Accumulation and Aggregation of Amyloid beta -Protein in Late Endosomes of Niemann-Pick Type C Cells*

Tsuneo YamazakiDagger , Ta-Yuan Chang§, Christian Haass, and Yasuo IharaDagger ||**

From the Dagger  Department of Neuropathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, the § Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, the  Adolf Butenandt-Institute, Department of Biochemistry, Laboratory for Alzheimer's Disease Research, Ludwig-Maximilians-University, 80336 Munich, Germany, and || Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan

Received for publication, October 20, 2000, and in revised form, November 16, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is growing evidence suggesting that cholesterol metabolism is linked to susceptibility to Alzheimer's disease by influencing amyloid beta -protein (Abeta ) metabolism. However, the precise cellular linkage sites between cholesterol and Abeta have not yet been clarified. To address this issue, we investigated Niemann-Pick type C (NPC) model cells and NPC mutant cells, which showed aberrant cholesterol trafficking. We observed a remarkable Abeta accumulation in late endosomes of both NPC model cells and mutant cells where cholesterol accumulates and a significant accumulation in the NPC mouse brain. This Abeta accumulation was independent of its constitutive secretion and production through an endocytic pathway. In addition, it is characterized by a marked predominance of Abeta 42 and insolubility in SDS, suggesting the presence of aggregated Abeta in late endosomes. Most importantly, Abeta accumulation is coupled with the cholesterol levels in late endosomes. Thus, late endosomes of NPC cells are a novel pool of aggregated Abeta 42 as well as cholesterol, suggesting a direct interaction between aggregated Abeta and cholesterol.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Formation of senile plaques composed of amyloid beta -protein (Abeta )1 is one of the hallmarks of Alzheimer's disease (AD) (1). Abeta , a small protein of Mr ~4,000, is cleaved out sequentially with beta - and gamma -secretases from a large membrane protein called beta -amyloid precursor protein (APP), and secreted into the extracellular space. There are two major species of Abeta that are defined by their C-terminal lengths; Abeta 40 terminates at Val-40, and Abeta 42 terminates at Ala-42. The latter has a much stronger tendency to aggregate into fibrils and accounts for less than 10% of secreted Abeta . However, Abeta 42 is the most predominant form found in senile plaques and is considered the initial species to be deposited in aged and AD brains (2, 3).

Three causative genes, APP, presenilin 1 (PS1), and PS2 have been identified in early onset familial AD (1), and their mutations appear to converge on an increased production of Abeta 42. However, the pathological mechanisms of sporadic AD, explaining more than 90% of AD patients, remain unknown. Interestingly, there is growing evidence for a significant linkage between Abeta and cholesterol metabolism. First, cholesterol loading or depletion affects Abeta generation both in vitro (4, 5) and in vivo (6). Second, the levels of total cholesterol and LDL-cholesterol, but not those of HDL-cholesterol, in the serum correlate with the amount of Abeta 42 in AD brains (7). Third, aggregated Abeta preferentially binds to cholesterol in vitro (8). Fourth, a substantial fraction of intracellular Abeta is localized in the detergent-insoluble membrane domain that is rich in glycosphingolipid and cholesterol (9, 10). Fifth, the presence of an E4 isoform of apolipoprotein E, an essential molecule for cholesterol metabolism, is a strong risk factor for developing AD (11). Sixth, high dietary cholesterol accelerates AD-related pathologies, including Abeta deposition, in a transgenic mouse model (12). However, no detailed mechanisms for clarifying Abeta -cholesterol interacting sites are known. We hypothesized that both cholesterol and Abeta metabolisms are linked intracellularly, and to examine this possibility, we focused on Niemann-Pick type C (NPC) disease.

NPC is an autosomal recessive neurovisceral storage disease (13), characterized by the presence of numerous foam cells in the bone marrow and visceral organs. Its hallmark is an intracellular accumulation of unesterified cholesterol and other lipids, especially sphingolipids, in vacuoles resembling late endosomes (14). A major gene (NPC1) responsible for NPC has recently been identified by positional cloning (15, 16). The cDNA sequence predicts a 1,278-residue polytopic integral membrane protein (NPC1) containing a putative cholesterol-sensing domain. The main features of the NPC phenotype can be mimicked by cultured cells exposed to a variety of reagents called class 2 amphiphiles, such as U18666A (17). It is now believed that these reagents act directly on the NPC1 protein (18). Based on these facts, we hypothesized that intracellular Abeta metabolism might be changed in these cholesterol-perturbed cells.

In this study, we investigated the intracellular Abeta in two cell lines. These were NPC model cells treated with a class 2 amphiphile, and a Chinese hamster ovary (CHO) cell mutant line that is deficient in NPC1 protein due to premature termination of its translation (19). We found that intracellular cholesterol levels strongly affect Abeta 42 accumulation and aggregation in late endosomes and that this Abeta 42 accumulation was also observed in NPC mouse brains.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines and Transfection-- CHO cells, stably transfected with wild-type (7WD10) and C-terminally truncated (Delta C) APP751 cDNAs (20), human embryonic kidney 293 cells transfected with APP695 cDNA (K295) (21), and CHO mutants 25RA and CT43 (19) were grown and maintained as described previously. For cholesterol depletion studies, monolayers of 7WD10 cells were washed twice with phosphate-buffered saline and cultured in cholesterol starvation medium (F-12 medium; Life Technologies, Inc.) with 10% delipidated fetal calf serum, 35 µM oleic acid (Sigma), 50 µM mevinolin, and 230 µM mevalonate (Sigma). Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, blocks the synthesis of steroidal and nonsteroidal isoprenoids (22). Delipidated fetal calf serum was prepared according to a procedure modified by Cadigan et al. (22). Water-soluble cholesterol and methyl-beta -cyclodextrin were purchased from Sigma. For transient transfection, APP695 cDNA was cloned into the vector pCXN (23) and transfected with LipofectAMINE (Life Technologies).

Subcellular Fractionation-- To avoid contamination of the cell lysate with the cell surface-adsorbed Abeta , cells were harvested by incubation with trypsin at 37 °C. A 1% Triton X-100 (Triton)-insoluble fraction of whole cell homogenates was prepared as described previously (10). Percoll (Amersham Pharmacia Biotech) gradient centrifugation was performed according to Lange et al. (24). Late endosomes were isolated according to van der Goot (25) using sucrose gradients containing 3 mM imidazole (pH 7.4). Three interfaces, at 8.5/25% (interface 1), 25/35% (interface 2), and 35/40.6% (interface 3) were carefully aspirated (800 µl each) and pelleted.

Western Blotting-- Each fraction obtained by subcellular fractionation was centrifuged at 540,000 × g for 20 min in a TLX ultracentrifuge (Beckman). The resulting pellets were extracted with 70% formic acid at room temperature for 30 min, and the extract was dried by Speed Vac (Savant Instruments). The proteins solubilized with the sample buffer were subjected to Western blotting as described previously (26). Cellular APP was extracted with 1% Triton in Tris saline (TS; 50 mM Tris-HCl, pH 7.4, 150 mM NaCl) and was subjected to Western blotting. In the present study, the samples were adjusted by their volumes and loaded on gels, except for that in Fig. 6A. The Abeta antibodies used were BAN50 (epitope: Abeta 1-10), 6E10 (Abeta 1-17: Senetek PLC), BA27 (specific for Abeta 40), and BC05 (specific for Abeta 42) (27). Monoclonal antibodies (5A3/1G7) recognize the midportion of APP (20). Antibodies to Rab7 and Rab5a were purchased from CytoSignal and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. The antibodies to caveolin 1 and BiP were from Transduction Laboratories and StressGen Biotechnologies Corp., respectively. The 6C4 anti-lysobisphosphatidic acid monoclonal antibody was a generous gift from Dr. J. Gruenberg (University of Geneva) (28).

Enzyme-linked Immunosorbent Assay (ELISA)-- ELISA for Abeta in the culture medium (29) and lysobisphosphatidic acid among fractions (30) was performed as described previously. BALB/c npcnih and wild-type BALB/c mice were obtained from the Jackson Laboratory. ELISAs for Abeta in mouse brains were performed by a procedure described elsewhere (31). The data were statistically analyzed using Student t test.

Flow Cytometric Analysis and Sorting-- For flow cytometry, samples from interface 1 were incubated with 50 µg/ml filipin (Sigma) for 30 min at 4 °C and immediately analyzed using a FACSVantage flow cytometer (Beckton Dickinson Immunocytometry systems). The flow rate for analysis and sorting was about 2,000 events/s. The nozzle tip was 50 µm in diameter.

Determination of the Cholesterol Content-- Lipids were extracted from the samples with hexane/isopropyl alcohol (3:2, v/v), and evaporated under nitrogen flow. The cholesterol content of each sample was determined using a cholesterol determination kit (Kyowa Medix, Tokyo).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Abeta Accumulates within the Cells Treated with U18666A-- We first assessed the Abeta levels within NPC model cells. CHO cells, stably expressing APP751 (7WD10), were treated with 3 µg/ml U18666A for 24 h (14). After harvesting by incubation with trypsin, the cells were homogenized with 1% Triton in TS, and the homogenates were centrifuged at 540,000 × g for 20 min. The resulting Triton-insoluble pellet was then extracted with 70% formic acid, and the Abeta levels in the extract were assessed by Western blotting (Fig. 1A). The U18666A treatment remarkably enhanced Abeta immunoreactivity in the Triton-insoluble fraction, whereas Abeta was barely detected in the same fraction of nontreated cells. Increased signal intensities of Abeta monomers at ~4 kDa and dimers at ~5-6 kDa on the blot were apparent (26) (Fig. 1A). Those signals in the high molecular mass range should represent nonspecific reactivities, because the secondary antibody alone labeled most of these bands. Compared with the BA27 immunoreactivity, there was an enormous increase in the BC05 immunoreactivity, indicating that the major Abeta species that accumulates following U18666A treatment is Abeta 42.



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Fig. 1.   Abeta accumulation in NPC model cells. A, CHO cells (7WD10), stably transfected with wild-type APP751 cDNA, were incubated with or without 3 µg/ml U18666A for 24 h. After homogenization, 1% Triton-insoluble fractions were pelleted and extracted with 70% formic acid, and Abeta in the extract was probed with various Abeta antibodies, as described under "Experimental Procedures." The immunoreactivities for all Abeta antibodies were greatly enhanced by the U18666A treatment. An increase in the level of Abeta 42 is apparently much greater than that of Abeta 40. Synthetic Abeta 42 (50 pg) is loaded in the right-most lane. B, a 1% Triton-soluble fraction of homogenates was subjected to Western blotting. The levels of Triton-soluble Abeta 42 also increased by the U18666A treatment, whereas that of Abeta 40 did not. Truncated Abeta species (asterisk) is weakly detected with BA27, and its level is not changed by the treatment. C, the U18666A treatment did not alter the expression levels of APP, as shown by Western blotting with 5A3/1G7 antibodies. Similar results were obtained in three independent experiments.

When Triton-soluble fractions were similarly analyzed, the signal for BC05 immunoreactivity was also increased by the treatment, whereas the intensities of BA27-immunoreactive bands at 4 and ~3 kDa (presumably representing a truncated species of Abeta ) were unchanged (Fig. 1B). Interestingly, the Abeta dimer, that was prominent in the Triton-insoluble fraction, was undetectable in the Triton-soluble fraction. We found that the Triton-soluble fraction consistently contained a greater amount of Abeta than did the Triton-insoluble fraction (data not shown). The U18666A treatment did not alter the expression level of APP (Fig. 1C); thus, an increase of Abeta was not a result of overproduction of its precursor.

To examine whether these phenomena are independent of the cell type, we applied the same experimental conditions to K295 cells and obtained the same results (data not shown).

Abeta Accumulates in Cholesterol-rich Compartments-- Next, we determined the particular cellular compartment in which Abeta accumulates. Since NPC cells accumulate mainly unesterified cholesterol, we simply speculated that Abeta resides in cholesterol-rich compartments. Thus, we fractionated cell homogenates on Percoll gradients according to an established protocol widely used for separating cholesterol-rich compartments (24). Abeta 42 was recovered only in fraction 1 in which free (unesterified) cholesterol was accumulated after the U18666A treatment (Fig. 2, A and B). However, due to the fractionation process, endosomal and lysosomal markers were distributed over the fractions (data not shown), and thus this protocol was judged not to be useful for determining the compartment in which Abeta accumulates.



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Fig. 2.   Abeta 42 accumulation in cholesterol-rich fractions. A, the homogenate of 7WD10 cells was fractionated on Percoll gradients, and the cholesterol content of each fraction was determined. Treatment of the cells with U18666A for 24 h shifted the distribution of free cholesterol to the buoyant region (fraction 1). This is a representative of three independent experiments. B, the homogenate of U18666A-treated cells was fractionated on Percoll gradients, and each fraction was centrifuged. The resulting pellets were extracted with 70% formic acid, and subjected to Western blotting using BC05. Immunoreactivity for BC05 was detected only in fraction 1. C, to isolate late endosomes, 7WD10 cells were homogenized, and the homogenates were centrifuged to equilibrium on sucrose gradients. Interfaces 1, 2, and 3 correspond to the 8.5/25, 25/35, and 35/40.6% sucrose interfaces, respectively (see "Experimental Procedures"). U18666A treatment induced cholesterol accumulation exclusively at interface 1. D and E, distribution of the markers specific for subcellular organelles: 6C4 antigen for late endosomes as shown by an ELISA assay (D), Rab7 for late endosomes, Rab 5a for early endosomes, caveolin for caveolae, and BiP for ER as shown by Western blotting (E). Interface 1 produces a late endosome-enriched fraction. After U18666A treatment, immunoreactivity for BC05, but not for BA27 was detected at interface 1, suggesting that the treatment induces Abeta 42 accumulation in late endosomes. Similar results were obtained in four independent experiments.

Because free cholesterol accumulates in late endosomes of NPC cells (14), we isolated late endosomes directly, using sucrose density gradient centrifugation. This protocol enabled us to fractionate late endosomes into interface 1 and early endosomes and plasma membrane into interfaces 2 and 3 (Fig. 2C) (25). As shown in Fig. 2C, a U18666A-induced increase of free cholesterol was observed in interface 1. To confirm the validity of this fractionation, each fraction was probed for specific markers including the 6C4 antigen (for late endosomes) (Fig. 2D), Rab7 (for late endosomes), and Rab 5a (for early endosomes) (Fig. 2E). The obtained results clearly showed that interface 1 indeed represents the late endosome-rich fraction, in which a remarkable increase in the level of Abeta 42 was observed by the U18666A treatment (Fig. 2E). These findings indicate that the U18666A treatment induces an accumulation of both Abeta 42 and free cholesterol in late endosomes. Abeta 40 was not always detectable in any of the three interfaces, even in the presence of U18666A; therefore, the intracellular Abeta levels were routinely assessed only with BC05.

Recently, we and others have reported that a significant fraction of intracellular Abeta resides in the detergent-insoluble membrane domain (9, 10). Caveolae are examples of such domains, but caveolin 1, a caveolae-resident protein, was located at interface 3 (Fig. 2E). Endoplasmic reticulum (ER) is assumed to be a site for Abeta 42 generation (4, 32). However, ER markers, BiP (Fig. 2E) and calnexin (data not shown), were undetectable in interface 1. Thus, caveolae and ER are not the sites in which the Abeta 42 accumulates following U18666A treatment.

The U18666A treatment itself did not alter the locations of several marker proteins examined (data not shown). All of the observations described above that were made in 7WD10 cells were reproduced in K295 cells (data not shown).

Abeta 42 Accumulates in Filipin-positive Granules-- To further confirm the colocalization of Abeta 42 with free cholesterol, we isolated cholesterol-rich vesicles from 7WD10 cells using a fluorescence-activated cell sorter (33). Because free cholesterol can be specifically and sensitively labeled with filipin, a fluorescent probe (34), interface 1 prepared from U18666A-treated cells was incubated with filipin, and the suspension was immediately analyzed using a fluorescence-activated cell sorter with a UV filter setting. There were two fluorescence peaks in filipin-labeled samples (Fig. 3A). The first peak (R1) was also present in the unlabeled samples; thus, R1 should represent autofluorescence from unlabeled membranes. The second peak (R2) consisted of a population of highly fluorescent vesicles. Sorted materials from the two peaks were separately pelleted, and each pellet was extracted with 70% formic acid and subjected to Western blotting. As shown in Fig. 3B, a BC05-positive signal, most intense at the Abeta dimer, was detected only in the R2 fraction, demonstrating that at least a part of the accumulated Abeta 42 indeed localizes in cholesterol-residing compartments.



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Fig. 3.   Western blotting of sorted filipin-labeled vesicles. Late endosome-rich membranous organelles (interface 1) were labeled with filipin and sorted by flow cytometry. R1 consisted of the fluorescence from membranous organelles that were not labeled with filipin (autofluorescence), while R2 represented a homogenous population of filipin-labeled highly fluorescent vesicles (see "Results"). Each fraction was pelleted and extracted with 70% formic acid and subjected to Western blotting using BC05. Abeta 42 monomers and dimers were detected only in the R2 fraction.

Intracellular Abeta Accumulation Does Not Affect Abeta Secretion-- We next examined whether the U18666A treatment causes any detectable alterations in constitutive Abeta secretion. 7WD10 cells were cultured with or without 3 µg/ml U18666A for 24 h. After a brief wash, the cells were maintained in culture for another 6 h with or without U18666A, and the levels of Abeta in the conditioned medium were quantified by ELISA. As shown in Table I, no statistical difference in the levels of secreted Abeta 40 and Abeta 42 between the treated and untreated cells was found. The ratio of Abeta 42 to the total Abeta was also not altered (Table I). These results indicate that Abeta 42 in late endosomes is not the source of secreted Abeta 42.


                              
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Table I
Effect of U18666A on Abeta secretion
7WD10 cells were cultured confluent with or without 3 µg/ml U18666A for 24 h. After washing, cells were continuously cultured in fresh medium with or without U18666A for another 6 h. The amount of Abeta in the conditioned medium was measured by ELISA. Average Abeta 42 ratios are shown in the right-most column.

Accumulated Abeta 42 Disappears upon Withdrawal of U18666A-- Since U18666A treatment has reversible effects on cholesterol transport (35, 36), we examined whether withdrawal of U18666A had an effect on Abeta accumulation and Abeta secretion. As shown in Fig. 4A, the accumulated Abeta 42 became completely undetectable 24 h after the withdrawal of U18666A. Any trace amount of Abeta was not detected in other interfaces (data not shown), indicating that accumulated Abeta 42 disappeared from cellular membranous compartments.



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Fig. 4.   Further characterization of Abeta 42 that accumulated in late endosomes. A, after incubation with U18666A for 24 h, 7WD10 cells were washed and further cultured in the absence of the reagent for 0, 24, and 48 h. Twenty-four hours after depletion of the reagent, the level of cholesterol (µg/mg protein) at interface 1 was dramatically reduced, and the immunoreactivity for Abeta 42 disappeared. B, interface 1 was carefully sampled and divided into halves and separately pelleted. One pellet was extracted with 2% SDS, while the other with 70% formic acid. The final volumes of both samples were adjusted, and the samples were subjected to Western blotting using BC05. While the SDS extract exhibited no Abeta -derived signals, the formic acid extract (FA) contained Abeta monomers, dimers, and higher order Abeta oligomers. C, CHO cells stably expressing Delta C-APP lacking the internalization signal were treated with or without 3 µg/ml U18666A for 24 h. After fractionation, interface 1 was taken and subjected to Western blotting using BC05. Even in this cell line, the treatment induced an intracellular accumulation of Abeta 42, indicating that the internalization of the cell surface APP is not required for Abeta accumulation in late endosomes.

We then measured Abeta levels by ELISA in the cultured medium after withdrawal of the reagent. After being exposed to U18666A for 24 h, 7WD10 cells were briefly washed and cultured for another 3 or 6 h in the regular medium. The Abeta content in the conditioned medium was compared with that of sister cultures incubated without U18666A (Table II). We did not observe any detectable change in the Abeta secretion level after the removal of U18666A.


                              
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Table II
Abeta secretion after U18666A treatment
After a 24-h treatment with U18666A (+), cells were washed and maintained in culture for the indicated chase time in the absence of the reagent. The amount of Abeta in conditioned media was measured by ELISA. For controls, sister cultures were incubated without U18666A (-) for 24 h. Average Abeta 42 ratios are shown in the right-most column.

Abeta 42 That Accumulates in Late Endosomes Is Aggregated-- Since the presence of intracellular, highly insoluble Abeta has been reported (37), we examined the insolubility characteristics of the Abeta that accumulated in late endosomes. Interface 1, prepared from U18666A-treated cells, was divided into halves and separately pelleted. One pellet was extracted with the 2% SDS sample buffer, while the other was extracted with 70% formic acid. As shown in Fig. 4B, accumulated Abeta was extracted only with formic acid, but not with SDS, although similar amounts of proteins other than Abeta were extracted with SDS and formic acid, as judged by Coomassie staining (data not shown). Thus, the Abeta 42 accumulated in late endosomes is most likely aggregated into an SDS-insoluble form.

Internalization of Cell Surface APP Is Not Required for U18666A-induced Abeta Accumulation-- Production and release of Abeta by 7WD10 cells depend primarily on internalization of APP from the cell surface, and its endocytosis signal is located in the cytoplasmic domain (20, 38). To determine whether this APP internalization is essential for the accumulation of Abeta 42 in late endosomes, CHO cells stably expressing APP751 lacking nearly the entire cytoplasmic domain (Delta C), were exposed to U18666A. The Delta C cells exhibited significant reductions in the APP internalization and in the production and secretion of Abeta (20, 38). As shown in Fig. 4C, remarkably increased levels of Abeta 42 were again observed in interface 1 following the U18666A treatment. These results may indicate that internalization of cell surface APP is not required for U18666A-induced Abeta accumulation in late endosomes.

Intracellular Cholesterol Levels Affect Abeta 42 Accumulation in Late Endosomes-- The above results strongly suggest a tight correlation between Abeta 42 accumulation and cellular cholesterol levels. To examine this possibility, 7WD10 cells were cultured in cholesterol starvation medium for 2 days. The cells were then further cultured in the cholesterol starvation medium with or without 3 µg/ml U18666A for 24 h. Although cholesterol starvation for 3 days resulted in an average 80% decrease in the total cellular cholesterol levels, the U18666A treatment somewhat increased the cholesterol level in interface 1 compared with that of untreated cells (Fig. 5). As shown in Fig. 5, under the cholesterol-depleted conditions, U18666A again induced Abeta 42 accumulation in interface 1. When the cells were cultured with 10 µg/ml exogenous water-soluble cholesterol (cholesterol and methyl-beta -cyclodextrin complex) for 24 h in the presence of U18666A, apparently greater amounts of Abeta 42 monomers and dimers were observed at interface 1. Methyl-beta -cyclodextrin alone (180 µg/ml) together with U18666A did not increase the accumulation as much. These data indicate that the Abeta 42 accumulation in late endosomes is under the influence of cellular cholesterol levels.



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Fig. 5.   Cellular cholesterol levels regulate Abeta accumulation in late endosomes. 7WD10 cells were cultured in the cholesterol starvation medium for 2 days and further cultured in the presence of indicated reagents for 24 h. After fractionation, interface 1 of each sample was prepared and subjected to Western blotting using BC05. Even under cholesterol-starved conditions, U18666A (3 µg/ml) induces Abeta accumulation in late endosomes. However, exogenous cholesterol apparently enhances the accumulation. Appreciable cholesterol levels at interface 1 are indicated. DLFC, delipidated calf serum; CD, methyl-beta -cyclodextrin. Similar results were obtained in three independent experiments.

NPC1-deficient CHO Cells Accumulate Abeta 42-- Although it is most likely that U18666A acts directly on the NPC1 protein, we examined whether a mutant cell that lacks the NPC1 gene can also accumulate Abeta . Mutant CT43 is a cholesterol-trafficking CHO cell mutant that carries a mutation in the NPC1 gene, resulting in production of a nonfunctional NPC1 protein (19). The parental cell 25RA has a gain-of-function mutation in the sterol regulatory element-binding protein and cleavage-activating protein. The line 25RA shows normal intracellular cholesterol trafficking and thus was used as a control. Both cell lines were transiently transfected with APP695 cDNA, and the levels of expressed APP were assessed by Western blotting followed by densitometric quantification. The Triton-insoluble fractions of the cell lysate, after normalization to the expression levels of APP, were assessed by Western blotting. We found that CT43 contained higher levels of Abeta 42 within the cell lysate than did 25RA (Fig. 6A). Thus, we concluded that both the cell lines deficient in NPC1 gene and the NPC model cells accumulated Abeta , and especially Abeta 42, in a similar manner within the cells.



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Fig. 6.   Abeta accumulates in NPC1-disrupted CHO mutant and in NPC1-deficient mouse brain. A, NPC1 mutant (CT43) and the parental (25RA) CHO cells were transiently transfected with APP695 cDNA. Two days later, cells were homogenized in 1% Triton in TS and spun down. The volumes of Triton-insoluble samples were adjusted to the expression levels of APP and loaded on the gel. A greater amount of Abeta 42 monomers and dimers are detected in CT43 cells. This is a representative of four independent experiments. B and C, brains of 6-week-old BALB/c npcnih (Homozygote (Homo)) and wild-type BALB/c mice (WT) (n = 3) were homogenized in TS. After centrifugation, TS-insoluble pellets were extracted with 6 M guanidine HCl and subjected to ELISA. The results were analyzed by Student's t test. The Abeta 42 (p = 0.033) and Abeta 40 levels (p = 0.049) are significantly increased in NPC1-deficient mice.

Abeta Accumulates in NPC Mouse Brains-- Finally, we examined whether Abeta 42 accumulates in vivo (i.e. in NPC mouse brains). We used a well characterized mutant mouse on a BALB/c strain, named BALB/c npcnih, in which the NPC1 gene is disrupted (16). This mouse exhibits progressive neurodegeneration and dies around 10 weeks of age. Brains from three wild-type BALB/c and three homozygote BALB/c npcnih mice, at the age of 6 weeks, were homogenized in TS. After centrifugation, TS-insoluble Abeta was extracted with 6 M guanidine HCl in 50 mM Tris-HCl, pH 7.6, and subjected to ELISA. As shown in Fig. 6, B and C, the Abeta 40 and Abeta 42 levels are significantly increased in NPC1-deficient mice compared with those in wild-type mice.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Several lines of evidence suggest a linkage between cholesterol metabolism and susceptibility to AD. Further, many reports indicate the correlation of cholesterol metabolism with Abeta secretion levels. Here, we have demonstrated the intracellular accumulation of Abeta , especially Abeta 42, along with free cholesterol in late endosomes in cholesterol-perturbed NPC cells. This cholesterol accumulation does not affect Abeta secretion levels, but the extent of Abeta accumulation in late endosomes is strongly influenced by their cholesterol levels. Importantly, this accumulated Abeta 42 in late endosomes appeared to be in an SDS-insoluble aggregate form. Thus, late endosomes of NPC cells are a novel pool of intracellular aggregated Abeta .

The mechanism of interaction between aggregated Abeta and cholesterol remains to be clarified. One possible explanation is that the observed Abeta 42 accumulation in late endosomes results from altered membrane trafficking induced by an abnormal lipid content in NPC cells. Cellular cholesterol levels (39) and lysobisphosphatidic acid, a late endosomal lipid, (28), are known to modulate endosomal membrane trafficking. In NPC cells, intracellular trafficking of lipids other than sterol is also disrupted. Thus, abnormal lipid accumulation, not only for cholesterol, may possibly affect intracellular membrane and Abeta /APP trafficking and may induce Abeta 42 aggregation in late endosomes. However, we prefer a more straightforward explanation; free cholesterol (together with other molecules) directly or indirectly (via other molecules) interacts with Abeta (especially an aggregated form of Abeta 42), and wherever free cholesterol accumulates within the cell, aggregated Abeta 42 also accumulates. Earlier studies have shown that the rate of degradation of 125I-labeled low density lipoprotein was not impaired in normal CHO cells treated with U18666A (36) or in mutant CHO cells defective in the NPC1 gene (19, 22). These results rule out the remote possibility that the massive accumulation of Abeta in NPC cells might be due to the capacity of the endosomes for protein degradation being compromised by the massive accumulation of cholesterol and/or other lipid in this compartment. It is notable that, even under cholesterol-depleted conditions, U18666A induced mobilization of remaining free cholesterol to late endosomes, which apparently accompany Abeta 42. One may argue that this assumption is contradictory to an in vivo observation; significant Abeta 42 accumulation occurs in NPC mouse brains where free cholesterol is reported not to be accumulated (40). However, it is quite possible that, even if free cholesterol levels are not increased in the brain, those in late endosomes may be significantly increased and coupled with Abeta 42 accumulation.

The effects of U18666A on cholesterol trafficking can be completely reversed when the reagent is withdrawn (35, 36). Free cholesterol that accumulates in late endosomes is sorted either to the plasma membrane/ER (19) or secreted into the culture medium (41). We took advantage of this property of U18666A to examine the relocation of the Abeta accumulated in late endosomes. Within 24 h after withdrawal of U18666A, the accumulated Abeta 42 disappeared completely from any of the membranous compartments. One may speculate that the accumulated Abeta is sorted to lysosomes for rapid degradation. Alternatively, the Abeta 42 may migrate to the plasma membrane/ER or be secreted into the culture medium, through interaction with cholesterol and/or other lipids. Because Abeta was observed neither in interface 2 nor in interface 3, where the plasma membrane and ER are localized (data not shown), it is possible that the accumulated Abeta is released into the culture medium. However, no increased secretion of Abeta after withdrawal of U18666A was detected by ELISA. There could be two explanations for this observation, which are not mutually exclusive. First, a change in the Abeta levels in the medium is so small that ELISA fails to detect it. Second, our ELISA system may exclusively detect Abeta monomer, but not Abeta dimers or oligomers (26). Thus, if the Abeta 42 that accumulates in late endosomes is secreted in an aggregated form, the ELISA cannot detect it.

As seen in Figs. 1A and 5, Abeta monomer and dimer appeared even in the absence of U18666A treatment. Thus, indiscernible amounts of Abeta must accumulate and aggregate even in non-NPC cells. Because cholesterol trafficking is normal in these cells, the aggregated form of Abeta might be shed in a constitutive manner into the extracellular space, along with cholesterol. These released Abeta aggregates may act as a seed for Abeta fibrillation, thereby leading to Abeta deposition in the brain (42). In fact, a recent report by Walsh et al. (43) supports this view, showing that intracellular Abeta aggregation is essential for the extracellular Abeta oligomerization. Conversely, in the NPC brain, intracellularly accumulated Abeta 42 cannot be efficiently secreted and accumulated within cells as seen in Fig. 6, B and C. This may possibly explain why no Abeta deposition is observed in the NPC brain.

Most interestingly, abundant neurofibrillary tangles, the other hallmark of AD, are observed in the brains of NPC patients with a slowly progressive clinical course (44). Those neurofibrillary tangles in the NPC brain are morphologically and biochemically indistinguishable from those in the AD brain (44). Thus, in addition to aberrant cholesterol trafficking, long term intracellular Abeta accumulation might accelerate neuronal degeneration, which eventually leads to neurofibrillary tangle formation in the NPC brain.


    ACKNOWLEDGEMENT

We thank H. Kuriki for operating the cell sorter.


    FOOTNOTES

* This work was supported in part by a research grant from the Ministry of Education, Science, Sports and Culture, Japan (to T. Y.).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.

** To whom correspondence should be addressed: Dept. of Neuropathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-3541; Fax: 81-3-5800-6852; E-mail: yihara@m.u-tokyo.ac.jp.

Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M009598200


    ABBREVIATIONS

The abbreviations used are: Abeta , amyloid beta -protein; AD, Alzheimer's disease; APP, beta -amyloid precursor protein; ER, endoplasmic reticulum; ELISA, enzyme-linked immunosorbent assay; NPC, Niemann-Pick type C; TS, Tris-saline; CHO, Chinese hamster ovary.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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