FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2072324061> ?p ?o ?g. }

• W2072324061 endingPage "15114" @default.
• W2072324061 startingPage "15110" @default.
• W2072324061 abstract "Deposition of aggregated amyloid β-protein (Aβ), a proteolytic cleavage product of the amyloid precursor protein (1Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3943) Google Scholar), is a critical step in the development of Alzheimer's disease (2Selkoe D.J. Annu. Rev. Cell Biol. 1994; 10: 373-403Crossref PubMed Scopus (742) Google Scholar). However, we are far from understanding the molecular mechanisms underlying the initiation of Aβ polymerization in vivo. Here, we report that a seeding Aβ, which catalyzes the fibrillogenesis of soluble Aβ, is generated from the apically missorted amyloid precursor protein in cultured epithelial cells. Furthermore, the generation of this Aβ depends exclusively on the presence of cholesterol in the cells. Taken together with mass spectrometric analysis of this novel Aβ and our recent study (3Mizuno T. Haass C. Michikawa M. Yanagisawa K. Biochim. Biophys. Acta. 1998; 1373: 119-130Crossref PubMed Scopus (48) Google Scholar), it is suggested that a conformationally altered form of Aβ, which acts as a “seed” for amyloid fibril formation, is generated in intracellular cholesterol-rich microdomains. Deposition of aggregated amyloid β-protein (Aβ), a proteolytic cleavage product of the amyloid precursor protein (1Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3943) Google Scholar), is a critical step in the development of Alzheimer's disease (2Selkoe D.J. Annu. Rev. Cell Biol. 1994; 10: 373-403Crossref PubMed Scopus (742) Google Scholar). However, we are far from understanding the molecular mechanisms underlying the initiation of Aβ polymerization in vivo. Here, we report that a seeding Aβ, which catalyzes the fibrillogenesis of soluble Aβ, is generated from the apically missorted amyloid precursor protein in cultured epithelial cells. Furthermore, the generation of this Aβ depends exclusively on the presence of cholesterol in the cells. Taken together with mass spectrometric analysis of this novel Aβ and our recent study (3Mizuno T. Haass C. Michikawa M. Yanagisawa K. Biochim. Biophys. Acta. 1998; 1373: 119-130Crossref PubMed Scopus (48) Google Scholar), it is suggested that a conformationally altered form of Aβ, which acts as a “seed” for amyloid fibril formation, is generated in intracellular cholesterol-rich microdomains. Aβ 1The abbreviations used are: Aβ, amyloid β-protein; APP, amyloid precursor protein; MDCK, Madin-Darby canine kidney cell; PBS, phosphate-buffered saline; EIA, enzyme immunoassay. 1The abbreviations used are: Aβ, amyloid β-protein; APP, amyloid precursor protein; MDCK, Madin-Darby canine kidney cell; PBS, phosphate-buffered saline; EIA, enzyme immunoassay. is physiologically secreted into the extracellular space; however, why and how soluble Aβ aggregates and forms amyloid fibrils remains to be elucidated. A great deal of effort has been made to clarify this issue, using mainlyin vitro systems. In most such experiments, it has been found that Aβ at much higher concentrations than those prevailing in biological fluids is needed for Aβ aggregation. Thus, it has been hypothesized that aggregation of soluble Aβ involves seeded polymerization (4Lansbury Jr., P.T. Neuron. 1997; 19: 1151-1154Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 5Harper J.D. Lansbury Jr., P.T. Annu. Rev. Biochem. 1997; 66: 385-407Crossref PubMed Scopus (1412) Google Scholar), although this assumption has not yet been provedin vivo. We have recently reported the detection of a novel Aβ in the apical compartment of cultures of MDCK cells that had been stably transfected with APP cDNA (ΔC MDCK cell) with a truncated cytoplasmic domain (ΔC APP) (3Mizuno T. Haass C. Michikawa M. Yanagisawa K. Biochim. Biophys. Acta. 1998; 1373: 119-130Crossref PubMed Scopus (48) Google Scholar). This Aβ species possesses unique molecular characteristics including its appearance as a smear on immunoblots and altered immunoreactivity. Significantly, these molecular characteristics disappeared dramatically following treatment of the cells with compactin or filipin, an inhibitor of de novocholesterol synthesis and a cholesterol-binding drug, respectively. Based on previously reported evidence for ΔC APP being missorted to the apical surface (6Haass C. Koo E.H. Capell A. Teplow D.B. Selkoe D.J. J. Cell Biol. 1995; 128: 537-547Crossref PubMed Scopus (123) Google Scholar) and the cholesterol concentrations of the apical plasma membrane and apical transport vesicles being higher than those in other cellular membranes (7Van A. Meer G. FEBS Lett. 1995; 369: 18-21Crossref PubMed Scopus (37) Google Scholar), we concluded that a novel Aβ is generated from apically missorted APP in a cholesterol-dependent manner. Regarding the involvement of cellular cholesterol in the generation of the pathogenic protein, it must be noted that cholesterol-rich lipid microdomains within cells, called caveolae-like domains, have been reported to be the likely sites of the conversion of the normal cellular form of prion protein (PrPc) to its pathogenic form (PrPsc) (8Vey M. Pilkuhn S. Wille H. Nixon R. DeArmond S.J. Smart E.J. Anderson R.G Taraboulos A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14945-14949Crossref PubMed Scopus (489) Google Scholar, 9Kaneko K. Vey M. Scott M. Pilkuhn S. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2333-2338Crossref PubMed Scopus (236) Google Scholar, 10Taraboulos A. Scott M. Semenov A. Avraham D. Laszlo L. J. Cell Biol. 1995; 129: 121-132Crossref PubMed Scopus (516) Google Scholar, 11Naslavsky N. Stein R. Yanai A. Friedlander G. Talaboulos A. J. Biol. Chem. 1997; 272: 6324-6331Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Thus, it would be of great interest to investigate whether the novel Aβ detected in our recent study (3Mizuno T. Haass C. Michikawa M. Yanagisawa K. Biochim. Biophys. Acta. 1998; 1373: 119-130Crossref PubMed Scopus (48) Google Scholar), the generation of which is exclusively dependent on the presence of cholesterol in the cell, has the potential to act as a seed for fibrillogenesis of soluble Aβ. Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum was used as the culture medium. We plated 2.5–3.0 × 105MDCK cells transfected with human APP695 cDNA, with full-length APP (wild-type MDCK cell) or APP with a truncated cytoplasmic domain (ΔC MDCK cell) (12Haass C. Koo E.H. Teplow D.E. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1564-1568Crossref PubMed Scopus (94) Google Scholar) onto 24-mm Transwell filters (Costar) and cultured these cells on the filters for 3 days. To determine the integrity of the cell monolayers that grew on the filters, measurement of the electric resistance between the apical and basolateral compartments of the MDCK cell culture was performed by immersing electrodes into each of the compartments. The MDCK cells culture media were changed 3 days after plating, and the cells were cultured 24 h longer. Thioflavin T assay was performed as described elsewhere (13Naiki H. Gejyo F. Nakakuki K. Biochemistry. 1997; 36: 6243-6250Crossref PubMed Scopus (98) Google Scholar), on a spectrofluorophotometer (RF-5300PC, Shimadzu, Kyoto, Japan). Optimum fluorescence measurements of amyloid fibrils were obtained at the excitation and emission wavelength of 446 nm and 490 nm, respectively, with the reaction mixture (1.0 ml) containing 5 μm thioflavin T (Nakalai tesque, Inc., Kyoto, Japan) and 50 mm of glycine-NaOH buffer, pH 8.5. Fluorescence was measured immediately after making the mixture and averaged for an initial 5 s. Synthetic Aβ (Aβ1–40, Bachem Switzerland) was initially dissolved in ice-cold distilled water at a concentration of 100, 200, and 300 μm, and then diluted with 9 volumes of PBS. Aliquots of the Aβ solutions were incubated in Eppendorf tubes at 37 °C. Every hour after the start of incubation, 10 μl of the solution of synthetic Aβ was taken and mixed with 990 μl of the reaction mixture. The lot number of the Aβ used in the experiment of Fig. 1 was 518765 and that of the Aβ used in the other experiments was 510313. Peak fluorescence was dependent on the concentration of Aβ. We used 20 μm Aβ peptide for this study. Congo red assay was performed as described elsewhere (14Choo-Smith L.-P. Garzon-Rodriguez W. Glabe C.G. Surewicz W.K. J. Biol. Chem. 1997; 272: 22987-22990Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Samples were spread on carbon-coated grids, negatively stained with 1% phosphotungstic acid, pH 7.0, then examined under a Hitachi H-7000 electron microscope with an acceleration voltage of 75 kV. The enzyme immunoassay (EIA) was performed essentially as described previously (3Mizuno T. Haass C. Michikawa M. Yanagisawa K. Biochim. Biophys. Acta. 1998; 1373: 119-130Crossref PubMed Scopus (48) Google Scholar, 15Suzuki N. Iwatsubo T. Odaka A. Ishibashi Y. Kitada C. Ihara Y. Am. J. Pathol. 1994; 145: 452-460PubMed Google Scholar). Briefly, 100 μl of the media of the MDCK cell cultures were diluted with 400 μl of buffer C (20 mm phosphate buffer (pH 7.0), 0.4m NaCl, 2 mm EDTA, 10% Block Ace (Dai-nippon, Tokyo, Japan), 0.2% bovine serum albumin, and 0.05% NaN3), and 100 μl of the mixture were subjected to the multiwell plates coated with 4G8, a monoclonal antibody specific for Aβ17–24 (16Kim K.S. Miller D.L. Sapienza V.J. Chen C.-M.J. Bai C. Grundke-Iqbal I. Currie J.R. Wisniewski H.M. Neurosci. Res. Commun. 1988; 2: 121-130Google Scholar). Appropriate amounts of synthetic Aβ40 (Aβ1–40, Bachem Switzerland) were applied to the multiwell plates for the construction of a standard curve. The plates were incubated at 4 °C overnight. After rinsing with PBS, loaded wells were reacted with appropriately diluted horseradish peroxidase-conjugated BA27, a monoclonal antibody specific for Aβ40, at 4 °C overnight. Bound enzyme activities were measured using the TMB Microwell peroxidase substrate system (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Aliquots (1.5 ml) of the medium from the transfected MDCK cell cultures were incubated with 4G8 (1.5 μg) at 4 °C overnight for Aβ immunoprecipitation 3 days after changing the medium. The mixtures were then incubated with protein G-Sepharose at 4 °C for 3 h and centrifuged. The pellets were washed thoroughly in RIPA buffer once, and then in Tris-saline buffer four times. The Aβ in the immunoprecipitates was extracted in 25 μl of a buffer containing 2% SDS by boiling. Following dilution with 975 μl of Tris-saline buffer, 5 μl of the solution (containing 1 pmol of the immunoprecipitated Aβ) was mixed with 10 μl of the synthetic Aβ solution (containing 2 nmol of Aβ1–40) and 85 μl of PBS buffer. The level of the immunoprecipitated Aβ was determined by enzyme immunoassay (data not shown), and the molecular ratio between synthetic Aβ and the immunoprecipitated Aβ was approximately 2,000:1. Amyloid fibril formation of synthetic Aβ was quantitatively determined by measuring fluorescence intensity at 2 and 6 h after starting the incubation as described before. The background fluorescence intensity was determined using an extract of protein G-Sepharose that was incubated with 4G8 in fresh medium. To inhibitde novo cholesterol synthesis, MDCK cells were incubated for 90 min with compactin (Sigma), a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, at concentrations of 0.5 and 1.0 μm, before changing the media to fresh ones. The cells were further cultured for 3 days with compactin, and then 1.5-ml aliquots of the medium from the apical and basolateral compartments of the culture were used for immunoprecipitation. MDCK cells were cultured in medium containing filipin (Sigma), a polyene antibiotic that specifically binds to cholesterol, at concentrations of 0.1 and 0.3 μg/ml for 90 min before changing the media to fresh ones. The cells were further cultured for 3 days with filipin, and then 1.5-ml aliquots of the medium from the apical and basolateral compartments of the culture were used for immunoprecipitation. To confirm that the seeding ability of the apical Aβ depends on the presence of cholesterol, we investigated whether the effect of compactin and filipin was reversed by the addition of exogenous free cholesterol. To determine the total level of cellular cholesterol in MDCK cells, cultures treated with compactin (1 μm) or filipin (0.3 μg/ml) for 2 days were washed 3 times in PBS and dried at room temperature. The samples were extracted with hexane/isopropanol (3:2 v/v) and dried with nitrogen. The total cholesterol levels in the samples were determined using a cholesterol determination kit (Kyowa Medical Co. Ltd., Tokyo, Japan). Protein concentration was determined using the BCA protein assay kit (Pierce), with bovine serum albumin as the standard. To determine the level of de novo cholesterol synthesis, MDCK cells were pretreated with compactin (1 μm) and filipin (0.3 μg/ml) in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum for 2 h. 2 μCi/ml [14C]acetate was added into the cultures treated with the compounds. After 3 h of incubation, the cultures were washed three times in PBS and dried at room temperature. The samples were then extracted with hexane/isopropanol (3:2 v/v) and dried with nitrogen. The samples were quantitatively spotted on thin-layer chromatography plates and developed in a solvent system of hexane/ethyl ether/acetic acid (80:30:1). The radioactivities of the spots were detected and quantified by the Bio-imaging Analyzer System-2500 Mac (Fuji Film Co. Ltd., Tokyo, Japan). Mass spectrometric analysis was performed essentially as described elsewhere (17Wang R. Sweeney D. Gandy S.E. Sisodia S.S. J. Biol. Chem. 1996; 271: 31894-31902Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Aliquots of medium (5 ml) from the apical and basolateral compartments were incubated with 4G8 (5 μg) at 4 °C overnight for immunoprecipitation of Aβ. The mixtures were then incubated with protein G-Sepharose at 4 °C for 3 h and centrifuged. The pellets were washed thoroughly with RIPA buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% SDS, 0.5% deoxycholic acid, 1.0% Nonidet P-40) once and then with distilled water four times. The immunoprecipitated Aβ was extracted with 10 μl of trifluoroacetic acid. 1 μl of the extracted solution was mixed with 10 μl of UV-laser desorption matrix (trifluoroacetic acid/water/acetonitrile (1:20:20, v/v/v), containing saturated α-cyano-4-hydroxycinnamic acid; 0.5 μl of this mixture was loaded onto the mass spectrometer sample probe and dried at room temperature. Mass spectra were measured using a UV-laser desorption/ionization time-of-flight mass spectrometer (Voyager Elite, PerSeptive Biosystem). MDCK cells were cultured as described previously (6Haass C. Koo E.H. Capell A. Teplow D.B. Selkoe D.J. J. Cell Biol. 1995; 128: 537-547Crossref PubMed Scopus (123) Google Scholar). Stable transfection of these cells with APP cDNA was performed as described elsewhere (12Haass C. Koo E.H. Teplow D.E. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1564-1568Crossref PubMed Scopus (94) Google Scholar). To investigate whether the novel Aβ in the medium of the apical compartment of the ΔC MDCK cell cultures, referred to as apical Aβ, potentially accelerates amyloid fibril formation of synthetic Aβ, we performed a thioflavin T assay as described previously (13Naiki H. Gejyo F. Nakakuki K. Biochemistry. 1997; 36: 6243-6250Crossref PubMed Scopus (98) Google Scholar), using the Aβ peptide immunoprecipitated from the conditioned media. As shown in Fig. 1a, when synthetic Aβ (Aβ1–40) was incubated with the apical Aβ derived from the ΔC MDCK cells, the fluorescence increased without a lag phase and proceeded to equilibrium hyperbolically. This time-course curve suggests that the apical Aβ acts as a seed in this experiment. A perfect linear semilogarithmic plot (r = 0.998) shown in Fig. 1b, indicates that F(t) satisfies a differential equation: F′(t) =B − CF(t), where Band C are constants (18Naiki H. Nakakuki K. Lab. Invest. 1996; 74: 374-383PubMed Google Scholar). Based on this differential equation, amyloid fibril formation from synthetic Aβ incubated with the apical Aβ can be explained by a first-order kinetic model;i.e. the extention of amyloid fibrils may proceed via the consecutive association of synthetic Aβ first onto the apical Aβ then onto the ends of growing fibrils (18Naiki H. Nakakuki K. Lab. Invest. 1996; 74: 374-383PubMed Google Scholar). Electron microscopic analysis showed the formation of typical amyloid fibrils with a diameter of approximately 10 nm and helical structure (Fig. 1c, left panel). Similar helical filament structure was observed when 400 μm of synthetic Aβ (Aβ1–40) was incubated at pH 7.5, 37 °C for 3 days (18Naiki H. Nakakuki K. Lab. Invest. 1996; 74: 374-383PubMed Google Scholar). When synthetic Aβ was incubated with other types of immunoprecipitated Aβ, the increase in the fluorescence was small and no fibrillar structures were observed with electron microscopic analysis (Fig. 1,a and c, right panel). Accelerated fibrillogenesis upon addition of the apical Aβ was further confirmed using Congo red assay (data not shown). The extent of enhancement of fibrillogenesis by the apical Aβ was statistically significant (Fig. 2a). We excluded the possibility that these results were caused by alteration in the amount of Aβ secreted from the cells by determining the level of Aβ by enzyme immunoassay (Fig. 2b). To investigate the molecular mechanism of generation of the apical Aβ with seeding ability, we first asked whether the generation is dependent on the cellular cholesterol because the level of cholesterol in the apical plasma membrane is higher than that of basolateral plasma membrane (7Van A. Meer G. FEBS Lett. 1995; 369: 18-21Crossref PubMed Scopus (37) Google Scholar). When we incubated the cells with compactin or filipin, acceleration of fibrillogenesis upon addition of the apical Aβ was substantially inhibited in a dose-dependent manner as shown in Fig. 3a. Again, we excluded the possibility that these results were caused by alteration in the amount of Aβ secreted from the cells by determining the level of Aβ by enzyme immunoassay (Fig. 3b). To further confirm that the seeding ability of the apical Aβ depended on the presence of cholesterol, we performed an experiment to see if the effect of compactin and filipin was reversed by the addition of exogenous cholesterol. As shown in Fig. 3c, the inhibition of the apical Aβ-induced acceleration of fibrillogenesis of synthetic Aβ following treatment with compactin and filipin, was dramatically reduced by the addition of exogenous cholesterol. Total cellular level of cholesterol was not dramatically decreased in cultures treated with compactin or altered at all in those treated with filipin, whereas thede novo cholesterol synthesis was markedly suppressed in cultures treated with compactin (Fig. 3d). These results suggest that generation of the seeding Aβ requires the presence of cholesterol in specific microdomains and does not depend on the total cellular level of cholesterol. The altered Aβ species was also generated in cultures that were grown in media not containing fetal bovine serum or supplemented with lipoprotein-deprived serum (data not shown), indicating that its generation does not depend on the presence of lipoprotein(s) or other factors in the serum. We then attempted to characterize the novel SDS-stable Aβ species. It was previously reported that Aβ can form SDS-stable oligomers (19Podlisny M.B. Ostaszewski B.L. Squazzo S.L. Koo E.H. Rydel R.E. Teplow D.B. Selkoe D.J. J. Biol. Chem. 1995; 270: 9564-9570Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar); thus, we attempted to determine its molecular mass using matrix-assisted laser desorption/ionization mass spectrometry. The molecular mass of the major peak for the apical Aβ was identical to that of human Aβ6–40 peptide (Fig. 4a), whereas that of the Aβ immunoprecipitated from the media of the basolateral compartment was identical to the molecular mass of Aβ5–40 (data not shown), which is consistent with a previous report (12Haass C. Koo E.H. Teplow D.E. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1564-1568Crossref PubMed Scopus (94) Google Scholar). Notably, the molecular mass of the apical Aβ did not change following treatment of the cells with compactin (Fig. 4b), whereas its smearing on the immunoblot was lost following compactin treatment (data not shown) as described previously (3Mizuno T. Haass C. Michikawa M. Yanagisawa K. Biochim. Biophys. Acta. 1998; 1373: 119-130Crossref PubMed Scopus (48) Google Scholar). To exclude the possibility of a different Aβ species being extracted in the experiment of mass spectrometry because of a difference in the used extracting buffer, we performed Western blotting analysis of the immunoprecipitates extracted with trifluoroacetic acid, which was used for the mass spectrometric study, but the smear appearance persisted (data not shown). This result indicates that the acquisition of these unique molecular characteristics by the apical Aβ is not due to the association of the Aβ with other molecules, but rather because of some conformational alteration. This assumption is also supported by our previous finding that the altered immunoreactivity of this novel Aβ was restored by treatment of the cells with compactin (3Mizuno T. Haass C. Michikawa M. Yanagisawa K. Biochim. Biophys. Acta. 1998; 1373: 119-130Crossref PubMed Scopus (48) Google Scholar). Furthermore, formic acid treatment of the apical Aβ abolished its ability to act as a seed (data not shown). Here we report, for the first time, that a seeding Aβ, which catalyzes the fibrillogenesis of soluble Aβ, is endogenously generated in cell culture. A noteworthy finding in this study is that generation of the seeding Aβ depends exclusively on the presence of cholesterol as shown in Fig. 3, a and c. Determination of the intracellular site of the generation of the seeding Aβ remains to be determined; however, lipid microdomains, called rafts (20Simon K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8091) Google Scholar), sharing a high content of cholesterol and glycosphingolipid with caveolae or caveolae-like domains are likely to be the best candidate for the following reasons: first, the axonally sorted APP, analogous to apically sorted APP in epithelial cells, is conveyed via caveolae-like domains in neurons (21Bouillot C. Prochiantz A. Rougon G. Allinquant B. J. Biol. Chem. 1996; 271: 7640-7644Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar); second, localization of APP in caveolae has recently been reported (22Ikezu T. Trapp B.D. Song K.S. Schlegel A. Lisanti M.P. Okamoto T. J. Biol. Chem. 1998; 273: 10485-10495Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar); third, further evidence to support the generation of Aβ in the cholesterol-rich microdomains is accumulating (23Simons M. Keller P. De Strooper B. Beyreuther K. Dotti C.G. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6460-6464Crossref PubMed Scopus (1082) Google Scholar, 24Lee S.J. Liyanage U. Bickel P.E. Xia W. Lansbury Jr., P.T. Kosik KS. Nat. Med. 1998; 4: 730-734Crossref PubMed Scopus (374) Google Scholar). At this point, it is extremely difficult to elucidate the molecular mechanism of acquisition by the apical Aβ of its unique molecular characteristics, including its seeding ability; however, it may be reasonable to assume that the apical Aβ adopts an altered conformation based on the following experimental results obtained from this and previous studies (3Mizuno T. Haass C. Michikawa M. Yanagisawa K. Biochim. Biophys. Acta. 1998; 1373: 119-130Crossref PubMed Scopus (48) Google Scholar): first, the immunoreactivity of the apical Aβ to BAN50, in addition to its smearing behavior on gel electrophoresis, changed following treatment of the cells with compactin without any alteration in its mass number (Ref. 3Mizuno T. Haass C. Michikawa M. Yanagisawa K. Biochim. Biophys. Acta. 1998; 1373: 119-130Crossref PubMed Scopus (48) Google Scholar and Fig. 4); second, the BAN50 immunoreactivity for the apical Aβ recovered following treatment of the Aβ with formic acid (3Mizuno T. Haass C. Michikawa M. Yanagisawa K. Biochim. Biophys. Acta. 1998; 1373: 119-130Crossref PubMed Scopus (48) Google Scholar). In the putative conformational alteration of the apical Aβ, auxiliary factors localized in the lipid microdomains may be involved, as is suggested in the conversion of the normal cellular form of prion protein (PrPc) to its pathogenic form (PrPsc) (9Kaneko K. Vey M. Scott M. Pilkuhn S. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2333-2338Crossref PubMed Scopus (236) Google Scholar). Among the candidates for such factors, we prefer to consider GM1 ganglioside for the following reasons: first, GM1 ganglioside is one of the main resident molecules in the microdomains (25Parton R.G. J. Histochem. Cytochem. 1994; 42: 155-166Crossref PubMed Scopus (453) Google Scholar); second, we have previously found GM1 ganglioside-bound Aβ in human brains in the early stages of Alzheimer's disease (26Yanagisawa K. Odaka A. Suzuki N. Ihara Y. Nat. Med. 1995; 1: 1062-1066Crossref PubMed Scopus (479) Google Scholar); third, Aβ undergoes alteration of its secondary structure via interaction with GM1 ganglioside (27McLaurin J. Chakrabartty A. J. Biol. Chem. 1996; 271: 26482-26489Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 28Choo-Smith L.-P. Surewicz W.K. FEBS Lett. 1997; 402: 95-98Crossref PubMed Scopus (167) Google Scholar); and fourth, it has recently been reported that amyloid fibril formation of Aβ is drastically accelerated in the presence of GM1 ganglioside (14Choo-Smith L.-P. Garzon-Rodriguez W. Glabe C.G. Surewicz W.K. J. Biol. Chem. 1997; 272: 22987-22990Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Thus, it is intriguing to speculate that the Aβ generated from the apically missorted APP undergoes conformational alteration via association with GM1 ganglioside in the lipid microdomains and then acts as a template for the consecutive conversion of a nascent soluble Aβ into a seeding Aβ. Recently, much attention has been focused on the conformational alteration of constitutive proteins in the brain in various neurodegenerative diseases (4Lansbury Jr., P.T. Neuron. 1997; 19: 1151-1154Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 5Harper J.D. Lansbury Jr., P.T. Annu. Rev. Biochem. 1997; 66: 385-407Crossref PubMed Scopus (1412) Google Scholar, 29Carrell R.W. Lomas D.A. Lancet. 1997; 350: 134-138Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar). In such processes, a constitutive protein in the brain undergoes minor perturbations of structure, leading to an increase in β-sheet content; it has been proposed that these disease processes be grouped into one new category, the conformational diseases (29Carrell R.W. Lomas D.A. Lancet. 1997; 350: 134-138Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar). Conformational conversion of the prion protein with resultant aggregation of its pathogenic form is a well-known example belonging to this category (30Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (857) Google Scholar). Although Alzheimer's disease could also be included in the conformational diseases group (4Lansbury Jr., P.T. Neuron. 1997; 19: 1151-1154Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 5Harper J.D. Lansbury Jr., P.T. Annu. Rev. Biochem. 1997; 66: 385-407Crossref PubMed Scopus (1412) Google Scholar, 29Carrell R.W. Lomas D.A. Lancet. 1997; 350: 134-138Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar), to date, no study has ever shown the generation of a conformationally altered isoform of Aβ with seeding ability. In this regard, our results present, for the first time, evidence for the generation of a seeding Aβ in cell culture, and furthermore, may be used to explain the molecular mechanism underlying initiation of amyloid fibril formation in vivo. Finally, from the results of this and other (31De Strooper B. Craessaerts K. Dewachter I. Moechars D. Greenberg B. Van Leuven F. Van den Berghe H. J. Biol. Chem. 1995; 270: 4058-4065Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) studies, one can consider the possibility that missorting of APP or altered intracellular trafficking of Aβ plays a role in the pathogenesis of Alzheimer's disease. Further studies, using polarized differentiated neurons should be carried out to investigate the consequences of generation of a seeding Aβ from axonal or presynaptic membranes. The authors thank Y. Hanai for preparing this manuscript and I. Yamaguchi for technical support in the electron microscopic analysis." @default.
• W2072324061 created "2016-06-24" @default.
• W2072324061 creator A5008709206 @default.
• W2072324061 creator A5015246993 @default.
• W2072324061 creator A5039416937 @default.
• W2072324061 creator A5049284705 @default.
• W2072324061 creator A5065084452 @default.
• W2072324061 creator A5066569735 @default.
• W2072324061 creator A5069337032 @default.
• W2072324061 date "1999-05-01" @default.
• W2072324061 modified "2023-09-29" @default.
• W2072324061 title "Cholesterol-dependent Generation of a Seeding Amyloid β-Protein in Cell Culture" @default.
• W2072324061 cites W135479848 @default.
• W2072324061 cites W1966815798 @default.
• W2072324061 cites W1966895389 @default.
• W2072324061 cites W1967237003 @default.
• W2072324061 cites W1975806945 @default.
• W2072324061 cites W1989269014 @default.
• W2072324061 cites W1994928619 @default.
• W2072324061 cites W2000764878 @default.
• W2072324061 cites W2013650907 @default.
• W2072324061 cites W2031343997 @default.
• W2072324061 cites W2032604948 @default.
• W2072324061 cites W2047435525 @default.
• W2072324061 cites W2050111346 @default.
• W2072324061 cites W2063880942 @default.
• W2072324061 cites W2073536737 @default.
• W2072324061 cites W2081852339 @default.
• W2072324061 cites W2081881251 @default.
• W2072324061 cites W2087932565 @default.
• W2072324061 cites W2094394444 @default.
• W2072324061 cites W2096601751 @default.
• W2072324061 cites W2121936687 @default.
• W2072324061 cites W2124342403 @default.
• W2072324061 cites W2130079515 @default.
• W2072324061 cites W2134139236 @default.
• W2072324061 cites W2137695170 @default.
• W2072324061 cites W2167182434 @default.
• W2072324061 cites W2167564565 @default.
• W2072324061 cites W4240929891 @default.
• W2072324061 doi "https://doi.org/10.1074/jbc.274.21.15110" @default.
• W2072324061 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10329717" @default.
• W2072324061 hasPublicationYear "1999" @default.
• W2072324061 type Work @default.
• W2072324061 sameAs 2072324061 @default.
• W2072324061 citedByCount "117" @default.
• W2072324061 countsByYear W20723240612012 @default.
• W2072324061 countsByYear W20723240612013 @default.
• W2072324061 countsByYear W20723240612014 @default.
• W2072324061 countsByYear W20723240612015 @default.
• W2072324061 countsByYear W20723240612016 @default.
• W2072324061 countsByYear W20723240612017 @default.
• W2072324061 countsByYear W20723240612018 @default.
• W2072324061 countsByYear W20723240612020 @default.
• W2072324061 countsByYear W20723240612022 @default.
• W2072324061 countsByYear W20723240612023 @default.
• W2072324061 crossrefType "journal-article" @default.
• W2072324061 hasAuthorship W2072324061A5008709206 @default.
• W2072324061 hasAuthorship W2072324061A5015246993 @default.
• W2072324061 hasAuthorship W2072324061A5039416937 @default.
• W2072324061 hasAuthorship W2072324061A5049284705 @default.
• W2072324061 hasAuthorship W2072324061A5065084452 @default.
• W2072324061 hasAuthorship W2072324061A5066569735 @default.
• W2072324061 hasAuthorship W2072324061A5069337032 @default.
• W2072324061 hasBestOaLocation W20723240611 @default.
• W2072324061 hasConcept C179104552 @default.
• W2072324061 hasConcept C185592680 @default.
• W2072324061 hasConcept C2777633098 @default.
• W2072324061 hasConcept C2778163477 @default.
• W2072324061 hasConcept C36248471 @default.
• W2072324061 hasConcept C54355233 @default.
• W2072324061 hasConcept C55493867 @default.
• W2072324061 hasConcept C6557445 @default.
• W2072324061 hasConcept C81885089 @default.
• W2072324061 hasConcept C86803240 @default.
• W2072324061 hasConceptScore W2072324061C179104552 @default.
• W2072324061 hasConceptScore W2072324061C185592680 @default.
• W2072324061 hasConceptScore W2072324061C2777633098 @default.
• W2072324061 hasConceptScore W2072324061C2778163477 @default.
• W2072324061 hasConceptScore W2072324061C36248471 @default.
• W2072324061 hasConceptScore W2072324061C54355233 @default.
• W2072324061 hasConceptScore W2072324061C55493867 @default.
• W2072324061 hasConceptScore W2072324061C6557445 @default.
• W2072324061 hasConceptScore W2072324061C81885089 @default.
• W2072324061 hasConceptScore W2072324061C86803240 @default.
• W2072324061 hasIssue "21" @default.
• W2072324061 hasLocation W20723240611 @default.
• W2072324061 hasOpenAccess W2072324061 @default.
• W2072324061 hasPrimaryLocation W20723240611 @default.
• W2072324061 hasRelatedWork W2051541351 @default.
• W2072324061 hasRelatedWork W2072324061 @default.
• W2072324061 hasRelatedWork W2129455651 @default.
• W2072324061 hasRelatedWork W2575859011 @default.
• W2072324061 hasRelatedWork W2594273137 @default.
• W2072324061 hasRelatedWork W3016007186 @default.
• W2072324061 hasRelatedWork W3081654908 @default.
• W2072324061 hasRelatedWork W4200048535 @default.
• W2072324061 hasRelatedWork W4238841048 @default.

• next