(Received for publication, November 8, 1995)
From the
Amyloid precursor protein (APP) and cholesterol metabolism are
genetically linked to Alzheimer's disease, the latter through
apolipoprotein E, a lipid and cholesterol transport protein. We have
examined the hypothesis that the processing of APP is disrupted by
elevated cholesterol, which is known to modulate the activity of
several transmembrane proteins. In the current study, cholesterol,
solubilized by methyl--cyclodextrin or ethanol, was added to the
culture media of APP 751 stably transfected HEK 293 cells. Radiolabeled
APP and APP
(the soluble N-terminal derivative following
-secretase cleavage) were precipitated from lysates and
conditioned media of stably transfected HEK 293 cells; the relative
levels were determined by quantitative densitometry following
separation by SDS-polyacrylamide gel electrophoresis. The data show
that cholesterol, solubilized by methyl-
-cyclodextrin, greatly
reduced the levels of APP
. Low doses of
ethanol-solubilized cholesterol similarly caused a dramatic reduction
of APP
. By contrast, levels of APP holoprotein remained
the same or increased. The large decrease seen in APP
production was not due to nonspecific inhibition of secretion
because several secreted proteins increased in level. Cholesterol,
which impedes membrane fluidity, may lower APP
production
by impeding the interaction of the substrate with its protease(s). If
APP
were to function trophically, as suggested by other
studies, the current conclusion suggests that changes in cellular
cholesterol levels in Alzheimer's disease could contribute to
neuronal degeneration by decreasing the production of
APP
.
Amyloid precursor protein (APP) ()can be degraded by
several different pathways. One pathway releases A
, a
39-43-amino acid peptide that is the main constituent of the
amyloid plaques in the brains of Alzheimer's disease (AD)
patients (Glenner and Wong, 1984; Masters et al., 1985). The
C-terminal cleavage of A
, termed
-secretase cleavage, occurs
in the putative transmembrane domain by an unknown mechanism (Kang et al., 1987). The N-terminal cleavage,
-secretase,
releases a soluble N-terminal derivative that is found in human
cerebrospinal fluid (Seubert et al., 1993). Another pathway,
termed the
-secretase pathway, cleaves within the A
segment
to release APP
, a nonamyloidogenic soluble N-terminal
derivative, also found in human cerebrospinal fluid (Esch et
al., 1990; Palmert et al., 1992). The exact cleavage site
is between residues 16 and 17 of A
(Anderson et al.,
1991; Wang et al., 1991). The soluble APP derivative found in
the conditioned media of the cell line used in this study, HEK 293
cells, is almost exclusively the result of
-secretase cleavage
(Wang et al., 1991).
Modulation of APP levels
may be of physiological consequence. APP
induces a 2-fold
increase in the phosphorylation of tau, a microtubule-associated
protein that is hyperphosphorylated in AD (Greenburg et al.,
1994). In addition, APP
has a trophic effect on cerebral
neurons in culture (Araki et al., 1991) and is mitogenic for
Swiss 3T3 cells (Schubert et al., 1989). APP
must be added to the growth medium of two different cell lines
with reduced APP production to restore normal cell proliferation
(LeBlanc et al., 1992; Ninomiya et al., 1993).
APP
also protects neurons against hypoglycemic damage and
glutamate toxicity, causing a rapid and prolonged reduction in
intracellular Ca
concentration (Mattson et
al., 1993).
Production of APP has been found to
be influenced by several agents. Augmented iron, phorbol
12,13-dibutyrate, interleukin 1, cholinergic agonists, estrogen,
cholinesterase inhibitors, and cellular depolarization all increase
-secretase cleavage (Bodovitz et al., 1995; Buxbaum et al., 1992; Caporaso et al., 1992a; Gillespie et al., 1992; Jaffe et al., 1994; Nitsch et
al., 1993). Decreases in
-secretase cleavage have been
observed with the iron chelator desferrioxamine (Bodovitz et
al., 1995) as well as agents of a less modulatory and more
disruptive manner, such as monensin (disruption of distal Golgi
cisternae), methylamine (alkalization of acidic intracellular
compartments), and site-directed mutagenesis (Caporaso et al.,
1992b; Sisodia, 1992; De Strooper et al., 1993; Usami et
al., 1993).
In this study, we have examined the possible
disruptive effects of elevated cholesterol on APP processing. Increases
in cholesterol previously have been shown to modify the function of
certain membrane proteins. Function has decreased, as in the case of
the Meta I-Meta II transition of rhodopsin (Mitchell et al.,
1990), or increased, as in the cases of the
Na-K
-ATPase, carrier-mediated lactate
transport, and the acetylcholine receptor (Yeagle, 1991; Grunze et
al., 1980; Craido et al., 1982; Fong and McNamee, 1986).
Several lines of evidence have linked alterations in cholesterol
metabolism and transport to AD. The E4 allele of ApoE is associated
with higher plasma cholesterol levels (Sing and Davignon, 1985), and
ApoE4 is present with increased frequency in patients with sporadic and
late onset familial AD (Strittmatter et al., 1993; Saunders et al., 1993; Corder et al., 1993). In addition,
hypercholesterolemia is one of the major risk factors for critical
coronary artery disease (cCAD), a condition that results in A
deposition 3-10 times more frequently than in non-heart disease
controls (Sparks et al., 1990, 1993). We report that
methyl-
-cyclodextrin-solubilized cholesterol increases the levels
of both mature and immature APP holoproteins in a dose-dependent
fashion while dramatically reducing the production of
APP
.
Conditioned media were used without modification and incubated with 22C11 or 4G8. Cells were rinsed once with PBS and removed from the plate with PBS, 0.02% EDTA. Cells were lysed in PBS plus 0.1% SDS, 1% Triton X-100, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and 100 kallikrein-inactivating units/ml of aprotinin (PBS-TDS). Lysates were incubated with antiserum 8256. In all cases the primary antibody-antigen complex was precipitated with Protein A-Sepharose (Pharmacia Biotech Inc.) in PBS-TDS. Immunoprecipitated proteins were resolved on a 10%/16% Tris-Tricine gel (Schagger and Von Jagow, 1987). Gels were fluorographed (DuPont NEN), dried, and exposed to Kodak X-Omat film.
Cholesterol, water solubilized by methyl--cyclodextrin
at a ratio of 46 mg of cholesterol/g of solid, was delivered to human
embryonic kidney (HEK) 293 cells stably transfected with APP 751. The
cholesterol exchange between cyclodextrin and membranes reaches
equilibrium in less than 1 min and is proportional to the concentration
of cyclodextrin used (Irie et al., 1992a).
Cyclodextrin-solubilized cholesterol, at the doses used here, has been
shown to be an effective delivery system for cell culture studies as
well as parenteral administration to animals (De Caprio et
al., 1992; Irie et al., 1992a, 1992b).
We used this
delivery system to determine if cholesterol modulation affected the
stability of APP, a transmembrane protein with a half-life of only
20-30 min (Kang et al., 1987; Weidemann et al.,
1989). Following a 24-h incubation to allow for intracellular
distribution and membrane turnover, the APP 751 stably transfected HEK
293 cells were labeled for 8 h with S-labeled
methionine/cysteine in the presence of cholesterol to obtain
steady-state measurements. During the entire procedure, at all
concentrations of cholesterol used, the cells remained attached to the
plates; at 240 mg/dl they were slightly rounded, but at 160, 80, and 40
mg/dl cholesterol they looked indistinguishable from controls (data not
shown). Cell lysates were used for immunoprecipitation of APP
holoproteins with C-terminal polyclonal antiserum 8256 , previously
characterized in Bodovitz et al.(1995). Antiserum 8256
immunoprecipitated two bands of
110 and 130 kDa, which correspond,
respectively, to immature and mature APP holoprotein (Fig. 1a). Both protein bands increased significantly
following the addition of cyclodextrin-solubilized cholesterol (Fig. 1a and Fig. 2, a and b).
Mature APP holoprotein increased 50 ± 10% (p <
0.001; n = 6) at 40 mg of cholesterol/dl of media. (All
values are normalized to no drug control and are given as ±S.E.; p values were determined by a Student's t test.) The peak increase of 160 ± 37% (p <
0.05; n = 4) occurred at 160 mg/dl cholesterol,
followed by a smaller increase of 70 ± 14% (p <
0.05; n = 6) at 240 mg/dl cholesterol. Immature APP
holoprotein showed a similar rate of increase, e.g. 46
± 10% at 40 mg/dl cholesterol and 140 ± 64% at 160 mg/dl
cholesterol (p < 0.001; n = 4), but was
still increasing 660 ± 190% (p < 0.001; n = 6) at 240 mg/dl cholesterol.
Figure 1:
Cholesterol modulation of mature and
immature APP holoproteins and APP. HEK 293 cells stably
transfected with APP 751 were preincubated with no drug or the
indicated concentration (mg/dl) of
methyl-
-cyclodextrin-solubilized cholesterol or
methyl-
-cyclodextrin alone. The cells were changed to
methionine/cysteine-free media containing the same concentration of
cholesterol or cyclodextrin and labeled for 8 h with
[
S]methionine/cysteine. Cellular lysate was used
to immunoprecipitate APP holoproteins with C-terminal antiserum 8256;
conditioned media were used to immunoprecipitate the N-terminal
-secretase derivative, APP
, with monoclonal antibody
22C11. Immunoprecipitates were separated by 10% Tris/Tricine SDS-PAGE
electrophoresis and visualized by autoradiography. Representative
autoradiograms are shown. a, both mature and immature APP
holoproteins are increased at 160 mg/dl cholesterol. Mature APP
decreases at 240 mg/dl cholesterol, but immature APP continues to
increase. c, APP
decreases dramatically at 160
and 240 mg/dl cholesterol. The control band had to be overexposed in
order for the autoradiogram to exhibit any APP
at 240
mg/dl cholesterol. b, methyl-
-cyclodextrin was added at 0
or 5200 mg/dl; the latter corresponds to the amount used to solubilize
240 mg/dl cholesterol. This solubilization agent did not affect levels
of immature or mature APP holoprotein. d, even at 5200 mg/dl,
methyl-
-cyclodextrin did not affect levels of APP
.
These data demonstrate that the modulation of APP processing seen on
the left was the result of cholesterol and not its solubilization
agent.
Figure 2:
Densitometric analysis of cholesterol
modulation of APP holoproteins and APP. Autoradiograms
were scanned into the computer, and bands corresponding to mature APP,
immature APP, and APP
were densitometrically analyzed
with the Metamorph imaging system. a and b, increased
cellular cholesterol loading increased the steady-state levels of
mature and immature APP holoproteins. The increase in mature protein
reached a peak and then began to decline, although still remaining
above control levels at the highest cholesterol dose used, 240 mg/dl.
By contrast, the levels of immature protein were still dramatically
increasing at 240 mg/dl cholesterol. c, increased cellular
cholesterol loading decreased APP
levels. The change in
level lagged behind the changes seen with the holoproteins at low doses
but reached a 93 ± 1% (p < 0.001; n = 6) decrease at 240 mg/dl cholesterol. (All values are
normalized to no drug control and are given as ±S.E.; p values were determined by a Student's t test; n = 4 for cholesterol doses of 80 and 160 mg/dl; n = 6 for cholesterol doses of 40 and 240 mg/dl.) *, p < 0.05;**, p < 0.01;***, p <
0.001.
The modulation of APP
holoproteins suggested that downstream catabolites would also be
modulated. To test this possibility we immunoprecipitated A and
the N-terminal
-secretase derivative, APP
(see below
for characterization), from the conditioned media of APP 751 stably
transfected HEK 293 cells using, respectively, A
monoclonal
antibody 4G8 (as characterized by Buxbaum et al.(1992)) and
N-terminal monoclonal antibody 22C11 (Boehringer Mannheim). Although
A
levels showed no consistent change (data not shown), APP
significantly and reproducibly decreased in the presence of
augmented cholesterol (Fig. 1c and 2c). The
decrease was 16 ± 8% at 40 mg/dl cholesterol, indicating that
APP
levels were not as sensitive as holoprotein levels to
low doses of cholesterol. The decrease was 65 ± 9% (p < 0.05; n = 4) at 160 mg/dl cholesterol and 93
± 1% (p < 0.001; n = 6) at 240 mg/dl
cholesterol, indicating a strong response to higher doses of
cholesterol.
The N-terminal soluble APP derivative can be released
into conditioned media by either - or
-secretase cleavage. In
order to determine the relative amounts of each cleavage in our system,
we immunoprecipitated the total N-terminal derivative from conditioned
media, split the precipitates in half, and resolved them on two
separate SDS-PAGE gels. The proteins were transferred to membrane and
probed with either 22C11, a monoclonal antibody against the N terminus
of APP, or 6E10, a monoclonal antibody against residues 1-16 of
A
(Pirtilia et al., 1995). 22C11 recognizes total
N-terminal derivative whereas 6E10 recognizes that resulting from
- but not
-secretase cleavage. Both antibodies recognized
bands of similar intensity (Fig. 3). This finding, in
conjunction with the sequence analysis of Wang et al.(1991),
demonstrates that the soluble N-terminal APP derivative released into
the conditioned media of HEK 293 cells is almost exclusively
APP
, the
-secretase cleavage derivative.
Figure 3:
Characterization of the N-terminal APP
secretase derivative released into the conditioned media. N-terminal
secretase derivative, in total, was immunoprecipitated from conditioned
media with monoclonal antibody 22C11. The precipitate was split in half
and resolved on two separate SDS-PAGE gels. The proteins were
transferred to membrane and probed with either 22C11, a monoclonal
antibody against the N terminus of APP, or 6E10, a monoclonal antibody
against residues 1-16 of A. 22C11 recognizes the total
N-terminal derivative whereas 6E10 recognizes that resulting from
- but not
-secretase cleavage. Both antibodies recognized
bands of similar intensity. This finding, in conjunction with the
sequence analysis of Wang et al.(1991), demonstrates that the
soluble N-terminal APP derivative released into the conditioned media
of HEK 293 cells is almost exclusively APP
, the
-secretase cleavage derivative.
In order
to determine that the modulation of APP processing by cholesterol was
not affected by methyl--cyclodextrin, the solubilization agent, we
added this agent to our stably transfected cell system and
immunoprecipitated APP holoproteins and APP
. The
concentration used, 5200 mg/dl, corresponded to the solubilization of
240 mg/dl cholesterol. Even at this high concentration,
methyl-
-cyclodextrin did not affect levels of APP holoprotein (Fig. 1b) or levels of APP
(Fig. 1d).
As a further control for the
methyl--cyclodextrin-solubilized cholesterol delivery system, we
immunoprecipitated APP holoproteins and APP
from cells
maintained in delipidated serum and treated with ethanol-solubilized
cholesterol. This delivery system, in the same concentration range used
here, has been shown to induce a significant linear increase in
cellular free cholesterol (Lasa et al., 1991).
Ethanol-solubilized cholesterol, in contrast to
cyclodextrin-solubilized cholesterol (Fig. 1a), did not
modulate APP holoprotein levels (Fig. 4a). The reason
for the difference between the two delivery systems is unknown but
could be a result of variation in the intracellular distribution of
cholesterol; differential distribution might be the result of
cyclodextrin, but not ethanol, mediating both cholesterol influx and
efflux at the plasma membrane (Irie et al., 1992a). However,
the impact of ethanol-delivered cholesterol on APP
was
dramatic, with reductions even more pronounced than seen with
cyclodextrin-solubilized cholesterol (Fig. 4b). Levels
of APP
were greatly reduced with as little as 40 mg/dl
cholesterol and all but gone at 80 mg/dl and higher (Fig. 4b). Both delivery systems thus support our major
finding that increased cellular cholesterol dramatically inhibits
-secretase cleavage.
Figure 4:
Ethanol-solubilized cholesterol modulation
of APP holoproteins and APP. As a control for the
methyl-
-cyclodextrin-solubilized cholesterol delivery system, we
immunoprecipitated APP holoproteins and APP
from cells
maintained in delipidated serum and treated with ethanol-solubilized
cholesterol. a, this delivery system, in contrast to
cyclodextrin-solubilized cholesterol, did not modulate APP holoprotein
levels. The reason for the difference between the two delivery systems
is unknown but could be a result of variation in the intracellular
distribution of cholesterol; differential distribution might be the
result of cyclodextrin, but not ethanol, mediating both cholesterol
influx and efflux at the plasma membrane. b, the two delivery
systems did, however, yield similar results on the modulation of
APP
, with ethanol-solubilized cholesterol generating an
even more dramatic reduction. Levels of APP
were greatly
reduced at as little as 40 mg/dl cholesterol and all but gone at 80
mg/dl and higher. Both delivery systems, despite the differences in
modulation of APP holoproteins, support our major finding that
increased cellular cholesterol dramatically inhibits
-secretase
cleavage.
As a final control for our cyclodextrin
delivery system, we examined the effects of
methyl--cyclodextrin-solubilized cholesterol on general production
of cellular and secreted proteins. We analyzed
[
S]methionine/cysteine-labeled proteins from
cell lysate and conditioned media by 10% Tris/Tricine SDS-PAGE gel
electrophoresis (Fig. 5). A small percentage of the cellular
proteins changed in level, but there were no significant global
differences in distribution or intensity between the spectra of labeled
proteins, even at 240 mg/dl cholesterol (Fig. 5a). This
lack of change indicates that cellular protein metabolism was largely
unaffected by cholesterol modulation. By contrast, there were several
changes in unimmunoprecipitated
[
S]methionine/cysteine-labeled secreted proteins (Fig. 5b). There was a decrease in a
120-kDa band
with increasing cholesterol concentration; this band was
APP
. Its elevated levels in conditioned media were the
result of the overexpression of the APP 751 gene in stably transfected
HEK 293 cells. There were no other significant differences in the
spectra of labeled secreted proteins between 0 and 160 mg/dl
methyl-
-cyclodextrin-solubilized cholesterol. At 240 mg/dl
cholesterol, however, one band at
65 kDa appeared, and several
bands in the 40-kDa range increased in intensity. The increase is in
opposition to the decrease in APP
levels, demonstrating
that the latter change is not due to nonspecific cholesterol modulation
of secreted proteins.
Figure 5:
Cholesterol modulation of general cellular
and secreted proteins. Aliquots of the cellular lysates and conditioned
media collected for use in the immunoprecipitation studies of Fig. 1were left unimmunoprecipitated, separated with 10%
Tris/Tricine SDS-PAGE electrophoresis, and visualized by
autoradiography. a, a small percentage of the
[S]methionine/cysteine-labeled cellular proteins
changed in level, but there were no significant global differences in
distribution or intensity between the spectra of labeled proteins at
cholesterol doses of 0, 160, or 240 mg/dl. This lack of change
demonstrates that general protein metabolism was unaffected by
cholesterol modulation. b, by contrast, there were several
changes in unimmunoprecipitated
[
S]methionine/cysteine-labeled secreted
proteins. There was a decrease in a
120-kDa band with increasing
cholesterol concentration; this band was APP
. Its
elevated levels in conditioned media were the result of the
overexpression of the APP 751 gene in stably transfected HEK 293 cells.
There were no other significant differences in the spectra of labeled
secreted proteins between 0 and 160 mg/dl cholesterol. At 240 mg/dl
cholesterol, however, one band at
65 kDa appeared, and several
bands in the 40-kDa range increased in intensity. The increase is in
opposition to the decrease in APP
levels, demonstrating
the latter change is not due to nonspecific cholesterol modulation of
secreted proteins.
The increase in APP stability with
methyl--cyclodextrin-solubilized cholesterol is consistent with
several experimental observations. APP is a transmembrane protein (Kang et al., 1987) that is tyrosine-sulfated and O- and N-glycosylated (Weidemann et al., 1989). APP only has
a half-life of 20-30 min (Weidemann et al., 1989) but is
found in many different intracellular membranes, including lysosomal
and plasma (Haass et al., 1992), suggesting rapid
intracellular movement. Increasing the cholesterol content of
phospholipid bilayers increases their rigidity by ordering the acyl
chain region and may slow down APP transport, in turn slowing down the
transitions from immature to mature and mature to degraded. Even a
small decrease in the rate of these transitions would have, given the
short half-life of APP, a large effect on the net steady-state levels
of mature and immature APP holoproteins.
Mature APP is cleaved by
-secretase to generate APP
, demonstrating a
precursor-product relationship (Weidemann et al., 1989), yet
augmenting methyl-
-cyclodextrin-solubilized cholesterol increases
levels of mature APP while decreasing levels of APP
.
-Secretase function thus is reduced, either directly or
indirectly. Indirectly, function would be reduced if enzyme and
substrate were compartmentalized and if cholesterol were to block APP
access to the protease-containing compartment. Cholesterol also could
have a direct effect on
-secretase activity, which appears to be
membrane-associated.
-Secretase cleavage requires that APP be
inserted into a membrane, and it cleaves APP at a fixed distance from
the membrane instead of at a specific amino acid sequence (Sisodia,
1992). Furthermore, cleavage is blocked by APP site-directed
mutagenesis of three adjacent lysines to glutamic acid residues,
located just carboxyl to the proposed transmembrane domain (Usami et al., 1993); this substitution demonstrated that
perturbation of the association of APP with the membrane inhibits
-secretase cleavage. Another perturbation may be the result of the
stiffening of the membrane due to cholesterol loading, possibly
inhibiting lateral movement and the required contact between enzyme and
substrate (Fig. 6). Modulation of membrane fluidity also affects
the external accessibility of membrane proteins, as evidenced by the
complex changes in ligand binding of the serotonin receptor as a
function of membrane fluidity (Heron et al., 1980). A change
in the external accessibility of either
-secretase or APP may
inhibit the enzyme activity by disrupting a cleavage event that occurs
at a fixed distance from the membrane.
Figure 6:
Possible mechanism of the inhibition of
-secretase cleavage by cholesterol. The stiffening of the membrane
due to cholesterol loading may decrease
-secretase cleavage of APP
by inhibiting lateral movement (indicated by horizontal
arrows) and the required contact between enzyme and
substrate.
If cholesterol were to
increase in the AD brain, our data from the cyclodextrin delivery
system suggest an increase in immature and mature APP holoprotein and a
decrease in APP. The significance of the former change
is, at present, unclear. APP may function as a receptor (Kang et
al., 1987; Ferreira et al., 1993; Nishimoto et
al., 1993) and may be associated with the heterotrimeric
G-protein, G
(Nishimoto et al., 1993); its
overproduction could lead to aberrant intracellular signaling.
Alternatively, the main function of APP may be to serve as a precursor
for APP
and other cleavage derivatives. A decrease in
APP
would be the reduction of a mitogenic and trophic
factor as well as a protective agent against hypoglycemic damage and
glutamate toxicity (Araki et al., 1991; Schubet et
al., 1989; Mattson et al., 1993). The loss of such a
protein could exacerbate the cell death in AD.
Some evidence is available to suggest that cholesterol levels increase in AD. There is a predisposition for AD associated with the E4 allele of the cholesterol and lipid transport protein ApoE (Strittmatter et al., 1993; Saunders et al., 1993; Corder et al., 1993). Lipoproteins associated with ApoE4 are cleared more efficiently than those containing either of the other two alleles, E3 or E2 (Poirier et al., 1993), yet the E4 allele is linked to higher plasma cholesterol levels (Sing and Davignon, 1985) and is the most common of the three alleles in hypercholesterolemia (Utermann, 1984). The discrepancy stems from the efficient clearing of ApoE4 in the liver, possibly leading to a down-regulation of the low density lipoprotein receptor and, hence, an elevation of serum low density lipoprotein cholesterol (for review, see Davignon et al.(1988)).
Hypercholesterolemia is one of the major risk factors for cCAD, a
condition that often results in A deposition similar to that found
in AD (Sparks et al., 1990, 1993). Cerebral A
plaques are
3-10 times more common in cCAD than in nonheart disease controls
(Sparks et al., 1993). In addition, hypercholesterolemia
induced in rabbits resulted in elevated immunoreactivity of A
and
ALZ-50, an epitope only found in mature brain afflicted by AD (Sparks et al., 1994). All of these data suggest that increased
cholesterol levels are a risk factor for AD. Our data provide a
mechanism, namely the direct disruption of APP processing.