Amyloidogenic Processing of the Alzheimer Beta-Amyloid Precursor Protein Depends on Lipid Rafts

Abstract

Formation of senile plaques containing the beta-amyloid peptide (A beta) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer's disease (AD). APP is cleaved either by beta-secretase or by alpha-secretase to initiate amyloidogenic (release of A beta) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating A beta generation. Reducing cholesterol levels in N2a cells decreased A beta production. APP and the beta-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of A beta in a cholesterol-dependent manner. A beta generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre. This inhibition could be overcome by antibody cross-linking. These observations suggest the existence of two APP pools. Although APP inside raft clusters seems to be cleaved by beta-secretase, APP outside rafts undergoes cleavage by alpha-secretase. Thus, access of alpha- and beta-secretase to APP, and therefore A beta generation, may be determined by dynamic interactions of APP with lipid rafts.

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The Journal of Cell Biology, Volume 160, Number 1, January 6, 2003 113–123
http://www.jcb.org/cgi/doi/10.1083/jcb.200207113
JCB
Article
113
Amyloidogenic processing of the Alzheimer

-amyloid
precursor protein depends on lipid rafts
Robert Ehehalt,
1
Patrick Keller,
1
Christian Haass,
2
Christoph Thiele,
1
and Kai Simons
1
1
Max Planck Institute of Molecular Cell Biology and Genetics, D-01307 Dresden, Germany
2
Adolf Butenandt Institute, Department of Biochemistry, Laboratory for Alzheimer’s and Parkinson’s Disease Research,
Ludwig Maximilians University, D-80336 Munich, Germany
ormation of senile plaques containing the

-amyloid
peptide (A

) derived from the amyloid precursor protein
(APP) is an invariant feature of Alzheimer’s disease
(AD). APP is cleaved either by

-secretase or by

-secretase to
initiate amyloidogenic (release of A

) or nonamyloidogenic
processing of APP, respectively. A key to understanding AD
is to unravel how access of these enzymes to APP is regulated.
Here, we demonstrate that lipid rafts are critically involved
in regulating A

generation. Reducing cholesterol levels in
N2a cells decreased A

production. APP and the

-site
APP cleavage enzyme (BACE1) could be induced to copatch
at the plasma membrane upon cross-linking with antibodies
F
and to segregate away from nonraft markers. Antibody
cross-linking dramatically increased production of A

in a
cholesterol-dependent manner. A

generation was dependent
on endocytosis and was reduced after expression of the
dynamin mutant K44A and the Rab5 GTPase-activating
protein, RN-tre. This inhibition could be overcome by anti-
body cross-linking. These observations suggest the existence
of two APP pools. Although APP inside raft clusters seems
to be cleaved by

-secretase, APP outside rafts undergoes
cleavage by

-secretase. Thus, access of

- and

-secretase
to APP, and therefore A

generation, may be determined by
dynamic interactions of APP with lipid rafts.
Introduction
Formation of senile plaques composed of a 4-kD small peptide,
the amyloid

-peptide (A

)* is one of the hallmarks of
Alzheimer’s disease (AD). A

derives from a large type I
transmembrane protein, the amyloid precursor protein
(APP) (for review see Selkoe, 2001). It is cleaved out sequen-
tially by enzymes termed

- and

-secretase. The

-site APP
cleavage enzyme
(BACE1) has been identified recently as a
novel membrane-bound aspartyl-protease (De Strooper and
Annaert, 2000; Esler and Wolfe, 2001) and cleaves APP in
its luminal domain to generate a secreted ectodomain
(

APP). The remaining 10-kD

-cleaved COOH-terminal
stub of APP (

CTF) fragment is subsequently the substrate
for

-secretase, which cleaves the transmembrane domain of
APP to release A

.

-Secretase is a multiprotein complex,
requiring presenilins-1 and -2 for activity (De Strooper and
Annaert, 2000; Weihofen et al., 2002). A third enzyme, the

-secretase, cleaves APP in the middle of the A

region to
generate a secreted ectodomain (

APP) and a shorter

-cleaved
COOH-terminal stub of APP (

CTF) that is also cleaved by

-secretase.

-secretase activity was shown to be associated
with members of the ADAM (a disintegrin and metallo-
protease) family (ADAM 9, 10, and 17) (Buxbaum et al.,
1998; Koike et al., 1999; Lammich et al., 1999).

-Cleavage
is the dominant processing step, and since it cuts APP
within the A

region it prevents A

formation. Since

- and

-cleavages directly compete for their substrate APP, the key
in understanding A

generation is to find out how access of
these enzymes to APP is regulated.
There is growing evidence that cholesterol is of particular
importance in regulating

- and

-cleavage (Simons et al.,
2001). The E4 allele of apolipoprotein E has been shown to
be a major risk factor for AD (Corder et al., 1993; Strittmatter
et al., 1993), levels of total cholesterol and LDL in serum
were reported to correlate with the amount of A

in AD
brains (Kuo et al., 1998), and there is epidemiological evidence
that elevated cholesterol levels during mid-life increase the
risk of developing AD (Kivipelto et al., 2001). Elevated di-
Address correspondence to Kai Simons, Max Planck Institute of Molecular
Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden,
Germany. Tel.: 49-351-2102800. Fax: 49-351-2102900.
E-mail: simons@mpi-cbg.de
*Abbreviations used in this paper: A

, amyloid

-peptide; AD, Alzheimer’s
disease; APP, amyloid precursor protein;

APP,

-cleaved ectodomain of
APP; BACE1,

-site APP cleavage enzyme; CMV, cytomegalovirus;

CTF,

-cleaved COOH-terminal stub of APP;

CTF,

-cleaved
COOH-terminal stub of APP; DRM, detergent-resistant membrane; FP,
fluorescent protein; M

CD, methyl-

-cyclodextrin; PLAP, placental
alkaline phosphatase; swAPP, Swedish mutant of APP; TfR, human
transferrin receptor; wtAPP, wild-type APP.
Key words: lipid rafts;

-amyloid; BACE; Alzheimer’s disease; endocytosis

The Journal of Cell Biology
114 The Journal of Cell Biology
|
Volume 160, Number 1, 2003
etary cholesterol uptake increased amyloid plaque formation
in rabbits and transgenic mice (Sparks et al., 1994; Refolo et
al., 2000), and cholesterol loading and depletion affected A

generation in cultured cells and in an animal model (Simons
et al., 1998; Fassbender et al., 2001). There is also a correla-
tion between cellular cholesteryl-ester levels and A

produc-
tion (Puglielli et al., 2001), and it was demonstrated that ag-
gregated A

binds cholesterol in vitro (Avdulov et al.,
1997). Interestingly, two independent retrospective studies
reported a strong decrease in the incidence of AD and de-
mentia in patients treated with 3-hydroxy-3-methylglutaryl–
coenzyme A reductase inhibitors (Jick et al., 2000; Wolozin
et al., 2000).
All of these studies point out that cholesterol is critically
involved in A

generation. However, little is known about
the mechanisms by which cholesterol affects this process.
We previously hypothesized that the association of APP with
lipid rafts determines A

production (Simons et al., 1998).
Rafts are lateral assemblies of sphingolipids and cholesterol
within the membrane (for review see Simons and Toomre,
2000). They are thought to form ordered platforms, which
float around in the liquid-disordered matrix of the cellular
membrane and represent versatile devices to compartmental-
ize membrane processes. Biochemically, the components of
lipid rafts are characterized by their insolubility in detergents
such as Triton X-100 or CHAPS at 4

C (Fiedler et al.,
1993; Brown and London, 1997). A fraction of APP and
BACE1 were shown to be associated with detergent-resistant
membranes (DRMs) in a cholesterol-dependent manner
(Bouillot et al., 1996; Simons et al., 1998; Riddell et al.,
2001).

-Secretase cleavage, on the other hand, was elevated
after inhibition of

-secretase activity by cholesterol deple-
tion, and ADAM 10, a putative

-secretase, was soluble af-
ter detergent extraction (Kojro et al., 2001). The most
straightforward interpretation of these data is that APP is
present in two cellular pools, one associated with lipid rafts
where A

is generated and another outside of rafts where

-cleavage takes place. This model of membrane compart-
mentalization would explain how the same protein could be
processed in two different mutually exclusive ways.
If cleavage of APP by BACE1 occurred in rafts, it would
be important to know how and where this interaction is reg-
ulated. Therefore, in this paper we have studied these rela-
tionships and provide evidence that A

generation critically
depends on lipid rafts for enzyme activation to occur.
Results
Cholesterol depletion inhibits

-cleavage in N2a cells
To study the significance of lipid rafts for APP processing, we
have used a neuronal cell line, mouse neuroblastoma N2a. We
first analyzed the effect of cholesterol depletion on APP pro-
cessing, which was done by a combination of lovastatin treat-
ment and methyl-

-cyclodextrin (M

CD) extraction. Lova-
statin in the presence of small amounts of mevalonate decreases
de novo synthesis of cholesterol by inhibiting 3-hydroxy-
3-methylglutaryl–coenzyme A reductase and M

CD extracts
plasma membrane cholesterol. N2a cells were grown for 36 h
in lipid-deficient FCS in the presence of lovastatin, and im-
mediately before metabolic labeling they were treated with 10
mM M

CD for 5–30 min. Depending on the time of extrac-
tion, total cellular cholesterol levels could be reduced to 15%
of control cells. Increasing time of exposure and concentra-
tions of M

CD or prolonged treatment with lovastatin
started to affect cell viability.
To easily monitor the influence of cholesterol depletion
on APP processing, N2a cells were infected with adenovi-
ruses to express either human wild-type APP (wtAPP) or the
Swedish mutant of APP (swAPP). swAPP is dominantly

cleaved, resulting in a several-fold higher production of A

than for wtAPP. After M

CD extraction, the cells were
metabolically labeled with [
35
S]methionine and chased for
up to 2 h. Immunoprecipitations from conditioned medium
revealed that A

production was dependent on cellular cho-
lesterol levels (Fig. 1 A). Decreasing cellular cholesterol by
85% totally abolished A

secretion. Remarkably, already
relatively small changes in total cellular cholesterol levels
were found to have strong effects. A 20–30% decrease
showed a 50–60% reduction in A

secretion.

CTF, which
Figure 1. Cholesterol depletion inhibits -cleavage. (A) N2a cells
were grown in the presence () or absence () of lovastatin/
mevalonate/delipidated FCS and 10 h after infection with adenoviruses
to express wtAPP treated with 10 mM MCD for the indicated times.
Cells were then labeled with [35S]methionine for 40 min and chased
for 2 h. Immunoprecipitations of extracellular medium (A; antibody
70JE) and cell lysate (total wtAPP; antibody IP60) revealed that A
secretion decreased after cholesterol depletion. The extent of
cholesterol depletion was determined as described in Materials and
methods. (B) 10 h after infection with adenoviruses to express
swAPP, N2a cells were labeled for 30 min and chased for 30 min in
the presence of 10 mM MCD (leading to an 40–50% decrease in
total cellular cholesterol). Immunoprecipitations from extracellular
medium (antibody 6E10) and cell lysate (antibody IP60) showed a
decreased production of A and the COOH-terminal fragment
generated by -cleavage (CTF). At the same time, the COOH-terminal
fragment generated by -cleavage (CTF) and the soluble ectodomain
generated by -cleavage (APPs) were increased.

The Journal of Cell Biology
A production depends on lipid rafts | Ehehalt et al. 115
is generated by -cleavage, was also clearly reduced. On the
other hand, the production of CTF by -cleavage was in-
creased (Fig. 1 B). As expected from results of Kojro et al.
(2001), the soluble ectodomain generated by -cleavage was
also strongly increased in cholesterol-depleted cells. In gen-
eral, processing of wtAPP and swAPP were similarly affected
by cholesterol depletion.
These results show that cholesterol depletion of N2a cells
inhibits -cleavage, whereas -cleavage is increased. They
suggest that cholesterol is critically involved in regulating the
access of - and -secretase to APP.
APP and BACE copatch with placental alkaline
phosphatase but segregate from transferrin receptor
Lipid rafts are most abundant in the plasma membrane. In
fibroblasts, individual rafts have a size of 50 nm, corre-
sponding to 3,500 sphingolipid molecules and probably
not more than 10–30 proteins (Pralle et al., 2000). This
means that two different species of raft proteins would rarely
be in the same raft. However, it was shown previously that
raft and nonraft markers could be cross-linked with antibod-
ies into distinct patches (Harder et al., 1998; Janes et al.,
1999; Prior et al., 2001). Raft markers copatch and segregate
away from nonraft markers. Cross-linking with antibodies
can thus be used as an assay for raft association. We tested
whether antibody cross-linking induced copatching of APP
and BACE1 with a raft marker, the glycosyl phosphatidyl-
inositol (GPI)-anchored protein placental alkaline phos-
phatase (PLAP). As a nonraft marker, we used a mutant hu-
man transferrin receptor (TfR), where the cytosolic aa 5–41
(TfR del 5–41) have been removed. This mutant is defective
in endocytosis due to the deletion of the sorting signal.
Patches of TfR del 5–41 were shown to be segregated from
components found in lipid rafts (Harder et al., 1998).
Our experiments were performed with BACE1A-CFP and
YFP-wtAPP. CFP and YFP are the cyan and yellow color
variants of the green fluorescent protein, respectively. Con-
trol experiments demonstrated that these fluorescent protein
(FP)-containing constructs showed the same proteolytic pro-
cessing and immunofluorescence behavior as the corre-
sponding untagged proteins (unpublished data). BACE1A-
CFP was cross-linked with the polyclonal antibody 7523
recognizing the NH2-terminal part of BACE1 (Capell et al.,
2002). YFP-wtAPP was cross-linked with antiserum KG77
or mouse monoclonal antibody 3E6, both directed against
the FP. Control experiments with wtAPP or BACE1A-
VSVG and anti-APP antibody 5313 or anti-BACE1 anti-
body 7523 showed essentially the same patching (unpub-
lished data). Also, we did not see significant differences in
staining of swAPP and wtAPP.
Both BACE1 and wtAPP colocalized with PLAP at the
plasma membrane in the majority of cells, but they clearly
segregated from TfR del 5–41 (Fig. 2). BACE1 and wtAPP
could also be localized to the same patches upon cross-link-
ing (Fig. 3). For quantitative analyses of the extent of
copatching, images of 10 randomly selected cells on one
coverslip were taken and assigned into four categories: (1)
coclustering (80% overlap); (2) partial coclustering (clearly
overlapping spots 30–80%); (3) random distribution, and
Figure 2. Copatching of PLAP and TfR del 5–41 with YFP-wtAPP
and BACE1A-CFP. 10 h after transient transfection, the cells were
incubated for 45 min at 10C with the primary antibodies. Patching
of YFP-wtAPP and BACE1A-CFP was achieved with polyclonal
antibodies KG77 and 7523, respectively. PLAP and TfR del 5–41
were patched with mouse monoclonal antibodies from Dako and
Roche, respectively. Thereafter, the cells were washed and incubated
for 45 min with mixed Cy5- and Cy3-labeled secondary antibodies.
(A) YFP-wtAPP and BACE1A-CFP segregate from TfR del 5–41.
(B) Colocalization of cross-linked YFP-wtAPP and BACE1A-CFP
with PLAP. Bar, 10 m.
Figure 3. Copatching of YFP-wtAPP and BACE1A-VSVG at the
cell surface. (A) Immunofluorescence of BACE1A-VSVG using
polyclonal antiserum 7523 against the NH2-terminal part of BACE1
(green) and YFP-wtAPP using mAb 3E6 against the FP (red). Both
proteins were randomly distributed at the cell surface when the
antibody was added after fixation. (B) Colocalization of YFP-wtAPP
(red) and BACE1A-VSVG (green) after antibody induced patching
with the same antibodies as above. Bar, 10 m.

The Journal of Cell Biology
116 The Journal of Cell Biology | Volume 160, Number 1, 2003
(4) segregation. The data from five independent experiments
(Fig. 4) indicate that cross-linked wtAPP and BACE1 co-
patched with the raft protein PLAP and segregated from the
nonraft protein TfR del 5–41.
Antibody induced cross-linking increases association to
detergent-resistant membranes
Different proteins associate with rafts with different kinetics
and partition coefficients. Antibody-induced patching may
stabilize association of raft proteins with DRMs (Harder et
al., 1998; Janes et al., 1999). Thus, oligomerization, i.e., by
antibody cross-linking, can be used to monitor specific raft
lipid–protein interactions. Therefore, we investigated the as-
sociation of APP and BACE1 with DRMs under cross-link-
ing conditions. Association of a protein with DRMs is
shown by its insolubility in detergents such as Triton X-100
or CHAPS at 4C (Fiedler et al., 1993; Brown and London,
1997), which leads to flotation to low densities in sucrose or
OptiPrep gradients.
Initial experiments revealed that in N2a cells only a minor
amount (5%) of both APP and BACE1 were resistant to
extraction with 1% Triton X-100. However, when the cells
were extracted with 20 mM CHAPS a significantly higher
amount of BACE1 and APP floated to the low density
membrane fraction in a cholesterol-dependent manner (un-
published data). Therefore, we used CHAPS-extracted
membranes to examine the effect of antibody-induced
patching on DRM association. N2a cells were infected with
adenoviruses to express YFP-swAPP or BACE1A-CFP, met-
abolically labeled for 2 h with [35S]methionine, and chased
for 2 h in the absence of antibody or in the presence of anti-
FP (KG77) or anti-BACE1 (7523) antibodies, respectively.
Cells were then extracted with 20 mM CHAPS, and the de-
tergent extracts were subjected to OptiPrep step gradient
centrifugation. A significantly higher fraction of APP and
Figure 4. Quantification of copatching of PLAP, TfR del 5–41,
BACE1A-CFP, and YFP-wtAPP. Patches of different proteins were
scored into four groups: (1) copatching (80% overlap), (2) partial
copatching (30–80%, clearly overlapping patches), (3) random
distribution, and (4) segregation. The percentages of cells belonging
to each group are expressed as mean  SD (n  5).
Figure 5. Effect of antibody cross-linking on association of
BACE1A-CFP and YFP-swAPP to DRMs. 10 h after adenovirus
infection to express BACE1A-CFP or YFP-swAPP, the cells were labeled
for 2 h with [35S]methionine and chased for 2 h in the presence of
antibody KG77 (anti-FP) or antibody 7523 (anti-BACE1). The cells
were subsequently lysed in 20 mM CHAPS/TNE at 4C. (A) After
flotation in an OptiPrep step gradient, BACE1A-CFP and YFP-swAPP
were immunoprecipitated with antibody KG77 from the collected
fractions. (B) Quantification; antibody-induced patching significantly
increased the amount of APP (n  3) and of BACE1 (n  4) in the top
two fractions (DRM associated). The amount in the top two fractions
was correlated to the total amount of protein in all fractions.

The Journal of Cell Biology
A production depends on lipid rafts | Ehehalt et al. 117
BACE1 floated with DRMs after antibody-induced patch-
ing (Fig. 5). Quantification revealed that without cross-link-
ing 18.0  2.6% of APP (n  3) and 24.6  2.3% of
BACE1 (n  4) were found in the upper two fractions
(DRMs). Antibody cross-linking increased the DRM-associ-
ated fraction to 25.1  1.2% (n  3) and 32.3  0.7%
(n  4) of APP and BACE1, respectively. Thus, both APP
and BACE1 increased their detergent resistance upon cross-
linking, probably reflecting increased raft affinity caused by
oligomerization. Similar results have been obtained for other
raft proteins, which increase their raft association by forming
oligomers (Simons and Toomre, 2000; Cheng et al., 2001).
Cross-linking with antibodies increases A formation
If -cleavage were to take place in cholesterol/sphingolipid-
enriched microdomains, then antibody cross-linking should
not only increase the association of APP and BACE1 with
DRMs and induce their copatching at the surface of living
cells, but cross-linking should also increase A production. To
find out whether this is the case, we analyzed the effect of anti-
body cross-linking on A secretion. Cells were infected with
adenoviruses to express YFP-wtAPP and BACE-VSVG. They
were metabolically labeled for 40 min with [35S]-methionine
and chased for 2 h in the presence of antibodies KG77 (anti-
FP), 7523 (anti-BACE1), or both. Antibody cross-linking in-
creased A secretion significantly (Fig. 6, A and B).
We next examined the effect of cholesterol depletion on
antibody-induced A production. Cells were grown in the
presence of lovastatin as before and treated immediately be-
fore labeling for 5 min with 10 mM MCD. This procedure
depleted total cellular cholesterol by 50–60%. Antibody
cross-linking did not stimulate A secretion in cholesterol-
depleted cells (Fig. 6, C and D). Thus, under conditions
where rafts were disrupted increased amyloidogenic process-
ing of APP was no longer detectable.
Endocytosis is essential for -cleavage
Antibody cross-linking might not only lead to copatching of
raft components, it could also alter endocytosis of cross-
linked proteins. Previous studies suggest that endocytosis is
required for A generation (Koo and Squazzo, 1994; Perez
et al., 1999; Huse et al., 2000; Daugherty and Green,
2001). Therefore, we decided to inhibit endocytosis by tran-
siently expressing RN-tre or the dynamin II mutant K44A
in N2a cells. RN-tre is a Rab5-specific GTPase-activating
protein and inhibits clathrin-dependent endocytosis (Lan-
zetti et al., 2000). Dynamin is involved in fission of vesicles
from the plasma membrane. It was shown that expression of
the mutant K44A inhibits both clathrin-dependent and
some clathrin-independent endocytotic pathways (Damke et
al., 1994; Henley et al., 1998).
N2a cells were transiently transfected with equal amounts
of plasmids encoding for swAPP, RN-tre, or dynamin K44A
and labeled for 1 h with [35S]methionine. Immunoprecipita-
Figure 6. Effect of antibody cross-linking and cholesterol depletion
on A secretion. (A) Cells were infected with adenoviruses to
express YFP-wtAPP and BACE1-VSVG, metabolically labeled for 40
min with [35S]methionine, and chased for 2 h in the presence of the
indicated antibodies. YFP-wtAPP was cross-linked with anti-FP
antibody KG77, and BACE1 was cross-linked with antibody 7523.
(B) Quantification of the two independent experiments of A. The
ratio was arbitrarily set to 100% in cells not cross-linked with
antibodies. Secreted A was normalized to the total amount of APP
found in the cell lysate. (C) A secreted from cross-linked/cholesterol-
depleted cells. N2a cells were grown in the presence (depletion)
or absence (depletion) of lovastatin/mevalonate/lipid-deficient
FCS. 10 h after infection with adenoviruses to express YFP-wtAPP,
the cells were treated for 5 min with 10 mM MCD, labeled for 40
min with [35S]methionine, and chased for 2 h in the presence (Ab)
or absence (Ab) of anti-FP antibody KG77. (D) Quantification of
five independent experiments. The amount of secreted A was
normalized to the total amount of APP present in the cell lysate. The
ratio was arbitrarily set to 100% in cells neither cross-linked nor
cholesterol depleted.

The Journal of Cell Biology
118 The Journal of Cell Biology | Volume 160, Number 1, 2003
tions from cell lysates with antibody IP60 (raised against the
COOH terminus of APP) and from media with antibody
70JE (A) were performed (Fig. 7 A). APP biosynthesis
was unchanged after expression of RN-tre or dynamin
K44A; however, the COOH-terminal fragment generated
by -cleavage (CTF) and secretion of A were significantly
reduced. Expression of dynamin K44A inhibited A secre-
tion by 80–90% (Fig. 7 A). Remarkably, the membrane-
bound fragment generated by -cleavage (CTF) was only
slightly increased (correlated to total APP). Thus, under
our experimental conditions endocytosis was essential for
-cleavage to occur, whereas -cleavage was not appreciably
stimulated by inhibiting endocytosis.
Antibody-induced cross-linking stimulates formation
of A at the cell surface
If endocytosis is required for -cleavage to occur and -cleav-
age critically depends on lipid rafts, one could perhaps stim-
ulate the cleavage already at the cell surface by cross-linking
APP and BACE1-containing rafts and thus overcome the
block of -cleavage caused by inhibition of endocytosis. N2a
cells were transfected with constructs to express dynamin
K44A, YFP-swAPP, and BACE1A-VSVG, respectively,
and pulse–chase experiments were performed as before. We
blocked endocytosis and cross-linked APP and BACE1 by
applying the respective ectodomain antibodies to living cells.
Copatching of APP and BACE1 resulted in a significant in-
crease in A secretion under conditions of strict inhibition
of endocytosis by dynamin K44A (Fig. 7 B). Cross-linking
in cells expressing RN-tre revealed similar results (unpub-
lished data). To control whether antibody cross-linking in-
creased endocytosis after inhibiting internalization with
dynamin K44A, we performed a control experiment using
human transferrin receptor as our probe. Indeed cross-link-
ing did not increase internalization of the transferrin recep-
tor (Fig. 7 C), supporting our conclusion that we can stimu-
late A production at the surface by raft patching.
Discussion
The data presented here strengthen the evidence that both
APP and BACE1 partition into lipid rafts in cellular mem-
branes and that amyloidogenic processing seems to occur
raft associated. First of all, we could demonstrate that A
production was critically dependent on the integrity of lipid
rafts. Lipid rafts are cholesterol- and sphingolipid-enriched
microdomains within cellular membranes, and removal of
raft lipids from cells leads to disruption of raft functions (Si-
mons and Toomre, 2000). Consistent with previous results,
we could demonstrate that a decrease of cellular cholesterol
levels inhibited A generation in N2a cells. The involve-
ment of rafts in A production is further supported by pre-
vious work showing that cholesterol and GM1 both bind to
A and facilitate amyloid fibril formation (Yanagisawa and
Ihara, 1998; Ariga et al., 2001; Kakio et al., 2001). Like
Figure 7. Endocytosis is essential for the generation of A, and
antibody cross-linking overcomes the block caused by inhibition of
endocytosis. (A) Inhibition of endocytosis and APP processing. After
transient transfection with YFP-swAPP and RN-tre or dynamin K44A,
N2a cells were metabolically labeled for 1 h with [35S]methionine.
Immunoprecipitations from cell lysate (swAPP, CTF, CTF;
antibody IP60) and medium (A; antibody 70JE) show a decrease of
fragments generated by -cleavage (CTF and A) upon expression
of endocytosis inhibitors. (B) The effect of copatching of APP and
BACE1 on A secretion under conditions where endocytosis is
inhibited. Cells were transfected with YFP-swAPP/BACE1A-VSVG/
empty vector (inhibition) or YFP-swAPP/BACE1A-VSVG/dynamin
K44A (inhibition), labeled with [35S]methionine for 40 min, and
chased for 2 h in the presence (Ab) or absence (Ab) of antibodies
KG77 (anti-FP) and 7523 (anti-BACE1). Quantification of three
independent experiments showed a significant increase in A
generation upon copatching under both conditions. The values are
normalized to the total amount of APP present in the cell lysate. The
ratio has been arbitrarily set to 100% in nonpatched cells transfected
with YFP-swAPP/BACE1A VSVG/empty vector. (C) Effect of antibody
cross-linking on internalization of biotin transferrin under conditions
where endocytosis is inhibited by expression of dynamin K44A.
Cells were transfected with human transferrin receptor (TfR) and
dynK44A-GFP, incubated on ice with biotin transferrin, and chased
for 10 min at 37°C in the presence (+Ab) or absence (Ab) of anti–
human TfR antibodies. Biotin transferrin was quantified on an Origen
M8 analyzer in cells where remaining surface transferrin was removed
by acid wash (black columns) or controls (no acid wash; grey
columns). Cross-linking did not increase internalization of transferrin.

The Journal of Cell Biology
A production depends on lipid rafts | Ehehalt et al. 119
many other raft-associated proteins, BACE1 is also palmi-
toylated at three cysteine residues within its transmembrane/
cytosolic tail (Benjannet et al., 2001) and is mainly apically
sorted in epithelial cells (Capell et al., 2002).
Another important result in support of raft association
was that after antibody-induced cross-linking both APP and
BACE1 copatched on the surface of living cells with each
other and with PLAP, a GPI-anchored raft-associated pro-
tein. All three proteins segregated from patches containing
cross-linked transferrin receptor, which served as a nonraft
marker. We and others have previously used this assay to
monitor how proteins associate with rafts at the cell surface
(Harder et al., 1998; Janes et al., 1999; Prior et al., 2001).
Antibody cross-linking of raft surface antigens leads to the
formation of large clusters, which are easily observable in the
light microscope. Copatching is dependent on cholesterol,
since patching is inhibited by cholesterol removal.
Importantly, antibody cross-linking increased the DRM
association of both APP and BACE1, as was previously
shown for other raft-associated proteins (Harder et al.,
1998). Because only a small fraction of APP and BACE1 as-
sociated with DRMs, these proteins are probably found (at
steady state) in two membrane pools, one raft associated and
another localized outside of rafts. How partitioning between
these two pools is regulated is not clear. It has been reported
that both APP and BACE1 can dimerize and that ho-
modimerization of APP increases A production (unpub-
lished data; Scheuermann et al., 2001). Oligomerization of
raft components can lead to increased raft affinity. Many
surface receptors, such as Fc() receptors and T and B cell
receptors, dimerize or oligomerize after ligand binding, and
this has been shown to increase association with DRMs
(Janes et al., 2000; Langlet et al., 2000; Cheng et al., 2001).
Therefore, dimerization might be important for regulating
raft association of APP and BACE1.
Partitioning of APP and BACE1 into rafts alone seems
not to be sufficient to induce -cleavage. Cleavage normally
also depends on endocytosis. This was demonstrated by our
experiments designed to inhibit endocytosis. In two ap-
proaches, we either expressed RN-tre, a Rab5 GTPase-acti-
vating protein (Lanzetti et al., 2000), or the dynamin mu-
tant K44A (Damke et al., 1994). The former perturbs
clathrin-dependent endocytosis (Lanzetti et al., 2000) and
the latter both clathrin-dependent and some clathrin-inde-
pendent endocytic pathways (Damke et al., 1994; Henley
et al., 1998). The results were clear cut: A generation was
strongly inhibited, whereas APP was still  cleaved. The lat-
ter was expected from reports demonstrating that -cleav-
age occurs at the cell surface (Haass et al., 1992; Parvathy et
al., 1999). Previous work has reported that -cleavage may
happen already late in the secretory pathway, or after deliv-
ery to the cell surface, and during endocytosis (Koo and
Squazzo, 1994; Perez et al., 1999; Huse et al., 2000;
Daugherty and Green, 2001; Kamal et al., 2001). Inhibi-
tion of endocytosis by our approaches suggests that, at least
in N2a cells, most of this cleavage occurs after internaliza-
tion. Since cholesterol depletion is also known to decrease
the rate of endocytosis (Rodal et al., 1999), this is also likely
to contribute to the decreased -cleavage. However, the in-
hibitory effect on A production by inhibition of endocy-
tosis could be overcome by cross-linking surface APP and/
or BACE1 with antibodies.
To account for these results, we envisage that -cleavage
would normally not take place at the cell surface because
surface APP and BACE1 are most likely present in separate
rafts (Fig. 8). Rafts are small and highly dispersed at the cell
surface and are suggested to contain only a subset of 10–
30 protein molecules (Pralle et al., 2000). Therefore, the
likelihood is low that APP and BACE1 are in the same indi-
vidual raft. For -cleavage to occur rafts would have to be
clustered to get APP and BACE1 into the same raft plat-
form. Thus, we hypothesize that APP and BACE1 meet af-
ter endocytosis by clustering and coalescence of APP- or
BACE1-containing rafts within endosomes (Fig. 8 A). How
and where clustering is accomplished during internalization
from the plasma membrane is not known. However, raft
clustering can be artificially induced at the cell surface by
cross-linking with antibodies (Fig. 8 B). This could lead to
the increased -cleavage in clusters/patches containing both
APP and BACE1 that we had observed. Remarkably, we did
not detect a dramatic increase in -secretase processing of
APP after inhibition of endocytosis. We assume that this is
due to a continued raft association of a fraction of cell sur-
face APP, which would not be accessible to -cleavage. On
the other hand, cholesterol depletion would shift the parti-
tioning of APP from lipid rafts to the surrounding lipid bi-
layer and lead to the observed increase of -cleavage.
The inhibition of -cleavage by cholesterol depletion sug-
gests that BACE1 processing of APP critically depends on
the lipid raft environment. In living cells, BACE1 seems to
require intact rafts for activity, and BACE1 outside of rafts
appears to be inactive. Other such examples of raft-depen-
dent processes are known. The conformational change of the
cellular prion protein (PrPc) to its pathogenic scrapie form
critically depends on the integrity of rafts (Taraboulos et al.,
1995; Baron et al., 2002). Membrane vesicle transport
(Klopfenstein et al., 2002), many signal transduction path-
ways, like Ras and GDNF signaling (Roy et al., 1999; Tan-
sey et al., 2000), or T and B cell activation and allergic re-
sponse mechanisms are raft dependent (Janes et al., 1999;
Langlet et al., 2000; Cheng et al., 2001). However, clearly
more work will be necessary to demonstrate whether
-cleavage of APP is indeed directly dependent on the raft
lipid environment as postulated in the hypothetical scheme
in Fig. 8. Also, the mechanisms regulating the trafficking
and clustering of APP and BACE1 need to be identified.
Nevertheless, our data support a crucial role for lipid rafts in
APP processing and A generation. Compartmentalization
by lipid rafts seems to be important in regulating the access
of APP to - and -secretases.
Also the -secretase complex was shown to be raft associ-
ated (Li et al., 2000; Wahrle et al., 2002). Moreover, the
-cleaved COOH-terminal fragment, the substrate for
-cleavage, is found in DRMs and so is the product A (Lee
et al., 1998; Riddell et al., 2001). How and where -secre-
tase acts to cleave out A is not known. Interestingly,
Yanagisawa and coworkers have in a series of publications
demonstrated that cholesterol-dependent sequestration of
A promotes fibrillogenesis of soluble A and suggested
that A associated with rafts undergoes a conformational

The Journal of Cell Biology
120 The Journal of Cell Biology | Volume 160, Number 1, 2003
change, which promotes amyloid plaque formation (Yana-
gisawa et al., 1995; Mizuno et al., 1999; Kakio et al., 2001).
Thus, the stage is set for a molecular dissection of how cho-
lesterol and lipid rafts contribute to amyloid plaque forma-
tion in the pathogenesis of AD.
Materials and methods
Cells
Mouse neuroblastoma N2a cells were cultured at 37C in DME supple-
mented with 10% FCS, 100 U/ml penicillin, 100 g/ml streptomycin, and
2 mM L-glutamine (Invitrogen). Cells used for microscopy were grown on
11-mm diameter collagen-coated coverslips in complete medium without
phenol red.
Reagents and antibodies
MCD, mevalonate, and cycloheximide were from Sigma-Aldrich, and
lovastatin was from Calbiochem. The following antibodies were used
against APP: rabbit polyclonal antibody IP60 (Ehehalt et al., 2002) directed
against the very COOH terminus of APP, mouse monoclonal antibody
6E10 (Senetek) that detects APP and A, rabbit polyclonal antibody 70JE
(Ehehalt et al., 2002) against aa 1–11 of A, and the rabbit polyclonal anti-
body 5313 (Steiner et al., 1999) directed against the NH2-terminal part of
the APP ectodomain. Antibodies against FP were rabbit polyclonal anti-
body KG77 raised against recombinant GFP expressed in bacteria and
mouse monoclonal antibody 3E6 (Molecular Probes). Rabbit polyclonal
antibody 7523 (Capell et al., 2002) was against the NH2-terminal end of
BACE1. The anti–human transferrin receptor monoclonal antibody was
from Roche. Mouse monoclonal and rabbit polyclonal anti-PLAP antibod-
ies were from Dako.
Constructs and generation of recombinant adenoviruses
The BACE1A-YFP/CFP constructs were described previously (Ehehalt et al.,
2002); BACE1A containing a VSVG tag (BACE1A-VSVG) was constructed
as follows. BACE1A-YFP in pGEMT (Promega) was digested with AflII and
NotI to release the YFP moiety. The VSVG epitope (MYTDIEMNRLGK)
was then added using two complementary oligonucleotides to reconstitute
the AflII and NotI restriction sites. The oligonucleotides used were
5- TTAAGGG TATGTATACTGATATCGAAATGAATCGATTGGGTAAGT-
GAGC-3 and 5-GGCCGCTCACTTACCCAATCGATTCATTTCGATAT-
CAGTATACATACCC-3. BACE1A-VSVG was subsequently transferred as a
SalI-NotI fragment into the mammalian expression vector pShuttle–
cytomegalovirus (CMV) (He et al., 1998).
The YFP-APP construct was obtained as follows. APP was tagged in the
ectodomain by replacing the naturally occurring Kunitz-type protease in-
hibitor domain with the FP. This was necessary because insertion of the FP
at the very NH2 terminus of APP resulted in a misfolded chimeric protein,
incapable of leaving the ER. YFP was amplified by PCR from pEYFP-N1
Figure 8. Model for raft clustering and -cleavage
of APP. (A) Under normal (steady state) conditions,
two cellular pools of APP and presumably also of
BACE1 exist at the plasma membrane. One is associated
with rafts (black), and another is outside of rafts. Rafts
are small and highly dispersed at the cell surface.
Because they contain only a few proteins, APP and
BACE1 are mainly localized in separate rafts at the
plasma membrane. Endocytosis is necessary for APP-
and BACE1-containing rafts to cluster for consecutive
-cleavage. We assume that raft APP is cleaved by raft
BACE1. (B) If endocytic clustering is inhibited, e.g., by
the expression of dynamin K44A, the block can be
relieved by copatching with antibodies against BACE1
and APP. This leads to coalescence of rafts at the
plasma membrane and results in -cleavage. These
conditions are postulated to mimic the process
normally taking place after internalization.

The Journal of Cell Biology
A production depends on lipid rafts | Ehehalt et al. 121
(CLONTECH Laboratories, Inc.) to add an XcmI site at the 5 end, an XhoI
site at the 3 end, and spacers on either side of YFP. The oligonucleotides
used were 5-ACCACAGAGTCTGTGGAAGAGGTGGTTCGAGGCG-
GCGGATCTACCGTGGGCAGCGCACCGGTCGCCACCATG-3 (the XcmI
site is underlined and the bolded sequence matches plasmid pEYFP-N1)
and 5-TCTCGAGATACTTGTCAACGGCATCAGGGGTACTGGCTGC-
TGTTGTAFGAACTCCGCCGCCGGTAGATGCGGTCACGCTGCCGGTG-
CCCTTGTACAGCTCGTCCATG-3 (the XhoI site is underlined, and the
bolded sequence matches plasmid pEYFP-N1). This PCR product was li-
gated as an XcmI-XhoI fragment into pGEMT-APP695 digested with XcmI
and XhoI. YFP-APP was subsequently transferred as a SalI-NotI fragment
into the mammalian expression vector pShuttle-CMV. Adenoviruses were
prepared as described (He et al., 1998).
PLAP under control of the Rous sarcoma virus promoter and the TfR
del 5–41 expression construct in pCMV5 were described previously
(Harder et al., 1998). Dynamin II K44A in pCMV5 was provided by S.
Schmid (Scripps Research Institute, La Jolla, CA) (Damke et al., 1994;
Fish et al., 2000), and the cDNA of RN-tre (Lanzetti et al., 2000) was pro-
vided by M. Zerial (Max Planck Institute of Molecular Cell Biology and
Genetics).
Transfection, viral infection, and cholesterol depletion
24 h after seeding of N2a cells into 3.5-cm dishes, they were infected with
recombinant adenoviruses for 0.5 h at 37C in complete medium. After a
change of medium, the cells were incubated for 10–16 h at 37C and then
used for biochemical assays.
Transient transfections using calcium phosphate precipitation were per-
formed with 1–3 g of each expression plasmid as described by Chen and
Okayama (1988). For cholesterol depletion, the cells were grown for 1 d in
normal medium and then for 1 d in either DME supplemented with 2 mM
L-glutamine, 10% lipid-deficient FCS, 2 M lovastatin, and 0.25 mM me-
valonate, or in complete medium. They were then infected and grown for
a further 12–16 h in the same medium. The cells were treated for 5–30 min
with 10 mM MCD in methionine-free medium (labeling medium) and
thereafter metabolically labeled with 100 Ci/dish of [35S]methionine
(NEN). Depending on the experiment, the cells were chased for 0.5–2 h in
labeling medium containing an excess of methionine (150 g/ml) and 20
g/ml cycloheximide to inhibit protein synthesis.
Cholesterol determinations were done with the Amplex Red Cholesterol
Assay kit (Molecular Probes), which revealed a depletion of up to 80% of
total cellular cholesterol after treatment with a combination of lovastatin
and MCD.
Immunoprecipitation and quantification
After metabolic labeling, the cell culture medium was collected and cell
extracts were prepared using PBS containing 2% NP-40, 0.2% SDS, and
25 g/ml each of chymostatin, leupeptin, antipain, and pepstatin A. Immu-
noprecipitates were recovered on protein A–Sepharose CL4B beads (Amer-
sham Biosciences) and analyzed either on 10% polyacrylamide (Laemmli,
1970) or 10–20% Tris-Tricine (Invitrogen) gels. Individual bands were
quantified using the Fujifilm BAS 1800II image plate reader and Science
Lab 99 Image Gauge v3.3 software (Raytest Isotopenmessgeraete).
Immunofluorescence and antibody-induced patching
For immunofluorescence microscopy, the cells were fixed for 4 min at 8C
with 4% paraformaldehyde in PBS followed by an incubation in methanol
for 4 min at –20C. Fixed cells were incubated for 1 h at RT with a proper
dilution of antibodies in PBS/0.2% gelatin. After three washes with PBS/
0.2% gelatine, they were incubated with the respective secondary antibod-
ies in PBS/0.2% gelatin for 1 h at RT.
To cluster raft proteins, the respective antibodies were diluted in CO2-
independent medium (Invitrogen) containing 20 mg/ml BSA. Antibodies
against PLAP were diluted 1:35, the anti–human transferrin receptor mono-
clonal antibody 1:100, the polyclonal anti-FP (KG77) and polyclonal anti-
BACE1 (7523) antibodies 1:100, and the monoclonal anti-FP (3E6) 1:50.
The cells were incubated for 45 min with the respective combination of
antibodies at 10C, briefly washed, and further incubated for 45 min at
10C with mixed fluorescently labeled secondary antibodies. Cy3-labeled
secondary antibodies were diluted 1:500, and the Cy5-labeled secondary
antibodies were diluted 1:100. The cells were fixed as described above.
Fluorescent images were acquired on an Olympus BX61 microscope.
Quantification of copatching
Because of variation in expression levels of transiently expressed proteins
and differences in cell shape, quantification of copatching was done as de-
scribed before (Harder et al., 1998). Briefly, images of 10 randomly se-
lected cells expressing the two marker proteins were taken from one cover-
slip. An individual not involved in recording scored the images in a blind
fashion into four categories: (1) coclustering (80% overlap), (2) partial
coclustering (clearly overlapping patches; 30–80%), (3) random distribu-
tion, and (4) segregation. The percentage of cells in each class from five in-
dependent experiments were expressed as mean  SD.
Preparation of DRMs
Detergent extraction with CHAPS was performed as described (Fiedler et
al., 1993). N2a cells were grown in 3.5-cm dishes, infected with adenovi-
ruses to express BACE1A-YFP or YFP-APP, labeled for 2 h with [35S]me-
thionine, and chased for 2 h in labeling medium containing an excess of
methionine (150 g/ml), 20 g/ml cycloheximide, and for some samples
antibodies 7523 or KG77 (1:100). The cells were washed once with PBS
and scraped on ice into 300 l 25 mM Tris-HCl, pH 7.4, 150 mM NaCl,
and 3 mM EDTA (TNE) buffer containing 25 g/ml each of chymostatin,
leupeptin, antipain, and pepstatin A. The cells were homogenized through
a 25 G needle and centrifuged for 5 min at 3,000 rpm. The postnuclear su-
pernatant was subjected to extraction for 30 min at 4C in 20 mM CHAPS/
TNE. The extracts were adjusted to 40% OptiPrep (Nycomed) and overlaid
in a TLS 55 centrifugation tube with 1.4 ml of 30% OptiPrep/TNE and 200
l TNE. After centrifugation for 2 h at 55,000 rpm, five fractions were col-
lected from the top, and BACE1A-YFP or YFP-APP were recovered by im-
munoprecipitation with antibody KG77.
Uptake of biotin transferrin
N2a cells seeded on coverslips were transfected with either human trans-
ferrin receptor (TfR) and dynK44A-GFP or with TfR and pEGFP-N1 (CLON-
TECH Laboratories, Inc.). On the next day, the coverslips were washed by
dipping in ice-cold PBS and then incubated for 20 min on ice with human
biotin transferrin (50 g/ml; Sigma-Aldrich) in 30 l of low carbonate
MEM supplemented with 2 mg/ml BSA (medium/BSA). After another wash
with ice-cold PBS, the coverslips were incubated for 20 min on ice with a
1:40 dilution of the monoclonal anti–human TfR antibody (Roche) in 30 l
of medium/BSA or with medium/BSA alone. The cells were washed again
in ice-cold PBS and subsequently incubated for 10 min at 37C in 50 l of
medium/BSA to allow internalization of biotin transferrin. Biotin transferrin
that remained on the surface was removed by three washes (2 min each)
with ice-cold 0.5 M acetic acid and 0.5 M NaCl. Control cells used to de-
termine the total amount of biotin transferrin present were incubated with
ice-cold PBS for the same time.
The cells were lysed in 200 l of PBS, 2% NP-40, and 0.2% SDS. Part
of the lysate was incubated with streptavidin-coated magnetic beads (Dy-
nal), a sheep antitransferrin antibody (Scottish Antibody Production Unit),
and a rabbit anti–sheep secondary antibody (Dianova) coupled to a ruthe-
nium trisbipyridine chelate (IGEN International, Inc.). These reagents have
been described previously by Horiuchi et al. (1997). Biotin transferrin was
then quantified on an Origen M8 analyzer (IGEN International, Inc.).
We thank Marino Zerial, Michel Bagnat, and Mikael Simons for critically
reading the manuscript. We would like to thank Marta Miaczynska for
help with the biotin transferrin uptake experiments.
R. Ehehalt was supported by grant EH196/1-1 from the Deutsche For-
schungsgemeinschaft (DFG), P. Keller was supported by a grant from the
Max Planck Gesellschaft, and K. Simons and P. Keller were supported by
the DFG Schwerpunktprogramm SPP 1085 “Zellulaere Mechanismen der
Alzheimer Erkrankung.”
Submitted: 22 July 2002
Revised: 20 November 2002
Accepted: 22 November 2002
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