Cholesterol-dependent Generation of a Seeding Amyloid beta -Protein in Cell Culture*

Tetsuya MizunoDagger , Makoto Nakata§, Hironobu Naiki, Makoto MichikawaDagger , Rong Wangparallel , Christian Haass**, and Katsuhiko YanagisawaDagger Dagger Dagger

From the Dagger  Department of Dementia Research, National Institute for Longevity Sciences, Gengo 36-3, Morioka, Obu, 474-8522, Japan, § Protein Research Foundation, Peptide Institute, Inc., 4-1-2 Ina, Minoh, Osaka 562, Japan,  Department of Pathology, Fukui Medical University, Fukui 910-1193, Japan, parallel  Laboratory for Mass Spectrometry, Rockefeller University, New York, New York 10021-6399, and ** Department of Molecular Biology, J5, Central Institute for Mental Health, 68159 Mannheim, Germany

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Deposition of aggregated amyloid beta -protein (Abeta ), a proteolytic cleavage product of the amyloid precursor protein (1), is a critical step in the development of Alzheimer's disease (2). However, we are far from understanding the molecular mechanisms underlying the initiation of Abeta polymerization in vivo. Here, we report that a seeding Abeta , which catalyzes the fibrillogenesis of soluble Abeta , is generated from the apically missorted amyloid precursor protein in cultured epithelial cells. Furthermore, the generation of this Abeta depends exclusively on the presence of cholesterol in the cells. Taken together with mass spectrometric analysis of this novel Abeta and our recent study (3), it is suggested that a conformationally altered form of Abeta , which acts as a "seed" for amyloid fibril formation, is generated in intracellular cholesterol-rich microdomains.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Abeta 1 is physiologically secreted into the extracellular space; however, why and how soluble Abeta aggregates and forms amyloid fibrils remains to be elucidated. A great deal of effort has been made to clarify this issue, using mainly in vitro systems. In most such experiments, it has been found that Abeta at much higher concentrations than those prevailing in biological fluids is needed for Abeta aggregation. Thus, it has been hypothesized that aggregation of soluble Abeta involves seeded polymerization (4, 5), although this assumption has not yet been proved in vivo.

We have recently reported the detection of a novel Abeta in the apical compartment of cultures of MDCK cells that had been stably transfected with APP cDNA (Delta C MDCK cell) with a truncated cytoplasmic domain (Delta C APP) (3). This Abeta 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 novo cholesterol synthesis and a cholesterol-binding drug, respectively. Based on previously reported evidence for Delta C APP being missorted to the apical surface (6) and the cholesterol concentrations of the apical plasma membrane and apical transport vesicles being higher than those in other cellular membranes (7), we concluded that a novel Abeta 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) (8-11). Thus, it would be of great interest to investigate whether the novel Abeta detected in our recent study (3), 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 Abeta .

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

MDCK Cell Culture-- 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 × 105 MDCK cells transfected with human APP695 cDNA, with full-length APP (wild-type MDCK cell) or APP with a truncated cytoplasmic domain (Delta C MDCK cell) (12) 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 and Congo Red Assay of Fibril Formation of Synthetic Abeta -- Thioflavin T assay was performed as described elsewhere (13), 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 Abeta (Abeta 1-40, 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 Abeta solutions were incubated in Eppendorf tubes at 37 °C. Every hour after the start of incubation, 10 µl of the solution of synthetic Abeta was taken and mixed with 990 µl of the reaction mixture. The lot number of the Abeta used in the experiment of Fig. 1 was 518765 and that of the Abeta used in the other experiments was 510313. Peak fluorescence was dependent on the concentration of Abeta . We used 20 µM Abeta peptide for this study. Congo red assay was performed as described elsewhere (14).

Electron Microscopy-- 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.

Enzyme Immunoassay-- The enzyme immunoassay (EIA) was performed essentially as described previously (3, 15). 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), 0.4 M 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 Abeta 17-24 (16). Appropriate amounts of synthetic Abeta 40 (Abeta 1-40, 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 Abeta 40, at 4 °C overnight. Bound enzyme activities were measured using the TMB Microwell peroxidase substrate system (Kirkegaard and Perry Laboratories, Gaithersburg, MD).

Effect of Addition of the Abeta Immunoprecipitated from the Medium of the MDCK Cells on the Fibril Formation of Synthetic Abeta -- 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 Abeta 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 Abeta 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 Abeta ) was mixed with 10 µl of the synthetic Abeta solution (containing 2 nmol of Abeta 1-40) and 85 µl of PBS buffer. The level of the immunoprecipitated Abeta was determined by enzyme immunoassay (data not shown), and the molecular ratio between synthetic Abeta and the immunoprecipitated Abeta was approximately 2,000:1. Amyloid fibril formation of synthetic Abeta 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.

Inhibition of de Novo Cholesterol Synthesis-- To inhibit de 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.

Treatment with Filipin-- 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.

Addition of Exogenous Cholesterol-- To confirm that the seeding ability of the apical Abeta depends on the presence of cholesterol, we investigated whether the effect of compactin and filipin was reversed by the addition of exogenous free cholesterol.

Determination of the Total Cellular Level of Cholesterol and the Level of de Novo Cholesterol Synthesis-- 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 Spectrometry-- Mass spectrometric analysis was performed essentially as described elsewhere (17). Aliquots of medium (5 ml) from the apical and basolateral compartments were incubated with 4G8 (5 µg) at 4 °C overnight for immunoprecipitation of Abeta . 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 Abeta 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 alpha -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).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

MDCK cells were cultured as described previously (6). Stable transfection of these cells with APP cDNA was performed as described elsewhere (12). To investigate whether the novel Abeta in the medium of the apical compartment of the Delta C MDCK cell cultures, referred to as apical Abeta , potentially accelerates amyloid fibril formation of synthetic Abeta , we performed a thioflavin T assay as described previously (13), using the Abeta peptide immunoprecipitated from the conditioned media. As shown in Fig. 1a, when synthetic Abeta (Abeta 1-40) was incubated with the apical Abeta derived from the Delta C MDCK cells, the fluorescence increased without a lag phase and proceeded to equilibrium hyperbolically. This time-course curve suggests that the apical Abeta 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 B and C are constants (18). Based on this differential equation, amyloid fibril formation from synthetic Abeta incubated with the apical Abeta can be explained by a first-order kinetic model; i.e. the extention of amyloid fibrils may proceed via the consecutive association of synthetic Abeta first onto the apical Abeta then onto the ends of growing fibrils (18).


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Fig. 1.   Seeding ability of the apical Abeta on the amyloid fibril formation. Panel a, effects of the immunoprecipitated Abeta on the kinetics of amyloid fibril formation in vitro. 50 µM synthetic Abeta 1-40 was incubated at a ratio of 2,000:1 with Abeta immunoprecipitated from the media of the apical () and basolateral (open circle ) compartment of Delta C MDCK cells, and the apical (black-square) and basolateral () compartment of wild-type MDCK cells. Amyloid fibril formed were quantified using the thioflavin T assay. Note that when synthetic Abeta was incubated with the apical Abeta derived from the Delta C MDCK cells (), the fluorescence increased without a lag phase and proceeded to equilibrium hyperbolically. Panel b, the semilogarithmical plot of the difference: A-F(t) versus incubation time. F(t) is the fluorescence as a function of time in the case of synthetic Abeta incubated with the apical Abeta derived from the Delta C MDCK cells, and A is the tentatively determined F(infinity ) shown as a broken line in panel a. Linear regression and correlation coefficient were calculated (r = 0.998). Note that F(t) is described by a differential equation: F'(t) = B - CF(t), that is, it follows a first-order kinetic model (see text). Panel c, electron micrographs of the mixture incubated for 3 h at 37OC containing 50 µM of synthetic Abeta plus Abeta immunoprecipitated from the media of the apical (left panel) or basolateral (right panel) compartment of Delta C MDCK cells. Note that typical amyloid fibrils were formed from synthetic Abeta plus the apical Abeta (left panel), whereas no fibrillar structures were formed from synthetic Abeta plus the basolateral Abeta (right panel). The bar indicates a length of 100 nm.

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 Abeta (Abeta 1-40) was incubated at pH 7.5, 37 °C for 3 days (18). When synthetic Abeta was incubated with other types of immunoprecipitated Abeta , 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 Abeta was further confirmed using Congo red assay (data not shown). The extent of enhancement of fibrillogenesis by the apical Abeta was statistically significant (Fig. 2a). We excluded the possibility that these results were caused by alteration in the amount of Abeta secreted from the cells by determining the level of Abeta by enzyme immunoassay (Fig. 2b).


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Fig. 2.   Statistical analysis of the acceleration of amyloid fibril formation by the apical Abeta . Panel a, accelerated fibril formation from 20 µM of synthetic Abeta 1-40 upon addition of Abeta immunoprecipitated from the media of the apical and basolateral compartments of the Delta C MDCK cells. Each column represents the average ±1 S.E. of 4 values, of the extent of increase in fluorescence over the background level obtained using protein G-Sepharose incubated in fresh media. Note that acceleration of the amyloid fibril formation was observed in the mixture containing Abeta immunoprecipitated from the medium of the apical compartment of the Delta C MDCK cells. *p < 0.01 (Student's t test). Panel b, determination of the level of Abeta 40 secreted into the media of the MDCK cells by EIA. EIA was performed as described previously (15). Note that the levels of Abeta 40 in the media were not correlated with the fluorescence intensities (panel a).

To investigate the molecular mechanism of generation of the apical Abeta 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 (7). When we incubated the cells with compactin or filipin, acceleration of fibrillogenesis upon addition of the apical Abeta 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 Abeta secreted from the cells by determining the level of Abeta by enzyme immunoassay (Fig. 3b). To further confirm that the seeding ability of the apical Abeta 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 Abeta -induced acceleration of fibrillogenesis of synthetic Abeta 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 the de novo cholesterol synthesis was markedly suppressed in cultures treated with compactin (Fig. 3d). These results suggest that generation of the seeding Abeta requires the presence of cholesterol in specific microdomains and does not depend on the total cellular level of cholesterol. The altered Abeta 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.


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Fig. 3.   Cholesterol-dependent seeding ability of the apical Abeta . Panel a, inhibition of the apical Abeta -induced acceleration of fibril formation of synthetic Abeta 1-40 following treatment of the cells with compactin or filipin. Each column represents the average ± 1 S.E. of 4 values of the fluorescence increase over the background level obtained using protein G-Sepharose incubated in fresh media. Panel b, determination of the level of Abeta 40 secreted into the media of the MDCK cells by EIA. EIA was performed as described previously (15). Note that the levels of Abeta 40 in the media were not correlated with the fluorescence intensities (panel a). Panel c, prevention of inhibition of the apical Abeta -induced acceleration of fibril formation by compactin and filipin, following the addition of exogenous cholesterol at the concentration indicated. Each column represents the average ± 1 S.E. of 4 values as described in panel a. Panel d, determination of the total cellular level of cholesterol and the de novo cholesterol synthesis in cultures treated with compactin and filipin. Each column represents the average ± 1 S.E. of 4 values. *p < 0.01; **p < 0.05 (Student's t test).

We then attempted to characterize the novel SDS-stable Abeta species. It was previously reported that Abeta can form SDS-stable oligomers (19); 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 Abeta was identical to that of human Abeta 6-40 peptide (Fig. 4a), whereas that of the Abeta immunoprecipitated from the media of the basolateral compartment was identical to the molecular mass of Abeta 5-40 (data not shown), which is consistent with a previous report (12). Notably, the molecular mass of the apical Abeta 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 (3). To exclude the possibility of a different Abeta 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 Abeta is not due to the association of the Abeta 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 Abeta was restored by treatment of the cells with compactin (3). Furthermore, formic acid treatment of the apical Abeta abolished its ability to act as a seed (data not shown).


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Fig. 4.   , Mass spectrometry of the apical Abeta . Matrix-assisted laser desorption/ionization mass spectrometry spectra of the Abeta immunoprecipitated from the medium of the apical compartment of Delta C MDCK cell cultures without (panel a) and with (panel b) compactin treatment. Peaks observed in the spectra were labeled with protonated molecular masses (M+H+), which corresponded to the calculated protonated molecular mass of 3712.2 Da for human Abeta 6-40. We did not detect significant peaks corresponding to Abeta 42, probably due to its presence in low amounts. Note that the molecular mass of the Abeta obtained from the compactin-treated cells was identical to that obtained from nontreated cells.

Here we report, for the first time, that a seeding Abeta , which catalyzes the fibrillogenesis of soluble Abeta , is endogenously generated in cell culture. A noteworthy finding in this study is that generation of the seeding Abeta 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 Abeta remains to be determined; however, lipid microdomains, called rafts (20), 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 (21); second, localization of APP in caveolae has recently been reported (22); third, further evidence to support the generation of Abeta in the cholesterol-rich microdomains is accumulating (23, 24).

At this point, it is extremely difficult to elucidate the molecular mechanism of acquisition by the apical Abeta of its unique molecular characteristics, including its seeding ability; however, it may be reasonable to assume that the apical Abeta adopts an altered conformation based on the following experimental results obtained from this and previous studies (3): first, the immunoreactivity of the apical Abeta 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. 3 and Fig. 4); second, the BAN50 immunoreactivity for the apical Abeta recovered following treatment of the Abeta with formic acid (3). In the putative conformational alteration of the apical Abeta , 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) (9). 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 (25); second, we have previously found GM1 ganglioside-bound Abeta in human brains in the early stages of Alzheimer's disease (26); third, Abeta undergoes alteration of its secondary structure via interaction with GM1 ganglioside (27, 28); and fourth, it has recently been reported that amyloid fibril formation of Abeta is drastically accelerated in the presence of GM1 ganglioside (14). Thus, it is intriguing to speculate that the Abeta 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 Abeta into a seeding Abeta .

Recently, much attention has been focused on the conformational alteration of constitutive proteins in the brain in various neurodegenerative diseases (4, 5, 29). In such processes, a constitutive protein in the brain undergoes minor perturbations of structure, leading to an increase in beta -sheet content; it has been proposed that these disease processes be grouped into one new category, the conformational diseases (29). Conformational conversion of the prion protein with resultant aggregation of its pathogenic form is a well-known example belonging to this category (30). Although Alzheimer's disease could also be included in the conformational diseases group (4, 5, 29), to date, no study has ever shown the generation of a conformationally altered isoform of Abeta with seeding ability. In this regard, our results present, for the first time, evidence for the generation of a seeding Abeta 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 (31) studies, one can consider the possibility that missorting of APP or altered intracellular trafficking of Abeta 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 Abeta from axonal or presynaptic membranes.

    ACKNOWLEDGEMENTS

The authors thank Y. Hanai for preparing this manuscript and I. Yamaguchi for technical support in the electron microscopic analysis.

    FOOTNOTES

* This study was supported by a research grant for Longevity Sciences (8A-1) and Brain Research Science from the Ministry of Health and Welfare and by Core Research for Evolutional Science and Technology, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed. Tel.: 81-562-44-5651 (ext. 834); Fax: 81-562-44-6594; E-mail: katuhiko{at}nils.go.jp.

    ABBREVIATIONS

The abbreviations used are: Abeta , amyloid beta -protein; APP, amyloid precursor protein; MDCK, Madin-Darby canine kidney cell; PBS, phosphate-buffered saline; EIA, enzyme immunoassay.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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