RESUMO
Epidemiological studies have documented a reduced prevalence of Alzheimer's disease among users of nonsteroidal anti-inflammatory drugs (NSAIDs). It has been proposed that NSAIDs exert their beneficial effects in part by reducing neurotoxic inflammatory responses in the brain, although this mechanism has not been proved. Here we report that the NSAIDs ibuprofen, indomethacin and sulindac sulphide preferentially decrease the highly amyloidogenic Abeta42 peptide (the 42-residue isoform of the amyloid-beta peptide) produced from a variety of cultured cells by as much as 80%. This effect was not seen in all NSAIDs and seems not to be mediated by inhibition of cyclooxygenase (COX) activity, the principal pharmacological target of NSAIDs. Furthermore, short-term administration of ibuprofen to mice that produce mutant beta-amyloid precursor protein (APP) lowered their brain levels of Abeta42. In cultured cells, the decrease in Abeta42 secretion was accompanied by an increase in the Abeta(1-38) isoform, indicating that NSAIDs subtly alter gamma-secretase activity without significantly perturbing other APP processing pathways or Notch cleavage. Our findings suggest that NSAIDs directly affect amyloid pathology in the brain by reducing Abeta42 peptide levels independently of COX activity and that this Abeta42-lowering activity could be optimized to selectively target the pathogenic Abeta42 species.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Fragmentos de Peptídeos/metabolismo , Sulindaco/análogos & derivados , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Doença de Alzheimer/etiologia , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Ácido Aspártico Endopeptidases , Encéfalo/metabolismo , Células CHO , Cricetinae , Modelos Animais de Doenças , Endopeptidases/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Ibuprofeno/farmacologia , Indometacina/farmacologia , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Prostaglandina-Endoperóxido Sintases/metabolismo , Sulindaco/farmacologia , Células Tumorais CultivadasRESUMO
Endocytosis of full-length beta-amyloid precursor protein (APP) from the plasma membrane contributes to beta-amyloid peptide (Abeta) secretion, and, hence, potentially contributes to the molecular pathogenesis of Alzheimer's disease. We recently have demonstrated that central neuronal APP is endocytosed in a common vesicular compartment with recycling synaptic vesicle integral membrane proteins, but is then sorted away from synaptic vesicles for retrograde transport to the neuronal soma. For this report, we explore whether recombinant adenovirus can be used to modulate APP expression in cultured central neurons to study APP processing by the endocytotic pathway in these cells. Using a replication-deficient recombinant adenovirus that expresses a lacZ reporter (Ad5/CMV-lacZ), we demonstrate high efficiency of transfection (30-35%) at low viral titer (10-20 MOI), with no significant neuronal toxicity or cytoarchitectural change. In addition, we demonstrate that infection with the control virus does not result in re-direction of endogenous neuronal APP from usual endocytotic pathways. We have prepared, using the same genomic background as the control virus, an adenoviral vector that expresses the neuronal isoform of human APP (Ad5/CMV-APP). Infection with Ad5/CMV-APP at 10-20 MOI results in significantly increased immunoreactivity for endocytosed APP with preservation of usual endocytotic trafficking. These results demonstrate that recombinant adenovirus at low titer is an appropriate and effective vector for protein trafficking/processing studies in cultured central neurons.
Assuntos
Adenoviridae , Precursor de Proteína beta-Amiloide/genética , Endocitose/genética , Vetores Genéticos , Neurônios/citologia , Infecções por Adenoviridae , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/imunologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Anticorpos Monoclonais , Células Cultivadas , Cerebelo/citologia , Genes Reporter , Humanos , Óperon Lac , Microscopia Eletrônica , Neurônios/metabolismo , Neurônios/ultraestrutura , Ratos , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
We recently demonstrated that the Alzheimer's beta-amyloid precursor protein (APP) is internalized from the axonal cell surface. In this study, we use biochemical and cell biological methods to characterize endocytotic compartments that participate in trafficking of APP in central neurons. APP is present in presynaptic clathrin-coated vesicles purified from bovine brain, together with the recycling synaptic vesicle integral membrane proteins synaptophysin, synaptotagmin, and SV2. In contrast, APP is largely excluded from synaptic vesicles purified from rat brain. In primary cerebellar macroneurons, cell-surface APP is internalized with recycling synaptic vesicle integral membrane proteins but is subsequently sorted away from synaptic vesicles and transported retrogradely to the neuronal soma. Internalized APP partially co-localizes with rab5a-containing compartments in axons and with V-ATPase-containing compartments in both axons and neuronal soma. These results provide direct biochemical evidence that an obligate sorting compartment participates in the regeneration of synaptic vesicles during exo/endocytotic recycling at nerve terminals but do not preclude concurrent "kiss-and-run" recycling. Moreover, APP is now, to our knowledge, the first demonstrated example of an axonal cell-surface protein that is internalized with recycling synaptic vesicle membrane proteins but is subsequently sorted away from synaptic vesicles.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Vesículas Sinápticas/fisiologia , Animais , Bovinos , Membrana Celular , Células Cultivadas , Cerebelo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Clatrina/metabolismo , Vesículas Revestidas/metabolismo , Endocitose , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Ratos/embriologiaRESUMO
Proton-translocating ATPases of the vacuolar class (V-ATPases) are found in a variety of animal cell compartments that participate in vesicular membrane transport, including clathrin-coated vesicles, endosomes, the Golgi apparatus, and lysosomes. The exact structural relationship that exists among the V-ATPases of these intracellular compartments is not currently known. In the present study, we have localized the V-ATPase by light and electron microscopy, using monoclonal antibodies that recognize the V-ATPase present in clathrin-coated vesicles. Localization using light microscopy and fluorescently labeled antibodies reveals that the V-ATPase is concentrated in the juxtanuclear region, where extensive colocalization with the Golgi marker wheat germ agglutinin is observed. The V-ATPase is also present in approximately 60% of endosomes and lysosomes fluorescently labeled using alpha 2-macroglobulin as a marker for the receptor-mediated endocytic pathway. Localization using transmission electron microscopy and colloidal gold-labeled antibodies reveals that the V-ATPase is present at and near the plasma membrane, alone or in association with clathrin. These results provide evidence that the V-ATPases of plasma membrane, endosomes, lysosomes, and the Golgi apparatus are immunologically related to the V-ATPase of clathrin-coated vesicles.
Assuntos
Membrana Celular/imunologia , Clatrina/imunologia , Membranas Intracelulares/imunologia , ATPases Translocadoras de Prótons/imunologia , Vacúolos/enzimologia , Animais , Anticorpos Monoclonais , Bovinos , Membrana Celular/ultraestrutura , Células Cultivadas , Clatrina/ultraestrutura , Imunofluorescência , Complexo de Golgi/imunologia , Complexo de Golgi/ultraestrutura , Membranas Intracelulares/ultraestrutura , Rim/citologia , Lisossomos/imunologia , Lisossomos/ultraestrutura , Microscopia Imunoeletrônica , Vacúolos/ultraestruturaRESUMO
In order to refine further our structural model of the coated vesicle (H+)-ATPase (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802), we have extended our structural analysis to identify peripheral and glycosylated subunits of the pump as well as to identify subunits which are in close proximity in the native (H+)-ATPase complex. Treatment of the purified, reconstituted (H+)-ATPase with 0.30 M KI in the presence or absence of ATP or MgATP results in the release of the 73-, 58-, 40-, 34-, and 33-kDa subunits, leaving behind the 100-, 38-, 19-, and 17-kDa subunits in the membrane. Because the former group of polypeptides is released from the membrane in the absence of detergent, they correspond to peripheral membrane proteins. To determine which subunits are in close proximity, cross-linking of the purified (H+)-ATPase was carried out using the cleavable, bifunctional amino reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by two-dimensional gel electrophoresis. These studies indicate that contact regions exist between the 73- and 58-kDa subunits as well as between the 17-kDa subunit and the 40-, 34-, and 33-kDa subunits. To test for glycosylation of the (H+)-ATPase, the detergent-solubilized complex was treated with neuraminidase followed by electrophoresis and blotting using a peanut lectin/horseradish peroxidase conjugate. Galactose-inhibitable staining of the 100-kDa subunit, together with affinity chromatography of the intact (H+)-ATPase on peanut lectin agarose, indicates that the 100-kDa subunit is glycosylated, most likely at a site exposed on the luminal side of the membrane. These results, together with those presented in the preceding paper (Adachi, I., Arai, H., Pimental, R., and Forgac, M. (1990) J. Biol. Chem. 265, 960-966), were used in the construction of a refined model of the coated vesicle (H+)-ATPase.