 |
INTRODUCTION |
Several different lines of evidence point to a potentially
important but not well understood interaction between Alzheimer's disease (AD)1 and cholesterol
(for reviews, see Refs. 1-4). Apolipoprotein E4, a protein that binds
and transports cholesterol and other lipids, has been identified as a
risk factor for familial and sporadic AD (5, 6). Patients on specific
inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase have a
lower risk of developing AD as compared with individuals not taking
those drugs (7, 8). A metabolite of brain cholesterol,
(24S)-hydroxycholesterol, was elevated in cerebral
spinal fluid of AD patients compared with control subjects (9).
Amyloid
-peptide (A
), the main component of neuritic plaques seen
in brains of AD patients, interacts with cholesterol. This interaction
is reciprocal. Cholesterol levels modulate amyloid precursor
protein and A
synthesis (10-13). Conversely A
alters cholesterol dynamics. Cholesterol modulates the actions of A
on
brain membrane fluidity (14-16). A
species (25-35, 1-40, and 1-42) increased the internalization of apolipoprotein E (apoE) complexed with cholesterol into neurons (17). A
-(1-42) increased apoE levels in astrocytes (18). Cholesterol efflux from rat hippocampal
neurons to cyclodextrin was enhanced by A
-(1-40), and these results
were attributed to redistribution of cholesterol to the plasma membrane
(19). The Golgi complex plays an important role in cholesterol
trafficking (20, 21), and A
could act on cholesterol homeostasis in
the Golgi complex. Chemical inhibitors known to act on the Golgi
complex alter cholesterol trafficking (20). Furthermore it has been
shown that phosphatidylcholine-phospholipase D (PC-PLD) contributes to
regulation of cholesterol efflux and that PC-PLD is associated with the
Golgi complex (20-24). The A
peptide fragment 25-35 increased PLD
activity in LA-N-2 cells (25). The effects of A
on cholesterol in
the Golgi complex may also be dependent on whether A
is fresh or
aged. Recently it was reported that oligomeric A
-(1-40), but not
monomeric A
-(1-40), stimulated release of cholesterol,
phospholipids, and GM1 ganglioside from neurons (26). Aged
A
-(1-40), but not fresh A
-(1-40), preferentially binds
cholesterol as compared with fatty acids or phosphatidylcholine (27)
that might enhance removal of lipids from cell compartments. Fresh
A
-(1-40) was reported to be located in the hydrophobic area of
synaptic plasma membrane, whereas A
-(1-40) aged for 48 h was
intercalated adjacent to the phospholipid polar head group region
(28).
The current study tested the hypotheses that A
-(1-42) modifies
Golgi complex cholesterol homeostasis in astrocytes and that the
effects of A
-(1-42) were dependent on whether A
-(1-42) was fresh or aged. Gel electrophoresis of fresh A
-(1-42) and
A
-(1-42) aged for 48 h showed that aged A
-(1-42) consisted
of ~74% of the protein as a tetramer, whereas the fresh A
-(1-42)
showed only 11% of the protein as a tetramer (28), and such structural differences could alter the behavior of A
. The effects of
A
-(1-42) on Golgi complex cholesterol could involve activity of
PC-PLD, and this possible action of A
-(1-42) was examined. In
addition, it has been reported that apoE levels were increased in
astrocytes treated with aged A
-(1-42) (18), and such effects might
alter cholesterol distribution in cells. ApoE levels also were
determined. Experiments were conducted using fresh and aged
A
-(1-42) in immortalized DITNC1 astrocytes (29). Two different
methods were used to determine the effects of A
-(1-42) on Golgi
cholesterol content of astrocytes: 1) confocal microscopy using the
fluorescent cholesterol analogue NBD-cholesterol and a fluorescent
marker for the Golgi complex, BODIPY TR ceramide (30); and 2)
isolation of the Golgi complex into two different fractions thought to
represent different regions of the Golgi complex using sucrose density
gradient centrifugation (31, 32). The results presented herein provide
new insights into interaction of A
and cholesterol. We show that
fresh A
-(1-42) has a strikingly different effect on cholesterol
levels in the Golgi complex of astrocytes as compared with aged
A
-(1-42). Incubation of astrocytes with fresh A
-(1-42)
significantly increased cholesterol levels, whereas aged A
-(1-42)
significantly reduced cholesterol levels in the Golgi complex as shown
by fluorescent probes and confocal microscopy. The reverse peptide
A
-(42-1), either fresh or aged, did not alter Golgi complex
cholesterol levels, which argues against a nonspecific action of
A
-(1-42). Isolation of the astrocyte Golgi complex into
different fractions revealed the novel finding that the effects of
fresh A
-(1-42) on cholesterol content were opposite on the two
Golgi fractions, whereas aged A
-(1-42) had effects similar
to those observed with confocal microscopy. PC-PLD activity, cell
membrane cholesterol, and apoE levels of astrocytes were associated
with effects of fresh A
-(1-42) on cholesterol levels but not with
effects of aged A
-(1-42), suggesting different mechanisms for
actions of fresh and aged A
-(1-42) on Golgi complex cholesterol.
Our data suggest that extracellular A
-(1-42) specifically targets
the Golgi complex and disrupts cholesterol homeostasis.
 |
EXPERIMENTAL PROCEDURES |
Materials--
NBD-cholesterol
(22-(N-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-23,24-bisnor-5-cholen-3-
-ol),
BODIPY TR ceramide, and the Amplex® Red
phosphatidylcholine-specific phospholipase D (A-12219) assay kit were
obtained from Molecular Probes (Eugene, OR). A
-(1-42) and
A
-(42-1) were purchased from Bachem California Inc. (Torrance, CA). Fetal bovine serum was obtained from HyClone (Logan, UT). UDP-[6-3H]galactose was purchased from Amersham
Biosciences. The A
-(1-42) enzyme-linked immunosorbent assay kit
(catalogue no. 17711) was obtained from Immuno-Biological
Laboratories (Minneapolis, MN). All other chemicals unless
specifically mentioned were purchased from Sigma.
Cell Culture--
DITNC1 rat astrocytes were purchased from
American Type Culture Collection (Manassas, VA). These cells have been
shown to have the phenotypic characteristics of type 1 astrocytes (29). Astrocyte cells were incubated in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum and 5% glutamate for 2 days until
confluence and maintained at 37 °C in 5% CO2 and at
90% relative humidity. The cells at 80-85% confluence were treated
with medium containing 1% lipoprotein-deficient serum replacing
the 10% fetal bovine serum and incubated for 16 h prior to
experimentation. All experiments were done using confluent astrocytes.
Incubation of Cells with Fluorescent Labeled Probes and
A
-(1-42)--
Astrocytes were grown on Lab-TekTM
chamber slides, incubated with NBD-cholesterol (8 µM) for
1 h, and then washed three times with 1 ml of phosphate-buffered
saline (PBS). Fresh A
-(1-42) (1 µM) or A
-(1-42)
(1 µM) preincubated for 48 h (aged) was added to
cells and incubated for different time periods (30, 60, and 120 min).
We have reported previously that incubation of A
-(1-42) for 48 h resulted in ~74% of the peptide in a tetrameric form (28). In some
experiments the reverse peptide A
-(42-1) (fresh or aged for 48 h) was incubated with cells. Following incubation with A
, cells were
rinsed three times with PBS, and the Golgi marker BODIPY TR ceramide (2 µM) was added and incubated with the cells for 1 h. Cells were then washed with PBS, fixed with 4%
paraformaldehyde, and mounted for confocal microscopy using Gel/Mount
(Biomeda Corp., Foster City, CA).
Laser Scanning Confocal Microscopy--
Confocal fluorescence
imaging was performed on an Olympus Fluoview laser scanning confocal
imaging system (Olympus America Inc., New York). Images were captured
using multiple photomultiplier tubes regulated by Fluoview 2.0 software
(Olympus). Excitation of the fluorescent probes was accomplished
using 15-milliwatt krypton-argon lasers with 5-milliwatt output. An
Olympus BX 50 fluorescent microscope was used to capture the images
using an oil immersion objective. NBD-cholesterol was excited at 488 nm, and emission was recorded at 540 nm. BODIPY TR ceramide was excited at 568 nm, and emission was recorded at 598 nm. The captured images for
the red and green channels were merged and appeared yellow, which is
indicative of colocalization. Red and green are additive, generating
yellow to orange in RGB (red-green-blue) color space (33).
Quantitative analysis of the colocalization of NBD-cholesterol and
BODIPY TR ceramide was determined by image processing using MetaMorph
imaging system V4.3 from Universal Imaging Corp. (Downingtown, PA) as
described previously (33, 34) and expressed as percent colocalization.
Isolation of Golgi Fractions--
Isolation of the Golgi complex
was accomplished using sucrose density gradient centrifugation (32).
Confluent astrocytes were treated with fresh and aged A
-(1-42) as
described above. Cells were harvested and washed twice with PBS. The
cells were suspended in G buffer (10 mM Tris-HCl, 0.25 M sucrose, 2 mM MgCl2, pH 7.4)
containing 10 mM CaCl2. Leupeptin and
phenylmethylsulfonyl fluoride were added to the cell suspension to
inhibit proteolytic enzymes prior to homogenization. The cells were
homogenized in a Potter-type homogenizer with 20-30 strokes using a
serrated homogenizing pestle. The homogenate was centrifuged at
5,000 × g for 10 min. The pellet was suspended and
homogenized in 1.4 M sucrose and overlayered with 0.8, 1.0, and 1.2 M sucrose gradients in ultracentrifuge tubes.
Samples were then centrifuged at 95,000 × g for
2.5 h in a SW28 rotor in a Beckman L8-70 ultracentrifuge. Each
interface was carefully removed, diluted with G buffer, and centrifuged
in an SS34 rotor at 45,900 × g for 20 min. Each pellet was suspended in buffer and used for the experiments. Protein concentrations of the fractions were determined using the Bradford assay. Cholesterol content in each fraction was determined
enzymatically in a microassay using a diagnostic kit from Roche
Molecular Biochemicals (35) and procedures reported by our
laboratory (36-38).
Galactosyltransferase Activity--
A marker enzyme of the Golgi
complex is galactosyltransferase, and activity of this enzyme was
measured using procedures reported previously (32, 39). The incubation
mixture contained 7 mg/ml ovalbumin, 2 mM ATP, 200 mM MgCl2, 0.2% Triton X-100, and 50 mM Tris-HCl (pH 6.8). The reaction was initiated by adding
[3H]UDP-galactose to samples and incubated for 30 min,
and the reaction was stopped by adding ice-cold 24% trichloroacetic
acid. The precipitate was pelleted at 20,800 × g and
washed three times with 12% trichloroacetic acid. Samples were
solubilized in 5% SDS, and disintegrations/min were counted using a
scintillation counter.
PC-PLD Activity--
PC-PLD activity of control astrocytes and
astrocytes that had been incubated with either fresh or aged
A
-(1-42) for different time periods (30, 60, 90, and 120 min) was
quantified using the Amplex Red PC-PLD assay kit (Molecular Probes).
The intensity of the Amplex Red complex formed was measured in a
fluorescence microplate reader (Spectra Max Gemini XS, Molecular
Devices, Sunnyvale, CA) using an excitation wavelength of 542 nm and an
emission wavelength of 590 nm.
Measurement of A
-(1-42)--
Quantitative determination of
A
-(1-42) in the G1 and G2 fractions was carried out using an
enzyme-linked immunosorbent assay kit from Immuno-Biological
Laboratories. This kit uses a solid phase sandwich enzyme-linked
immunosorbent assay protocol with anti-human A
-(38-42) rabbit IgG
as primary antibody in a precoated plate and horseradish
peroxidase-conjugated anti-human A
-(11-28) mouse IgG as secondary
conjugated antibody. Briefly, standards and test samples were diluted
in a buffer consisting of 1% bovine serum albumin and 0.05% Tween 20 in PBS. Test samples as well as sample and reagent blanks were added to
the precoated plate and incubated overnight at 4 °C. After binding,
the wells were washed (seven times) prior to adding conjugated
secondary antibody and incubated for 1 h at 4 °C. After washing
(nine times), the chromogen containing tetramethylbenzidine was added
and incubated for 30 min at room temperature. Color reaction was
terminated by the stop solution, and measurement was performed at 450 nm using a Packard Fusion AlphaScreen microplate analyzer (Packard Instrument Co.). In each assay, a standard curve was constructed after
subtracting absorbance of the test sample blank. Data for test samples
were converted to ng of A
-(1-42)/mg of protein.
ApoE Isolation--
Media of control cells and cells
treated with either fresh or aged A
-(1-42) were removed, and
phenylmethylsulfonyl fluoride was added as a protease inhibitor. The
media were centrifuged at 800 × g for 5 min, and the
resulting supernatant was treated with 15% trichloric acid to
precipitate protein, washed with cold methanol, dried, and kept at
20 °C for later use. Cells were harvested with trypsin/EDTA, and
protein values were determined using the Bradford method with ovalbumin
standards. Cells were treated with lysing buffer containing 8 M urea, 5% SDS, and 5%
-mercaptoethanol. For Western
blot analysis of lysates and conditioned media, 1 µl of 1 M dithiothreitol and 1 µl of 0.2% bromphenol solution
was added to every 50 µl of lysing buffer before use, and samples
were kept at 70 °C for 30 min. 100-200 µg of protein were
electrophoresed on a 7.5% SDS, Tricine, HCl precast gel
(Bio-Rad), and proteins were transferred to a nitrocellulose membrane
and incubated with monoclonal mouse anti-rat apoE (1:250) from BD Laboratory (San Diego, CA). Horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000) was used as a secondary antibody (Pierce). Immunoreactivity was visualized with SuperSignal West Pico Chemiluminescent substrate (Pierce). Band density was quantitated by densitometry using the Eagle Eye II video system and EagleSight software (Stratagene, La Jolla, CA).
 |
RESULTS |
A
-(1-42) Modifies Cholesterol Distribution in the Golgi Complex
as Revealed by Confocal Microscopy--
Experiments in this study
tested the hypothesis that A
-(1-42) would modify cholesterol
distribution in the Golgi complex of astrocytes and that the effects of
A
-(1-42) on cholesterol distribution would be dependent on whether
A
-(1-42) was fresh or aged. Two different methods were used to
determine cholesterol distribution in the Golgi complex. 1) A
fluorescent cholesterol analogue, NBD-cholesterol, and a fluorescent
marker for the Golgi complex, BODIPY TR ceramide, were used and imaged
with confocal microscopy; and 2) sucrose density gradient
centrifugation was used to isolate two different fractions of the Golgi
complex, and cholesterol levels were determined enzymatically.
BODIPY TR ceramide, NBD-cholesterol, and confocal microscopy were used
to initially examine the effects of fresh and aged A
-(1-42) on
Golgi cholesterol content of astrocytes. Cells were incubated with
A
-(1-42) for different time periods (30, 60, and 120 min). Cell
viability as measured by trypan blue was not altered by incubation of
cells with fresh or aged A
-(1-42) for 120 min. Means ± S.E.
of the percentage of viable cells for control astrocytes, astrocytes
incubated with fresh A
-(1-42), and astrocytes incubated with aged
A
-(1-42) were 96.83 ± 0.58, 94.2 ± 1.5, and 97.83 ± 0.6, respectively. Confocal images of colocalization of
NBD-cholesterol and BODIPY TR ceramide in control astrocytes and
astrocytes incubated with fresh and aged A
-(1-42) for 120 min are
shown in Fig. 1. Panel C shows
colocalization of the two fluorescent probes in control astrocytes. It
can be seen in panel F that colocalization was greater in
astrocytes treated with fresh A
-(1-42), whereas aged A
-(1-42)
(panel I) reduced colocalization as compared with control
astrocytes and astrocytes incubated with fresh A
-(1-42). Analysis
of colocalization data using MetaMorph software and expressed as
percent colocalization shows that fresh A
-(1-42) (p
0.001) increased colocalization in the Golgi complex of astrocytes
as compared with control astrocytes (38 versus 23%) at the
120-min incubation period (Fig. 2). The
percent colocalization of the fluorescent probes was significantly
(p
0.01) less when astrocytes were incubated with
aged A
-(1-42) (15 versus 23%) for 120 min as compared
with control astrocytes (Fig. 2). Shorter incubation times with
A
-(1-42) did not significantly alter colocalization, although it
can be seen in Fig. 2 that fresh A
-(1-42) was increasing colocalization and aged A
-(1-42) was reducing colocalization at the
shorter incubation times. Colocalization was not altered by a lower
concentration (1 nmol) of fresh A
-(1-42) or aged A
-(1-42). Means ± S.E. of percent colocalization of NBD-cholesterol and BODIPY TR ceramide were 23.71 ± 3.7, 25.82 ± 4.7, and
23.33 ± 2.1 for control astrocytes, astrocytes incubated with
fresh A
-(1-42), and astrocytes incubated with aged A
-(1-42),
respectively. To determine whether the effects of A
-(1-42) were
specific and not attributable to a nonspecific perturbation of the
Golgi complex we incubated astrocytes for 120 min with the reverse
peptide A
-(42-1) that was either fresh or aged for 48 h.
Neither fresh nor aged A
-(42-1) had an effect on the percent
colocalization of BODIPY TR ceramide and NBD-cholesterol when compared
with control astrocytes or with each other (Fig. 2).

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Fig. 1.
Localization of cholesterol in the Golgi
complex of astrocytes incubated with fresh and aged
A -(1-42) for 120 min. Shown are confocal
images of control astrocytes labeled with NBD-cholesterol (panel
A, green) and BODIPY TR ceramide (panel B,
red). Colocalization is shown in panel C. Shown
are confocal images of astrocytes treated with fresh A -(1-42),
NBD-cholesterol (panel D, green), and BODIPY TR
ceramide (panel E, red). Colocalization is shown
in panel F. Shown are confocal images of astrocytes treated
with aged A -(1-42), NBD-cholesterol (panel G,
green), and BODIPY TR ceramide (panel H,
red). Colocalization is shown in panel I.
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Fig. 2.
Percent colocalization of BODIPY TR ceramide
and NBD-cholesterol in astrocytes incubated with fresh and aged
A -(1-42) for different time periods and
astrocytes incubated with A -(42-1) for 120 min. Quantitative measurements of colocalization of
NBD-cholesterol and BODIPY TR ceramide were determined using MetaMorph
software and expressed as percent colocalization. Data are means ± S.E. (n = 3 separate experiments). *,
p 0.01; **, p 0.001 as compared
with control astrocytes.
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UDP-galactosyltransferase Activity and Fluorescence of BODIPY TR
Ceramide in Golgi Complex Fractions--
Confocal microscopy revealed
that A
-(1-42) altered the colocalization of NBD-cholesterol and
BODIPY TR ceramide, a marker for the Golgi complex, and these data were
interpreted as A
-(1-42) altering Golgi cholesterol levels. The
Golgi complex is heterogenous in structure and function and may not be
equally affected by A
-(1-42). To further define the effects of
fresh and aged A
-(1-42) on cholesterol distribution in the Golgi
complex of astrocytes, the Golgi complex was isolated using sucrose
density centrifugation, and two different fractions were obtained that
are thought to represent different regions of the Golgi complex (32):
1) the light Golgi fraction (G1), which is the band at the 0.8-1.0
sucrose gradient interface; and 2) the Golgi fraction 2 (G2), which is
the band at the 1.0-1.2 sucrose gradient interface. Enrichment of
UDP-galactosyltransferase activity and fluorescence of BODIPY TR
ceramide have been used previously as markers for the Golgi complex
(30, 32, 40, 41), and these markers were used in the present study. The
means ± S.E. of UDP-galactosyltransferase activity (nmol/30
min/mg of protein) in astrocyte homogenate, G1 fraction, and G2
fraction were 0.019 ± 0.004, 2.751 ± 0.086, and 1.972 ± 0.129, respectively. UDP-galactosyltransferase activity was
significantly higher (p < 0.01) in the G1 fraction as
compared with the G2 fraction and was several orders of magnitude
higher in both the G1 and G2 fractions in contrast to the homogenate.
Enrichment of UDP-galactosyltransferase activity in the G1 fraction as
compared with the G2 fraction has been reported previously (32). BODIPY
TR ceramide, a fluorescent Golgi complex marker, had a similar
distribution in the G1 and G2 astrocyte fractions as was observed for
UDP-galactosyltransferase activity (data not shown).
A
-(1-42) Modifies Cholesterol Distribution in the Golgi Complex
Fractions as Revealed by Sucrose Density Centrifugation--
We next
examined the effects of A
-(1-42) on cholesterol content of the G1
and G2 fractions. The effects of fresh and aged A
-(1-42) on
cholesterol in the G1 fraction were similar to effects we observed
using confocal microscopy. Fresh A
-(1-42) significantly (p < 0.0001) increased cholesterol, whereas aged
A
-(1-42) significantly (p < 0.001) reduced
cholesterol in the G1 fraction (Fig. 3).
The magnitude of effects of fresh A
-(1-42) on cholesterol in the G1
fraction was greater than that of aged A
-(1-42), and these results
were similar to those observed with confocal microscopy.

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Fig. 3.
Changes in the distribution of cholesterol in
the Golgi complex of astrocytes incubated with fresh and aged
A -(1-42). Astrocytes were incubated with
A -(1-42) for 120 min after which time cells were harvested, and the
Golgi complex was isolated using sucrose density centrifugation as
described under "Experimental Procedures." The G1 fraction is the
band at the 0.8-1.0 M sucrose gradient, and the G2
fraction is the band at the 1.0-1.2 M sucrose gradient.
Cholesterol was determined enzymatically, and data are expressed as
percentage of control. Values are means ± S.E. (n = 3). *, p 0.001; **,
p 0.0001 as compared with control fractions.
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|
Fig. 3 shows that fresh A
-(1-42) had an opposite effect on
cholesterol in the G2 fraction as compared with the G1 fraction. There
was a significant (p < 0.001) reduction in cholesterol
in the G2 fraction when incubated with fresh A
-(1-42). On the other hand, aged A
-(1-42) had a similar effect on cholesterol in the G2
fraction as observed in the G1 fraction, resulting in a significant (p < 0.001) reduction in cholesterol (Fig. 3).
Differences in effects of fresh and aged A
-(1-42) on cholesterol
levels in the Golgi complex could be attributed to differences in
amounts of fresh and aged A
-(1-42) that reach the Golgi complex.
Levels of fresh A
-(1-42) were higher in the G1 and G2 fractions
(0.21 ± 0.03 and 0.22 ± 0.1 ng/mg of protein, respectively)
as compared with aged A
-(1-42) in the G1 and G2 fractions
(0.11 ± 0.05 and 0.16 ± 0.04 ng/mg of protein,
respectively). These differences in A
-(1-42) levels, however, were
not statistically significant.
Both colocalization data and data of the G1 fraction showed that fresh
A
-(1-42) increased cholesterol and aged A
-(1-42) reduced
cholesterol in the Golgi complex of astrocytes. The cell membrane is
enriched in cholesterol and could be a source of cholesterol in cells
treated with fresh A
-(1-42). Table I
shows that fresh A
-(1-42) significantly reduced both total and free
cholesterol in the cell membrane as compared with control astrocytes.
Total cholesterol was reduced by 34% and free cholesterol by 30% when treated with fresh A
-(1-42). Aged A
-(1-42) also
significantly reduced total and free cholesterol (17 and 11%,
respectively) in the cell membrane (Table I), but effects were much
less than those observed for fresh A
-(1-42). The ratio
of free cholesterol to total cholesterol was used as an estimate of
esterified cholesterol. Data in Table I show that the ratios were
similar among the three groups and did not differ significantly. It
also was noted that cholesterol content of the astrocyte homogenate
fraction did not differ among the three groups.
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Table I
Total cholesterol, free cholesterol, and the molar ratio of free to
total cholesterol in cell membranes of astrocytes incubated with
fresh and aged A -(1-42)
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A
-(1-42) Inhibits PC-PLD Activity--
PC-PLD has been shown
to be involved in regulating cholesterol efflux from cells (42) and in
release of secretory vesicles from the Golgi complex (23, 43), and an
isoform of PC-PLD has been reported to be associated with the Golgi
complex (20, 22). To begin to address a potential mechanism whereby
A
-(1-42) alters cholesterol content in the Golgi complex, we
examined the effects of fresh and aged A
-(1-42) on activity of
PC-PLD in astrocytes. Fig. 4
(panels A and B) reveals that fresh and aged
A
-(1-42) significantly (p < 0.001) inhibited
PC-PLD activity at each incubation time period. There was a small
diminution of effects of A
-(1-42) on PC-PLD activity with
increasing incubation time. PC-PLD activity was significantly
(p < 0.001) higher after 90 and 120 min of incubation versus 30 min of incubation for fresh A
-(1-42) and after
120 min for aged A
-(1-42) (Fig. 4, panels A
and B). The effects of fresh A
-(1-42) on PC-PLD activity
were consistent with the increase in cholesterol in the Golgi complex,
but PC-PLD activity does not explain the effects of aged A
-(1-42)
on Golgi complex cholesterol.

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Fig. 4.
Fresh and aged
A -(1-42) inhibit PC-PLD activity in
astrocytes. Astrocytes were incubated with fresh A -(1-42)
(panel A) and aged A -(1-42) (panel B) for
different time periods (30, 60, 90, and 120 min) after which time cells
were harvested as described under "Experimental Procedures." Cells
were placed in 96-well plates, and PC-PLD was determined using the
Amplex Red PC-specific PLD assay kit (Molecular Probes). Fluorescence
intensity was measured with a microplate reader using an excitation
wavelength of 542 nm and an emission wavelength of 590 nm. Values are
means ± S.E. (n = 3). *, p < 0.001 as compared with control cells; +, p < 0.001 as
compared with 30-min incubation time.
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|
Effects of Fresh and Aged A
-(1-42) on ApoE Levels in
Astrocytes--
It has been reported previously that A
-(1-42)
increased astrocyte activation, increased levels of apoE and apoJ in
the cell, and reduced levels of the two apolipoproteins in the
medium (18). The effects of activation were proposed to
primarily involve apoE and its receptors. Changes in
A
-(1-42)-induced levels of apoE and release of apoE could have an
effect on cholesterol content by sequestering cholesterol in the cell.
Fig. 5 (inset) shows that apoE
under reducing conditions can be seen as approximately a 34-kDa
monomer, and this finding is consistent with apoE expression in
astrocytes (18). Fresh A
-(1-42) significantly (p
0.01) increased apoE levels by 32% in astrocytes as compared with
control cells (Fig. 5). Aged A
-(1-42) appeared to reduce apoE
levels, but this difference was not statistically significant when
compared with control cells. ApoE was not detected in the conditioned
media from control and A
-(1-42)-treated cells (data not shown).

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Fig. 5.
Fresh A -(1-42) but
not aged A -(1-42) increases apoE levels in
astrocytes. Cells were treated as described under
"Experimental Procedures." Levels of apoE were quantitated from
Western blots. Data of cells treated with A -(1-42) are the levels
of apoE relative to control cells and are the means ± S.E. from
three to four independent experiments. The inset shows a
Western blot from a representative sample. *, p 0.01 as compared with control. Ctrl, control.
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|
 |
DISCUSSION |
Several different lines of evidence show that A
and cholesterol
interact (for reviews, see Refs. 4 and 44-47). This interaction is
reciprocal given that cholesterol modulates expression of amyloid precursor protein and A
(10-13) and cholesterol dynamics such as
cholesterol trafficking are affected by A
(15, 17-19). The Golgi
complex plays an important role in cholesterol trafficking (21, 48),
and we proposed that A
-(1-42) would alter cholesterol trafficking
in the Golgi complex and that the effects of A
-(1-42) would be
dependent on whether the peptide was fresh or aged. Two different
methods, confocal microscopy and isolation of the Golgi complex by
sucrose density centrifugation, were used to test the hypothesis that
fresh and aged A
-(1-42) would have different effects on cholesterol
in the Golgi complex of astrocytes. Results of this study showed that
A
-(1-42) modifies cholesterol distribution in the Golgi complex of
astrocytes. However, the effects of A
-(1-42) on cholesterol
distribution were dependent on whether A
-(1-42) was fresh or aged.
Fresh A
-(1-42) increased cholesterol content in the Golgi complex,
and aged A
-(1-42) reduced cholesterol content in the Golgi complex.
Moreover isolation of the Golgi complex into two fractions revealed the
novel finding that fresh A
-(1-42) increased cholesterol in the G1
fraction and reduced cholesterol in the G2 fraction. Aged A
-(1-42)
reduced cholesterol in both fractions. PC-PLD activity, cell membrane
cholesterol, and apoE levels were associated with effects of fresh
A
-(1-42) on cholesterol levels but not with effects of aged
A
-(1-42), arguing against a common mechanism.
A
-(1-42) altered cholesterol distribution in the Golgi complex of
astrocytes. Maximal effects of both fresh and aged A
-(1-42) were
observed after 120 min of incubation and a peptide concentration of
10
6 M. Shorter incubation times modified
cholesterol levels in the Golgi complex, but these effects were not
significantly different. An A
-(1-42) concentration of
10
9 M did not modify cholesterol levels.
A
-(1-42)-induced changes in Golgi complex cholesterol levels were
not simply attributable to a nonspecific perturbation of the Golgi
complex or cell viability. The reverse peptide A
-(42-1), either
fresh or aged, did not alter Golgi complex cholesterol levels compared
with control astrocytes. Viability of cells did not differ among the
control and cells treated with fresh or aged A
-(1-42).
Fresh A
-(1-42) increased astrocyte cholesterol in the G1 fraction
by 59% but reduced cholesterol in the G2 fraction by 29%. Earlier
work has indicated that the G1 fraction may represent the
trans-Golgi region and that the G2 fraction is
representative of the medial and cis regions (31,
32). Fresh A
-(1-42) may stimulate movement of cholesterol from the
cis and medial regions of the Golgi to the
trans-Golgi region. Mechanisms of anterograde intra-Golgi
transport have focused on cisternal maturation/progression and
vesicular/tubular transport, but the principal mechanism of intra-Golgi
transport including transport of lipids has not been resolved (for a
review, see Ref. 49). Movement of cholesterol from the G2 to the G1
fraction induced by fresh A
-(1-42) would account for only 29% of
the increase in cholesterol observed in the G1 fraction. Our data
showing that there was a significant reduction in cholesterol content
in the cell membrane of astrocytes treated with fresh A
-(1-42)
(34%) as compared with control astrocytes suggest that fresh
A
-(1-42) stimulates transport of cholesterol from the cell membrane
to the trans-Golgi region. There is evidence that cycling of
lipids including cholesterol and proteins occurs between the cell
surface membrane and the Golgi complex that is clathrin-independent
(50, 51). It has been suggested that this cell membrane-Golgi pathway
may regulate lipid raft distribution and function with recycling
occurring continuously (51). Amyloid precursor protein and A
are
associated with cholesterol-rich low density membrane domains (52-55).
Extracellular A
that is not highly aggregated may target lipid
rafts, promoting cycling of lipid rafts to the Golgi complex.
Aged A
-(1-42) reduced cholesterol levels in the Golgi complex but
also reduced cholesterol levels in the cell membrane by 16% as
compared with the 34% reduction by fresh A
-(1-42). The destinations and mechanisms of the cell membrane cholesterol removed by
fresh and aged A
-(1-42) may differ. Aged A
-(1-42) may complex with cholesterol in the cell membrane and transport cholesterol to the
cell interior. Support for this notion are data showing that indeed
aged A
-(1-40), but not fresh A
-(1-40), complexed with lipids
with cholesterol
stearic acid > PC (27). Once removed, the
aged A
-(1-42)-cholesterol complex may act as a type of lipid
droplet in the cell cytosol. Synucleins that had been chemically
cross-linked formed oligomers within cells that complexed with lipid
droplets and cell membranes (56). It is clear that fresh A
-(1-42)
and aged A
-(1-42) are acting differently on cholesterol homeostasis.
Cholesterol levels were increased in the Golgi complex by incubation
with fresh A
-(1-42), and this increase may be due in part to
cycling of cholesterol from the cell membrane to the Golgi complex. We
also propose that the elevation in cholesterol levels in the Golgi
complex involves PC-PLD function. PC-PLD plays a role in cholesterol
trafficking, it is located in the Golgi complex, and PC-PLD is also
involved in vesicular trafficking from the Golgi complex (20-23). Our
findings that fresh A
-(1-42) increased cholesterol content in the
Golgi complex and inhibited activity of PC-PLD are in agreement with
the purported role of PC-PLD in cholesterol trafficking. Inhibition of
PC-PLD reduces cholesterol efflux (22, 42). Activation of PC-PLD
enhanced the release of secretory vesicles from the
trans-Golgi complex (43). Inhibition of PC-PLD by fresh
A
-(1-42) may in turn reduce release of secretory vesicles with
their lipid and protein cargoes. We found that fresh A
-(1-42)
inhibited PC-PLD activity, but another study reported that the A
peptide fragment 25-35 increased PLD activity in LA-N-2 cells (57).
Differences in effects of A
on PLD activity between that study and
the present study may have resulted from dissimilarities in peptide
structure and peptide concentration (A
-(1-42) at 1 µM
versus A
-(25-35) at 100 µM). Inhibition of
PC-PLD activity by fresh A
-(1-42) was in agreement with our
findings that cholesterol levels were elevated in the Golgi complex in
the presence of fresh A
-(1-42). However, we also observed that aged
A
-(1-42) significantly inhibited PC-PLD activity but that
cholesterol levels were reduced in the Golgi complex of astrocytes
incubated with aged A
-(1-42). Both fresh and aged A
-(1-42)
inhibit PC-PLD activity, but the mechanism of effects on cholesterol
levels in the Golgi complex may differ as discussed in the preceding
paragraph. Aged A
-(1-42) may remove cholesterol by forming
complexes, whereas fresh A
-(1-42) induces movement of cholesterol
within the Golgi complex and from the cell membrane to the Golgi
complex and inhibits the recycling of cholesterol that involves
PC-PLD function.
The effects of fresh A
-(1-42) on Golgi complex cholesterol were
associated with apoE levels. Fresh A
-(1-42) significantly increased
apoE levels in astrocytes by ~32% as compared with control cells. On
the other hand, aged A
-(1-42) reduced apoE levels, but this
reduction did not differ significantly from that seen in control
cells. Previously it was found that aged A
-(1-42) increased apoE
levels in rat astrocytes from primary cell culture (18). In the same
study it also was reported that aged A
-(1-42) reduced levels of
apoE in the medium (18). We did not detect apoE in the media of
control astrocytes and cells incubated with either fresh or aged
A
-(1-42). Two major differences between our study and the earlier
report were the incubation time of 2 versus 12 h and
the A
-(1-42) concentration of 1 versus 10 µM, respectively. An important observation of the earlier
study was that A
-(1-42) did not alter steady-state levels of apoE
mRNA, and that may indicate that A
-(1-42) was not acting on
transcriptional regulation but could have been acting
post-translationally on apoE turnover (18). Whether the observed
increase in apoE levels in astrocytes incubated with fresh A
-(1-42)
plays a role in effects of the peptide on cholesterol in the Golgi
complex is unclear. ApoE is associated with the Golgi complex (58), and
changes in apoE levels may modify intracellular cholesterol distribution.
We have shown using two different methods that A
-(1-42) modifies
the cholesterol content of the Golgi complex of astrocytes. The effects
of A
-(1-42) on cholesterol levels were dependent on whether
astrocytes were treated with fresh or aged A
-(1-42). Fresh
A
-(1-42) increased cholesterol levels of the Golgi complex, whereas
aged A
-(1-42) reduced cholesterol levels. However, the direction of
effects of fresh A
-(1-42) on Golgi cholesterol levels was
contingent on the region of the Golgi complex suggesting that fresh
A
-(1-42) may also be acting on intra-Golgi cholesterol transport.
The Golgi complex has been described as a major site for protein and
lipid sorting and release of lipid rafts (59, 60). Either increasing or
decreasing cholesterol levels could impact on important Golgi complex
functions such as protein sorting, sphingomyelin synthesis, and lipid
trafficking resulting in cellular pathophysiology. Our data suggest
that the recycling of cholesterol between the cell membrane and the
Golgi complex was altered and that such changes could modify membrane
structure and function. Fresh A
may inhibit the formation of lipid
rafts in the Golgi complex including caveolin-organized rafts that are
transported to the plasma membrane and form caveolae. A
peptides are
located in detergent-insoluble membrane domains that are thought to be caveolae (54, 61) and in cholesterol domains not containing caveolin
(52). PC-PLD activity, cell membrane cholesterol, and apoE expression
of astrocytes were associated with effects of fresh A
-(1-42) on
Golgi complex cholesterol but not with effects of aged A
-(1-42),
arguing against a common mechanism. There is a reciprocal synergy
between A
and cholesterol. Changes in cholesterol content alter A
levels, and A
acts on cholesterol homeostasis. The dynamic
interaction of A
and cholesterol that includes targeting of the
Golgi complex may be an important factor that contributes to cellular
dysfunction occurring with AD.