Sono/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hipóxia/metabolismo , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismoRESUMO
Loquat (Eriobotrya japonica) leaves contain many bioactive components such as ursolic acid (UA) and amygdalin. We investigated the effects of loquat leaf powder and methanol extract in human neuroglioma H4 cells stably expressing the Swedish-type APP695 (APPNL-H4 cells) and C57BL/6 J mice. Surprisingly, the extract greatly enhanced cellular amyloid-beta peptide (Aß) 42 productions in APPNL-H4 cells. Administration of leaf powder increased Aß42 levels after 3 months and decreased levels after 12 months compared to control mice. Leaf powder had no effect on working memory after 3 months, but improved working memory after 12 months. Administration of UA decreased Aß42 and P-tau levels and improved working memory after 12 months, similar to the administration of leave powder for 12 months. Amygdalin enhanced cellular Aß42 production in APPNL-H4 cells, which was the same as the extract. Three-month administration of amygdalin increased Aß42 levels slightly but did not significantly increase them, which is similar to the trend observed with the administration of leaf powder for 3 months. UA was likely the main compound contained in loquat leaves responsible for the decrease in intracerebral Aß42 and P-tau levels. Also, amygdalin might be one of the compounds responsible for the transiently increased intracerebral Aß42 levels.
Assuntos
Amigdalina , Eriobotrya , Humanos , Animais , Camundongos , Eriobotrya/química , Pós/análise , Camundongos Endogâmicos C57BL , Folhas de Planta/química , Extratos Vegetais/química , Peptídeos beta-Amiloides/análise , Ácido UrsólicoRESUMO
Octodon degus is said to be one of the most human-like rodents because of its improved cognitive function. Focusing on its high sociality, we cloned and characterized some sociality-related genes of degus, in order to establish degus as a highly socialized animal model in molecular biology. We cloned degus Neurexin and Neuroligin as sociality-related genes, which are genetically related to autism spectrum disorder in human. According to our results, amino acid sequences of Neurexin and Neuroligin expressed in degus brain, are highly conserved to that of human sequences. Most notably, degus Neuroligin4 is highly similar to human Neuroligin4X, which is one of the most important autism-related genes, whereas mouse Neuroligin4 is known to be poorly similar to human Neuroligin4X. Furthermore, our work also indicated that testosterone directly binds to degus Neurexin and intercepts intercellular Neurexin-Neuroligin binding. Moreover, it is of high interest that testosterone is another key molecule of the higher incidence of autism in male. These results indicated that degus has the potential for animal model of sociality, and furthermore may promote understanding toward the pathogenic mechanism of autism.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Octodon/metabolismo , Receptores de Superfície Celular/metabolismo , Testosterona/metabolismo , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Proteínas de Ligação ao Cálcio/química , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Hipocampo/metabolismo , Humanos , Masculino , Moléculas de Adesão de Célula Nervosa/química , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Receptores de Superfície Celular/química , Globulina de Ligação a Hormônio Sexual/química , Testosterona/farmacologiaRESUMO
After publication of the article [1], it was brought to our attention that an acknowledgement was missing from the original version.
RESUMO
BACKGROUND: Multiple sclerosis is a neuroinflammatory demyelinating and neurodegenerative disease of the central nervous system characterized by recurrent and progressive demyelination/remyelination cycles, neuroinflammation, oligodendrocyte loss, demyelination, and axonal degeneration. Cyclic phosphatidic acid (cPA) is a natural phospholipid mediator with a unique cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. We reported earlier that cPA elicits a neurotrophin-like action and protects hippocampal neurons from ischemia-induced delayed neuronal death. We designed, chemically synthesized, and metabolically stabilized derivatives of cPA: 2-carba-cPA (2ccPA), a synthesized compound in which one of the phosphate oxygen molecules is replaced with a methylene group at the sn-2 position. In the present study, we investigated whether 2ccPA exerts protective effects in oligodendrocytes and suppresses pathology in the two most common mouse models of multiple sclerosis. METHODS: To evaluate whether 2ccPA has potential beneficial effects on the pathology of multiple sclerosis, we investigated the effects of 2ccPA on oligodendrocyte cell death in vitro and administrated 2ccPA to mouse models of experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination. RESULTS: We demonstrated that 2ccPA suppressed the CoCl2-induced increase in the Bax/Bcl-2 protein expression ratio and phosphorylation levels of p38MAPK and JNK protein. 2ccPA treatment reduced cuprizone-induced demyelination, microglial activation, NLRP3 inflammasome, and motor dysfunction. Furthermore, 2ccPA treatment reduced autoreactive T cells and macrophages, spinal cord injury, and pathological scores in EAE, the autoimmune multiple sclerosis mouse model. CONCLUSIONS: We demonstrated that 2ccPA protected oligodendrocytes via suppression of the mitochondrial apoptosis pathway. Also, we found beneficial effects of 2ccPA in the multiperiod of cuprizone-induced demyelination and the pathology of EAE. These data indicate that 2ccPA may be a promising compound for the development of new drugs to treat demyelinating disease and ameliorate the symptoms of multiple sclerosis.
Assuntos
Anti-Inflamatórios/uso terapêutico , Doenças Desmielinizantes/tratamento farmacológico , Ácidos Fosfatídicos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Transformada , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores da Monoaminoxidase/toxicidade , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Excitotoxicity is the pivotal mechanism of neuronal death. Prostaglandins (PGs) produced during excitotoxicity play important roles in neurodegenerative conditions. Previously, we demonstrated that initial burst productions of PGD2, PGE2, and PGF2α are produced by cyclooxygenase-2 (COX-2) in the hippocampus following a single systemic kainic acid (KA) administration. In addition, we showed that blocking of all PG productions ameliorated hippocampal delayed neuronal death at 30 days after KA administration. To investigate the role of individual PGs in the delayed neuronal death, we performed intracerebroventricular injection of PGD2, PGE2, or PGF2α in rats whose hippocampal PG productions were entirely blocked by pretreatment of NS398, a COX-2 selective inhibitor. Administration of PGD2 and PGF2α had a latent contribution to the delayed neuronal death, sustained over 30 days after a single KA treatment. Furthermore, PGD2 enhanced microglial activation, which may be involved in the delayed neuronal death in the hippocampus. These findings suggest that excitotoxic delayed neuronal death is mediated through microglia activated by PGD2.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Hipocampo/metabolismo , Ácido Caínico/administração & dosagem , Prostaglandina D2/biossíntese , Animais , Morte Celular/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Nitrobenzenos/administração & dosagem , Prostaglandina D2/metabolismo , Ratos , Sulfonamidas/administração & dosagemRESUMO
Sleep-disordered breathing produces cognitive impairments, and is possibly associated with Alzheimer disease (AD). Intermittent hypoxia treatment (IHT), an experimental model for sleep-disordered breathing, results in cognitive impairments in animals via unknown mechanisms. Here, we exposed mice to IHT protocols, and performed biochemical analyses and microarray analyses regarding their hippocampal samples. In particular, we performed gene ontology (GO)-based microarray analysis to elucidate effects of IHT on hippocampal functioning, which were compared with the effects of various previously-reported experimental conditions on that (ref. Gene Expression Omnibus, The National Center for Biotechnology Information). Our microarray analyses revealed that IHT and aging shared alterations in some common GO, which were also observed with kainic acid treatment, Dicer ablation, or moderate glutamate excess. Mapping the altered genes using the Kyoto Encyclopedia of Genes and Genomes PATHWAY database indicated that IHT and aging affected several pathways including "MAPK signaling pathway", "PI3K-Akt signaling pathway", and "glutamatergic synapse". Consistent with the gene analyses, in vivo analyses revealed that IHT increased phosphorylated tau, reflecting an imbalance of kinases and/or phosphatases, and reduced proteins relevant to glutamatergic synapses. In addition, IHT increased phosphorylated p70 S6 kinase, indicating involvement of the mammalian target of rapamycin signaling pathway. Furthermore, IHT mice demonstrated hyperactivity in Y-maze tests, which was also observed in AD models. We obtained important data or something from the massive amount of microarray data, and confirmed the validity by in vivo analyses: the IHT-induced cognitive impairment may be partially explained by the fact that IHT increases phosphorylated tau via biological processes common to aging. Moreover, as aging is a major risk factor for AD, IHT is a novel model for investigating the pathological processes contributing to AD onset.
Assuntos
Envelhecimento/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Hipóxia/metabolismo , Hipóxia/patologia , Proteínas tau/metabolismo , Animais , Ontologia Genética , Masculino , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Análise de Componente Principal , Sinapses/metabolismoRESUMO
Down syndrome (DS), the most common genetic disorder, is caused by trisomy 21. DS is accompanied by heart defects, hearing and vision problems, obesity, leukemia, and other conditions, including Alzheimer's disease (AD). In comparison, most cancers are rare in people with DS. Overexpression of dual specificity tyrosine-phosphorylation-regulated kinase 1A and a regulator of calcineurin 1 located on chromosome 21 leads to excessive suppression of the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, resulting in reduced expression of a critical angiogenic factor. However, it is unclear whether the calcineurin-NFAT signaling pathway is involved in AD pathology in DS patients. Here, we investigated the association between the calcineurin-NFAT signaling pathway and AD using neuronal cells. Short-term pharmacological stimulation decreased gene expression of tau and neprilysin, and long-term inhibition of the signaling pathway decreased that of amyloid precursor protein. Moreover, a calcineurin inhibitor, cyclosporine A, also decreased neprilysin activity, leading to increases in amyloid-ß peptide levels. Taken together, our results suggest that a dysregulation in calcineurin-NFAT signaling may contribute to the early onset of AD in people with DS.
Assuntos
Doença de Alzheimer/metabolismo , Calcineurina/metabolismo , Fatores de Transcrição NFATC/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Inibidores de Calcineurina/farmacologia , Ionóforos de Cálcio/farmacologia , Linhagem Celular Tumoral , Ciclosporina/farmacologia , Proteínas de Ligação a DNA , Síndrome de Down/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ionomicina/farmacologia , Luciferases/genética , Luciferases/metabolismo , Proteínas Musculares/genética , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/genética , Neprilisina/genética , Neprilisina/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Proteínas tau/genéticaRESUMO
The γ-secretase complex comprises presenilin (PS), nicastrin (NCT), anterior pharynx-defective 1 (Aph1), and presenilin enhancer 2 (Pen2). PS has two homologues, PS1 and PS2. Aph1 has two isoforms, Aph1a and Aph1b, with the former existing as two splice variants Aph1aL and Aph1aS. Each complex consists of one subunit each, resulting in six different γ-secretases. To better understand the functional differences among the γ-secretases, we reconstituted them using a yeast system and compared Notch1-cleavage and amyloid precursor protein (APP)-cleavage activities. Intriguingly, PS2/Aph1b had a clear substrate specificity: APP-Gal4, but not Notch-Gal4, was cleaved. In HEK cell lines expressing defined γ-secretase subunits, we showed that PS1/Aph1b, PS2/Aph1aL, PS2/Aph1aS and PS2/Aph1b γ-secretase produced amyloid ß peptide (Aß) with a higher Aß42+Aß43-to-Aß40 (Aß42(43)/Aß40) ratio than the other γ-secretases. In addition, PS2/Aph1aS γ-secretase produced less Notch intracellular domain (NICD) than did the other 5 γ-secretases. Considering that the Aß42(43)/Aß40 ratio is relevant in the pathogenesis of Alzheimer's disease (AD), and that inhibition of Notch cleavage causes severe side effect, these results suggest that the PS2/Aph1aS γ-secretase complex is a potential therapeutic target in AD.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Western Blotting , Endopeptidases , Células HEK293 , Humanos , Proteínas de Membrana/genética , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/genética , Presenilina-1/genética , Presenilina-2/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
Protein interacting with C kinase 1 (PICK1) is a synaptic protein interacting with the AMPA receptor subunits GluA2/3. The interaction between GluA2 and PICK1 is required for the removal of GluA2 from the synaptic plasma membrane during long-term depression (LTD). It has been suggested that glycogen synthase kinase 3ß (GSK-3ß) is activated during LTD, but the relationships between GluA2, PICK1, and GSK-3ß are not well understood. In particular, the substrate(s) of GSK-3ß have not yet been determined. Here we showed that PICK1 is a substrate of GSK-3ß. We found that Ser(339), Ser(342), Ser(412), and Ser(416) of PICK1 were putative GSK-3ß-mediated phosphorylation sites. Among these sites, Ser(416) played a crucial role in regulating the interaction between GluA2 and PICK1. We showed that replacing Ser(416) with Ala disrupted the GluA2-PICK1 interaction, whereas substituting Ser(416) with Glu or Asp retained this interaction. However, deletion of Ser(416) did not affect the GluA2-PICK1 interaction, and substitution of Ser(416) with Ala did not alter the PICK1-PICK1 interaction. Using image analysis in COS-7 cells with AcGFP1-fused PICK1, we showed that substitution of Ser(416) with Ala increased the formation of AcGFP1-positive clusters, suggesting an increase in the association of PICK1 with the membrane. This may have resulted in the dissociation of the GluA2-PICK1 complexes. Our results indicated that GSK-3ß-mediated phosphorylation of PICK1 at Ser(416) was required for its association with the AMPA receptor subunit. Therefore, the GSK-3ß-mediated phosphorylation of PICK1 may be a regulating factor during LTD induction.
Assuntos
Proteínas de Transporte/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Proteínas do Citoesqueleto , Ácido Glutâmico/química , Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Imunoprecipitação , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Receptores de AMPA/metabolismo , Homologia de Sequência de Aminoácidos , Serina/químicaRESUMO
Multiple sclerosis is a chronic demyelinating disease of the central nervous system leading to progressive cognitive and motor dysfunction, which is characterized by neuroinflammation, demyelination, astrogliosis, loss of oligodendrocytes, and axonal pathologies. Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. cPA elicits a neurotrophin-like action and protects hippocampal neurons from ischemia-induced delayed neuronal death. In this study, we investigated the effects of cPA on cuprizone-induced demyelination, which is a model of multiple sclerosis. Mice were fed a diet containing 0.2% cuprizone for 5 weeks, which induces severe demyelination, astrocyte and microglial activation, and motor dysfunction. Simultaneous administration of cPA effectively attenuated cuprizone-induced demyelination, glial activation, and motor dysfunction. These data indicate that cPA may be a useful treatment to reduce the extent of demyelination and the severity of motor dysfunction in multiple sclerosis. cPA is a potential lead compound in the development of drugs for the treatment of this devastating disease.
Assuntos
Cuprizona/toxicidade , Doenças Desmielinizantes/prevenção & controle , Modelos Animais de Doenças , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Transtornos das Habilidades Motoras/prevenção & controle , Ácidos Fosfatídicos/uso terapêutico , Animais , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Compostos Heterocíclicos com 1 Anel/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Transtornos das Habilidades Motoras/induzido quimicamente , Transtornos das Habilidades Motoras/patologia , Ácidos Fosfatídicos/farmacologia , Resultado do TratamentoRESUMO
The pathogenesis of Alzheimer's disease (AD) is correlated with the toxicity of amyloid ß-peptide (Aß), especially Aß42. γ-Secretase modulators (GSMs) are compounds that alter production of Aß42 without interfering with the physiological function of γ-secretase. Aß42-lowering GSMs have been studied with the hope of using them as therapeutic or prophylactic drugs for AD. However, the mechanism of action of GSMs is not well defined. We examined the effect of Aß42-lowering GSMs on model cells producing large amounts of Aß42: CHO cells expressing CTF1-51, a precursor peptide of Aß that is mainly cleaved into Aß42. Our results indicate that the effect of GSM in the model was weak. Thus, we conclude that CTF1-51 cleavage mainly yields Aß42 and suppresses the effects of some GSMs, a phenomenon that may be related to their mechanism of action.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/genética , Ibuprofeno/farmacologia , Fragmentos de Peptídeos/genética , Sulindaco/análogos & derivados , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/metabolismo , Sulindaco/farmacologiaRESUMO
Alzheimer's disease (AD) is characterized by senile plaques caused by amyloid-ß peptide (Aß) accumulation. It has been reported that Aß generation and accumulation occur in membrane microdomains, called lipid rafts, which are enriched in cholesterol and glycosphingolipids. Moreover, the ablation of cholesterol metabolism has been implicated in AD. Neprilysin (NEP), a neutral endopeptidase, is one of the major Aß-degrading enzymes in the brain. Activation of NEP is a possible therapeutic target. However, it remains unknown whether the activity of NEP is regulated by its association with lipid rafts. Here we show that only the mature form of NEP, which has been glycosylated in the Golgi, exists in lipid rafts, where it is directly associated with phosphatidylserine. Moreover, the localization of NEP in lipid rafts is enhanced by its dimerization, as shown using the NEP E403C homodimerization mutant. However, the protease activities of the mature form of NEP, as assessed by in vitro peptide hydrolysis, did not differ between lipid rafts and nonlipid rafts. We conclude that cholesterol and other lipids regulate the localization of mature NEP to lipid rafts, where the substrate Aß accumulates but does not modulate the protease activity of NEP.
Assuntos
Microdomínios da Membrana/enzimologia , Neprilisina/metabolismo , Peptídeos beta-Amiloides/metabolismo , Linhagem Celular Transformada , Colesterol/metabolismo , Dimerização , Endopeptidases/metabolismo , Humanos , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Mutação/genética , Neprilisina/genética , Transfecção , beta-Ciclodextrinas/farmacologiaRESUMO
γ-Secretase is composed of at least four proteins, presenilin (PS), nicastrin (NCT), Aph1, and Pen2. PS is the catalytic subunit of the γ-secretase complex, having aspartic protease activity. PS has two homologs, namely, PS1 and PS2. To compare the activity of these complexes containing different PSs, we reconstituted them in yeast, which lacks γ-secretase homologs. Yeast cells were transformed with PS1 or PS2, NCT, Pen2, Aph1, and artificial substrate C55-Gal4p. After substrate cleavage, Gal4p translocates to the nucleus and activates transcription of the reporter genes ADE2, HIS3, and lacZ. γ-Secretase activity was measured based on yeast growth on selective media and ß-galactosidase activity. PS1 γ-secretase was â¼24-fold more active than PS2 γ-secretase in the ß-galactosidase assay. Using yeast microsomes containing γ-secretase and C55, we compared the concentration of Aß generated by PS1 or PS2 γ-secretase. PS1 γ-secretase produced â¼24-fold more Aß than PS2 γ-secretase. We found the optimal pH of Aß production by PS2 to be 7.0, as for PS1, and that the PS2 complex included immature NCT, unlike the PS1 complex, which included mature NCT. In this study, we compared the activity of PS1 or PS2 per one γ-secretase complex. Co-immunoprecipitation experiments using yeast microsomes showed that PS1 concentrations in the γ-secretase complex were â¼28 times higher than that of PS2. Our data suggest that the PS1 complex is only marginally less active than the PS2 complex in Aß production.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Núcleo Celular/metabolismo , Complexos Multienzimáticos/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Transporte Ativo do Núcleo Celular/genética , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Núcleo Celular/genética , Endopeptidases , Regulação Fúngica da Expressão Gênica/genética , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microssomos/metabolismo , Complexos Multienzimáticos/genética , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Presenilina-1/genética , Presenilina-2/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
γ-Secretase catalyzes the cleavage of the intramembrane region of the Alzheimer amyloid precursor protein (APP), generating p3, amyloid-ß peptide (Aß), and the APP intracellular domain (AICD). Although a γ-secretase inhibitor has been shown to cause an accumulation of the APP C-terminal fragments (CTFs) α and ß and to decrease levels of p3 or Aß and AICD, we found that treatment with a lysosomotropic weak base, such as chloroquine or ammonium chloride, caused simultaneous accumulation of both CTFs and AICD, suggesting that lysosomal proteases are also involved in processing of APP. This observation was reinforced by the results that cysteine protease inhibitor E-64d and cathepsin B specific inhibitor CA-074Me caused the accumulation of both CTFs and AICD with no change in known secretase activities. γ-Secretase preferentially cleaved phosphorylated CTFs to produce Aß, but cathepsin B degraded CTFs regardless of phosphorylation. Our results suggest that cathepsin B plays novel roles in the metabolism of APP and that an inhibition of APP phosphorylation is an attractive therapeutic target for Alzheimer's disease.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Catepsina B/metabolismo , Glioma/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Animais , Catepsina B/antagonistas & inibidores , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Presenilinas/metabolismo , Estrutura Terciária de ProteínaRESUMO
The amyloid-beta (Abeta) peptide, widely known as the causative molecule of Alzheimer disease (AD), is generated by the sequential cleavage of amyloid precursor protein (APP) by the aspartyl proteases BACE1/beta-secretase and presenilin/gamma-secretase. Inhibition of BACE1, therefore, is a promising strategy for preventing the progression of AD. However, beta-secretase inhibitors (BSIs) exhibit unexpectedly low potency in cells expressing "Swedish mutant" APP (APPswe) and in the transgenic mouse Tg2576, an AD model overexpressing APPswe. The Swedish mutation dramatically accelerates beta-cleavage of APP and hence the generation of Abeta; this acceleration has been assumed to underlie the poor inhibitory activity of BSI against APPswe processing. Here, we studied the mechanism by which the Swedish mutation causes this BSI potency decrease. Surprisingly, decreased BSI potency was not observed in an in vitro assay using purified BACE1 and substrates, indicating that the accelerated beta-cleavage resulting from the Swedish mutation is not its underlying cause. By focusing on differences between the cell-based and in vitro assays, we have demonstrated here that the potency decrease is caused by the aberrant subcellular localization of APPswe processing and not by accelerated beta-cleavage or the accumulation of the C-terminal fragment of beta-cleaved APP. Because most patients with sporadic AD express wild type APP, our findings suggest that the wild type mouse is superior to the Tg2576 mouse as a model for determining the effective dose of BSI for AD patients. This work provides novel insights into the potency decrease of BSI and valuable suggestions for its development as a disease-modifying agent.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Mutação , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/isolamento & purificação , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/isolamento & purificação , Ácido Aspártico Endopeptidases/metabolismo , Bovinos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sistema Livre de Células/metabolismo , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismoRESUMO
gamma-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial gamma-secretase substrates containing amyloid precursor protein or Notch fragments. The gamma-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4p release from the membrane-bound substrates, i.e. growth of yeast on histidine and adenine, or beta-galactosidase assay. We screened and evaluated gamma-secretase mutants using this reconstitution system in yeast, which does not possess endogenous gamma-secretase activity. When we introduced familial Alzheimer mutants of PS1 in this system, their activities were shown to be loss of function. Although the protease activity of wild type PS1 depends on the other three subunits introduced, we obtained 15 new PS1 mutants, which are active in the absence of NCT. They possessed a S438P mutation at the ninth transmembrane domain (TM9) together with one missense mutation distributed through transmembrane and loop regions. These mutations were not related to familial Alzheimer mutations of PS1 as identified so far. The S438P mutant was partially active but required other mutations for full activation. Results of the beta-galactosidase assay suggested that they have wild type protease activities, which were further confirmed by the endoproteolysis of PS1, amyloid beta peptides, and Notch intracellular domain production in mammalian cells. These results suggest that NCT is dispensable for the protease activity of gamma-secretase.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Glicoproteínas de Membrana/metabolismo , Mutação/genética , Presenilinas/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Immunoblotting , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Presenilinas/metabolismo , Nexinas de Proteases , Receptores de Superfície Celular , Receptores Notch/genética , Receptores Notch/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
gamma-Secretase is composed of at least four transmembrane proteins, presenilin (PS) 1/2, nicastrin, anterior pharynx-1 (Aph-1) and presenilin enhancer-2 (Pen-2), and cleaves amyloid precursor protein (APP) to produce amyloid beta peptides (Abeta) that is deposited in the brains of Alzheimer disease. However, the mechanism of gamma-secretase-mediated cleavage remains unclear. To examine the enzymatic properties of gamma-secretase, we established an in vitro assay system using Saccharomyces cerevisiae, which does not possess homologs of human PS1/2, nicastrin, Aph-1, or Pen-2. We transformed these subunits and the substrate in pep4Delta cells with vacuole proteases inactivated, and microsome was isolated for in vitro assay. In the assay, Abeta40, Abeta42, and Abeta43 were produced with an optimal pH of approximately 7.0. We also detected Abeta-production by yeast endogenous protease(s), which was abolished by the addition of phosphatidyl choline. This novel system will facilitate the analysis of substrate recognition by gamma-secretase.