Oxysterol-binding protein-1 (OSBP1) modulates processing and trafficking of the amyloid precursor protein.

Abstract

:
Evidence from biochemical, epidemiological and genetic findings indicates that cholesterol levels are linked to amyloid-beta (Abeta) production and Alzheimer's disease (AD). Oxysterols, which are cholesterol-derived ligands of the liver X receptors (LXRs) and oxysterol binding proteins, strongly regulate the processing of amyloid precursor protein (APP). Although LXRs have been studied extensively, little is known about the biology of oxysterol binding proteins. Oxysterol-binding protein 1 (OSBP1) is a member of a family of sterol-binding proteins with roles in lipid metabolism, regulation of secretory vesicle generation and signal transduction, and it is thought that these proteins may act as sterol sensors to control a variety of sterol-dependent cellular processes.
We investigated whether OSBP1 was involved in regulating APP processing and found that overexpression of OSBP1 downregulated the amyloidogenic processing of APP, while OSBP1 knockdown had the opposite effect. In addition, we found that OSBP1 altered the trafficking of APP-Notch2 dimers by causing their accumulation in the Golgi, an effect that could be reversed by treating cells with OSBP1 ligand, 25-hydroxycholesterol.
These results suggest that OSBP1 could play a role in linking cholesterol metabolism with intracellular APP trafficking and Abeta production, and more importantly indicate that OSBP1 could provide an alternative target for Abeta-directed therapeutic.

Full text

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BioMed Central
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Molecular Neurodegeneration
Open Access
Research article
Oxysterol-binding protein-1 (OSBP1) modulates processing and
trafficking of the amyloid precursor protein
Celina V Zerbinatti†1, Joanna M Cordy†2, Ci-Di Chen†3, Maria Guillily2,
Sokreine Suon1, William J Ray1, Guy R Seabrook1, Carmela R Abraham3 and
Benjamin Wolozin*2
Address: 1Department of Alzheimer's Research, Merck & Co., Inc., West Point, PA, 19486, USA, 2Department of Pharmacology, Boston University
School of Medicine, Boston, MA, 02118, USA and 3Department of Biochemistry, Boston University School of Medicine, Boston, MA, 02118, USA
Email: Celina V Zerbinatti - celina_zerbinatti@merck.com; Joanna M Cordy - jocordy@bu.edu; Ci-Di Chen - cidichen@bu.edu;
Maria Guillily - mguillil@bu.edu; Sokreine Suon - sokreine_suon@merck.com; William J Ray - james_ray@merck.com;
Guy R Seabrook - guy_seabrook@merck.com; Carmela R Abraham - cabraham@bu.edu; Benjamin Wolozin* - bwolozin@bu.edu
* Corresponding author †Equal contributors
Background: Evidence from biochemical, epidemiological and genetic findings indicates that
cholesterol levels are linked to amyloid-β (Aβ) production and Alzheimer's disease (AD).
Oxysterols, which are cholesterol-derived ligands of the liver X receptors (LXRs) and oxysterol
binding proteins, strongly regulate the processing of amyloid precursor protein (APP). Although
LXRs have been studied extensively, little is known about the biology of oxysterol binding proteins.
Oxysterol-binding protein 1 (OSBP1) is a member of a family of sterol-binding proteins with roles
in lipid metabolism, regulation of secretory vesicle generation and signal transduction, and it is
thought that these proteins may act as sterol sensors to control a variety of sterol-dependent
cellular processes.
Results: We investigated whether OSBP1 was involved in regulating APP processing and found
that overexpression of OSBP1 downregulated the amyloidogenic processing of APP, while OSBP1
knockdown had the opposite effect. In addition, we found that OSBP1 altered the trafficking of
APP-Notch2 dimers by causing their accumulation in the Golgi, an effect that could be reversed by
treating cells with OSBP1 ligand, 25-hydroxycholesterol.
Conclusion: These results suggest that OSBP1 could play a role in linking cholesterol metabolism
with intracellular APP trafficking and Aβ production, and more importantly indicate that OSBP1
could provide an alternative target for Aβ-directed therapeutic.
Background
One of the major pathological hallmarks of AD is the dep-
osition of extracellular plaques composed predominantly
of the 4 kDa amyloid-β peptide (Aβ) [1,2]. This peptide is
derived from proteolytic processing of the amyloid pre-
cursor protein (APP) by two distinct enzymatic activities,
β- and γ-secretases. A third enzyme, named α-secretase, is
also able to cleave APP but within the Aβ region, preclud-
ing Aβ formation. Several different α-secretases have been
identified, including ADAM-9, -10 and ADAM-17/TACE
Published: 18 March 2008
Molecular Neurodegeneration 2008, 3:5doi:10.1186/1750-1326-3-5
Received: 26 October 2007
Accepted: 18 March 2008
This article is available from: http://www.molecularneurodegeneration.com/content/3/1/5
© 2008 Zerbinatti et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[3-5]. Cleavage of APP by α-secretase has both a constitu-
tive and a regulated component. The regulated compo-
nent is mediated by TACE and is stimulated by both
protein kinase C (PKC) [6] and extracellular signal-regu-
lated kinase (ERK) [7]. The β-cleavage of APP occurs via
the action of BACE1, a membrane-bound aspartyl pro-
tease primarily localized to the Golgi and endosomes
[8,9]. Following α- or β-cleavage, the C-terminal fragment
of APP (APP-CTFα/β) is subsequently cleaved by γ-secre-
tase, which exists mainly in the Golgi and endoplasmic
reticulum (ER) and is a complex made up of four proteins:
presenilin, nicastrin, Aph-1 and Pen2 [10]. γ-secretase also
has many other substrates, including the cell surface
receptor Notch and its homologue Notch2 [11,12].
APP processing by the secretases is modulated by many
factors including oxidative stress, ceramide, NSAIDs,
sphingolipids and cholesterol [13-16]. Increasing evi-
dence suggests that the metabolism of cholesterol, APP
and Aβ are strongly interdependent. Many studies show
that decreasing cellular cholesterol disrupts lipid rafts and
interferes with APP processing [17], and recent studies
have shown that Aβ, APP and presenilin can also regulate
production of cholesterol and sphingomyelin [18]. Oxys-
terols, products of cholesterol oxidation, have also been
shown to reduce Aβ secretion [19,20] and therefore could
provide a potential link between cellular cholesterol levels
and Aβ secretion.
Oxysterol-binding protein-1 (OSBP1) is the original
member of a family of lipid-binding proteins, which con-
sists of 12 members in humans. The other members of the
family are referred to as OSBP-related proteins (ORPs)
[21]. Most of the ORPs have a plextrin homology (PH)
domain at the N-terminal end, while all have a ligand
binding domain (LBD) at the C-terminal end [21]. In the
case of OSBP1, the LBD binds cholesterol and its catabolic
derivative, 25-hydroxycholesterol (25OH) [22,23]. Many
other ORPs also bind 25OH [24], while others have been
shown to bind different lipids including phosphoi-
nositides and phosphatidic acid [25,26]. Most ORPs local-
ize mainly to the cytosol, but association with membrane
organelles can occur and is regulated by lipid binding to
the LBD. Several ORPs also present alternatively spliced
variants [21].
The LBD of OSBP1 contains two separate binding sites,
one for cholesterol and one for 25OH, which enables
OSBP1 to regulate the extracellular ERK signalling path-
way [22]. When bound to cholesterol alone, OSBP1 exists
in the cytoplasm and ER, where it also binds two phos-
phatases, the serine/threonine phosphatase PP2A and a
PTPPBS class tyrosine phosphatase. Binding of these two
phosphatases reduces their availability in the cytoplasm/
ER and leads to increased activation of ERK [22]. Binding
of 25OH to OSBP1 or removal of cholesterol leads to
translocation to the Golgi apparatus, release of the phos-
phatases and reduced ERK activity.
The functions of OSBP1 and ORPs appear to be diverse,
with potential roles including control of lipid metabo-
lism, regulation of secretory vesicle generation and con-
trol of signaling pathways [27,28]. Recently it has been
suggested that ORPs' main role is to act as sterol sensors
and therefore control a wide variety of sterol-dependent
cellular processes [29]. Consistent with the high choles-
terol content of the CNS, OSBP1 and many ORPs are
highly expressed in several brain regions [30,31], and we
recently demonstrated that oxysterols can inhibit APP
processing [32]. In the present study we investigated
whether OSBP1 itself has a role in APP processing. We
found that cellular levels of OSBP1 modulated the amy-
loidogenic processing of APP and altered the subcellular
localization of APP-Notch2 heterodimers.
Results
Overexpression of OSBP1 inhibited APP processing and
sAPP secretion
To determine the effects of OSBP1 overexpression on APP
processing, H4 neuroglioma cells overexpressing wildtype
APP (H4-APP) were transiently transfected with myc-
tagged OSBP1. APP processing was evaluated by measur-
ing the steady-state levels of APP C-terminal fragments
(APP-CTFα/β) and soluble secreted APP species (sAPPα/
β) that are produced following α- and β-cleavage, respec-
tively. Overexpressing OSBP1 in H4-APP cells increased
APP-CTFα levels by ~65% (Figure 1A). Since the expres-
sion level of full-length APP was not significantly altered
by OSBP1 overexpression, increased APP-CTFα levels
were likely a result of OSBP1-mediated modulation of
APP processing. Interestingly, we found that OSBP1 over-
expression did not alter constitutive sAPPα secretion, and
it unexpectedly hampered the increase in secreted sAPPα
levels when the regulated α-cleavage pathway was
induced by PMA treatment (Figure 1B, middle panel). In
addition, OSBP1 overexpression was unable to further
alter the concomitant increase in APP-CTFα levels
induced by PMA treatment alone (Figure 1B, lower
panel). These results suggest that OSBP1 may modulate
APP processing via complex mechanisms, perhaps by
affecting both the non-amyloidogenic trafficking of APP
and the clearance of sAPPα.
Processing of APP via the amyloidogenic pathway was
found to be down-regulated by increased OSBP1 levels. In
contrast to the unpredictable changes observed within the
α-cleaved APP metabolites, all β-cleaved metabolites were
consistently altered. A 30% decrease in cellular levels of
APP-CTFβ and secreted sAPPβ were observed with OSBP1
overexpression in H4-APP cells (Figure 2A). The effect of

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OSBP1 overexpression on Aβ40 and Aβ42 secretion was
investigated in HEK cells stably expressing APPNFEV, a
mutated human APP exhibiting 100-fold enhanced
BACE1 cleavage rate relative to the APP wild type substrate
[33], therefore allowing for Aβ detection in the media by
ELISA. As expected, secreted Aβ40 and Aβ42 levels were
significantly reduced in HEK-APPNEFV cells expressing
OSBP1 (Figure 2B). Similar to the findings in H4-APP
cells, OSBP1 overexpression in HEK-APPNFEV cells did
not produce significant changes in the steady-state levels
of total APP nor led to changes in cell surface distribution
of APP as assessed by cell surface biotinylation experi-
ments (Figure 2C). Levels of sAPPα in the media meas-
ured by ELISA in HEK-APPNFEV were also unexpectedly
reduced by 24% in HEK-APPNEFV overexpressing OSBP1
(data not shown), suggesting again that OSBP1 overex-
pression impacts the non-amyloidogenic processing of
APP in a more complex manner.
OSBP1 overexpression increased the steady-state levels of α-secretase cleaved C-terminal fragment of APP (CTFα)
Figure 1
OSBP1 overexpression increased the steady-state levels of α-secretase cleaved C-terminal fragment of APP
(CTFα).A, H4 cells stably overexpressing APP (H4-APP) were transfected with OSBP1 cDNA as described in Methods. Cell
lysates from untransfected cells and those transiently overexpressing OSBP1 were immunoblotted with antibodies to the C-
terminus of APP (upper panel), c-myc (middle panel), which detects the myc-tagged OSBP1, and actin, used for loading control.
B, OSBP1 overexpression decreased PMA-regulated sAPPα secreted levels. Cells were treated with PMA as described in
Methods. Proteins from cell lysates and media were separated by SDS-PAGE and lysates were immunoblotted with anti-myc
antibody (upper panel). Media samples were analyzed for sAPPα using the 6E10 antibody (middle panel). Cell lysates were also
analyzed for CTFα using an antibody to the C-terminus of APP (lower panel). Densitometric analysis of Western blots is
shown on the right. *p < 0.05 and **p < 0.01 by Student's t test.

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Knockdown of OSBP1 increased β-site APP processing and
Aβ secretion
To investigate whether knockdown of OSBP1 would
result in opposite effects to that of OSBP1 overexpression,
three siRNA sequences were designed to specifically target
OSBP1. siRNA sequences targeting APP itself and BACE1
were used as positive controls, and media from trans-
fected cells were assayed for Aβ40 and Aβ42 by ELISA. As
expected, knockdown of BACE1 or APP markedly
decreased Aβ40 and Aβ42 secretion compared to that of
non-targeting siRNA control HEK-APPNFEV cells. Con-
versely to OSBP1 overexpression, knockdown of OSBP1
significantly increased Aβ40 and Aβ42 levels in the media
by 35% and 20%, respectively (Figure 3A). The effects of
OSBP1 knockdown on APP-CTFβ levels in HEK-APPNFEV
cells were consistent with changes in Aβ levels (Figure 3B),
i.e., APP-CTFβ was increased when compared to non-tar-
geting siRNA treated cells. While OSBP1 siRNA treatment
did not cause significant changes in full-length immature
APP (Figure 3B), a decrease in the mature, fully glyco-
sylated APP band was noticed and likely associated with
increased APP β-cleavage. However, APP-CTFα was not
decreased with OSBP1 knockdown, which is in concert
with our previous inconsistent results observed within α-
OSBP1 overexpression inhibited β-secretase cleaved APP-CTF (CTFβ) and decreased sAPPβ, Aβ40, and Aβ42 secreted levels, without affecting total or cell surface steady-state levels of full-length APP
Figure 2
OSBP1 overexpression inhibited β-secretase cleaved APP-CTF (CTFβ) and decreased sAPPβ, Aβ40, and Aβ42
secreted levels, without affecting total or cell surface steady-state levels of full-length APP.A, H4-APP cells were
transfected with OSBP1. After 48 h, medium was removed and replaced with fresh optiMEM. Following 3 h incubation, media
were collected and concentrated using Vivaspin columns. Proteins from lysates and media were separated by SDS-PAGE and
media samples were immunoblotted using the 192 wt antibody, against the β-cleaved epitope of APP (top panel); lysates were
blotted with anti c-myc antibody to confirm OSBP1 expression (middle panel) and with 6E10 antibody, which recognizes APP-
CTFβ (lower panel). Densitometric analysis is shown on the right. B, Secreted Aβ40 and Aβ42 levels in 24 h conditioned
media from mock transfected and OSBP1-overexpressing HEK-APPNFEV cells were measured by ELISA. C, HEK-APPNFEV
cells were transfected with OSBP1 cDNA as described in Methods. After 48 h incubation, cell monolayers were biotinylated
prior to lysis. Total and cell-surface biotinylated proteins were separated and analyzed by Western blotting using antibodies to
OSBP1, APP and actin as loading control. *p < 0.05 and **p < 0.01 by Student's t test.

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cleaved metabolites when OSBP1 was overexpressed. In
contrast to OSBP1, BACE1 siRNA led to increased levels of
mature APP, markedly decreased APP-CTFβ and increased
APP-CTFα levels (Figure 3B), which are all in agreement
with reduced APP β-cleavage. Western blotting of cell
lysates confirmed that transfection of OSBP1 siRNA pro-
duced effective knockdown of OSBP1 expression in HEK-
APPNFEV when compared with non-targeting siRNA
pool, or to siRNA pools targeted to APP or BACE1 (Figure
3C). Taken all together, our findings support the hypoth-
esis that OSBP1 overexpression regulates the processing of
APP, most likely by modulating its subcellular trafficking.
Knockdown of OSBP1 expression using siRNA increased secreted levels of Aβ40 and Aβ42 and the steady-state levels of CTFβ
Figure 3
Knockdown of OSBP1 expression using siRNA increased secreted levels of Aβ40 and Aβ42 and the steady-
state levels of CTFβ. Cells were transfected with the 3 siRNA sequences as described in Methods. Non-targeting siRNA
(NT) and siRNA against APP and BACE1 were used as controls. A, Media samples were assayed for Aβ40 and Aβ42 by ELISA.
B, Lysates from HEK-APPNFEV cells were separated by SDS-PAGE and immunoblotted with an antibody to the APP C-termi-
nus for detection of full length APP and APP-CTFs, and to actin as loading control. C, Lysates from HEK-APPNFEV cells and
immunoblotted with antibodies to OSBP1 and actin to confirm effective knockdown. *p < 0.01 and **p < 0.05 by ANOVA.

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Overexpressing OSBP1 modulated the distribution of APP
dimers
Since no apparent changes in cell surface distribution of
APP were detected (Figure 2C), we investigated the poten-
tial effects of OSBP1 overexpression on the subcellular
localization of APP. Co-expressing OSBP1 with an APP-
GFP construct indicated that OSBP1 did not affect the
gross intracellular distribution of APP, in the presence or
absence of OSBP1 ligand 25OH (Figure 4A). In addition,
25OH treatment did not alter the overall distribution of
APP-GFP expressed alone (Figure 4A), and no noticeable
changes in the subcellular localization of BACE1-GFP
were observed with OSBP1 co-expression (data not
shown). We also performed sucrose gradient fractionation
of APP-overexpressing cells transiently transfected with
OSBP1 or empty vector (Figure 4B and 4C). The majority
of OSBP1 coincided with the ER marker GRP78, detected
in fractions 11 and 12 (Figure 4B, right panel and 4C).
APP was found throughout fractions 4–12, and this distri-
Subcellular localization of total APP was not affected by OSBP1 overexpression
Figure 4
Subcellular localization of total APP was not affected by OSBP1 overexpression.A, H4 cells were transfected with
GFP-tagged APP with or without OSBP1 and then treated with 1 μM 25OH or vehicle for 24 h. Cells were examined under
40× magnification. B, H4 cells overexpressing APP were transfected with vector only (left panel) or OSBP1 (right panel); 48 h
later cells were homogenized and separated by sucrose gradient fractionation. Gradient fractions were analyzed by Western
blotting using antibodies to the APP C-terminus and to OSBP1. C, Fractions were also blotted with an antibody to the ER
marker GRP78.

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bution was not altered by overexpression of OSBP1 (Fig-
ure 4B).
We then postulated that OSBP1 might modulate a specific
intracellular pool of APP. To investigate this hypothesis
we used bimolecular fluorescence complementation
(BiFC), by which we have recently been able to identify
interactions between APP-APP homodimers and APP-
Notch heterodimers [34,35]. Using BiFC we demon-
strated that OSBP1 appeared to modulate the distribution
of APP-Notch2 heterodimers (Figure 5A). Constructs con-
sisting of Notch2 with a C-terminal deletion fused to the
N-terminus of YFP (N2C50YN) and APP fused to the C-
terminus of YFP (APPYC) were made as described previ-
ously [34]. H4 cells were transfected with APPYC plus
N2C50YN in the absence or presence of overexpressed
OSBP1 and then treated with 1 μM 25OH or vehicle for 24
h. APPYC plus N2C50YN only yielded BiFC fluorescence
showing diffuse staining with a modest amount in a com-
partment previously shown to be the Golgi apparatus
(Figure 5B, panel a). Cells co-transfected with OSBP1
showed almost exclusive fluorescence localization to the
Golgi apparatus (Figure 5B, panel b). Treatment of cells
overexpressing OSBP1 with 25OH shifted the distribution
of APP-N2C50 hetero-dimers to a distribution consistent
with ER and plasma membrane localization (Figure 5B,
panel c), whereas treatment with 25OH had no effect on
heterodimer localization in cells not overexpressing
OSBP1 (Figure 5B, panel d). Finally, we correlated the
subcellular distribution of OSBP1 to that of the APP-
Notch2 heterodimers. Cells were transfected with OSBP1
cDNA, treated with 1 μM 25OH or vehicle for 24 h, fixed,
and OSBP1 protein localization was determined by
immunofluorescence. OSBP1 labeling exhibited a pattern
consistent with cytoplasmic and ER distribution, shifting
to exclusively Golgi upon treatment with 25OH (Figure
5C) [36]. Thus, OSBP1 and APP-Notch2 heterodimers
localized to non-overlapping compartments and transi-
tion of OSBP1 from ER to Golgi induced by 25OH treat-
ment correlated with transition
heterodimers from Golgi to ER.
of APP-Notch2
Discussion
There is increasing evidence from biochemical, epidemio-
logical and genetic findings that cholesterol levels are
linked to Aβ production and AD; however, the mecha-
nisms involved are currently poorly understood. Our pre-
vious data demonstrate that the catabolic oxidation
products of cholesterol, termed oxysterols, potently
inhibit secretion of Aβ and sAPP [32]. Oxysterols are
known to bind two different targets, LXRs and ORPs.
From the three major naturally occurring oxysterols, 27-
hydroxycholesterol (27OH), 24(S)-hydroxycholesterol
(24OH) and 25-hydroxycholesterol (25OH), only 25OH
has been reported to bind ORPs. The brain specific 24OH
and the peripherally synthesized 27OH have been shown
to be primarily LXR ligands [37]. Several studies have
examined the effect of LXR agonists on APP processing
[19,38,39]. Some of these studies demonstrate that stim-
ulation of LXRs inhibits Aβ production in cell culture and
reduces Aβ deposition in vivo, although present data in the
literature are not entirely consistent [19,38,39]. We
hypothesized that 25OH binding to ORPs might be an
additional mechanism by which oxysterols modulate APP
processing in cells. In support of this hypothesis, the
CH25H gene encoding for cholesterol 25-hydroxylase and
located within the major AD-linkage region of chromo-
some 10, is ranked third in a list of genes with strongest
association with the disease [40]. 25OH-mediated activa-
tion of ORPs would likely result in a distinct modulation
of APP processing than that of LXRs, since ORPs regulate
cholesterol distribution and organelle trafficking, while
LXRs are transcription factors which primarily induce
genes involved in cholesterol efflux [41,42]. However, it
has been recently reported that ORP8 modulates expres-
sion levels of the cholesterol-efflux pump ATP-binding
cassette A1 (ABCA1) in macrophages [43], and therefore
it is possible that ORPs also contribute to the activity of
LXRs by modulating cellular levels of LXR-target genes.
The present study investigated whether OSBP1, the arche-
typal member of the family, could alone modulate APP
processing.
We found that overexpression of OSBP1 in cells stably
expressing APP led to downregulation of the amyloidog-
enic processing shown by a decrease in APP-CTFβ and
sAPPβ levels, as well as Aβ40 and Aβ42 secretion. As
expected, knockdown of OSBP1 had the opposite effect in
these metabolites. However, the effects of OSBP1 on the
non-amyloidogenic processing of APP appear to be more
complex. While OSBP1 overexpression increased and
OSBP1 knockdown decreased APP-CTFα, sAPPα levels in
the media were decreased in OSBP1 overexpressing cells.
One possible explanation for these findings is that OSBP1
overexpression also upregulates cellular binding sites for
sAPPα, and that this effect is even greater than its effect on
APP processing. sAPPa has neuroprotective and neuro-
trophic functions and it is several orders of magnitude
more potent than sAPPβ because it contains a heparin-
binding domain at the C-terminus, enabling binding to
several proteoglycans involved in cell adhesion, cell-cell,
or cell-matrix interactions, cell growth, and synaptic plas-
ticity [44,45]. Therefore, OSBP1 upregulation could be
beneficial by both decreasing the amyloidogenic process-
ing of APP and favoring the neuroprotective actions of
sAPPα.
OSBP1 and other members of the ORP family have previ-
ously been implicated in the control of intracellular traf-
ficking. For example, ORP2 has been shown to enhance

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Localization of APP-N2C50 heterodimers was modulated by OSBP1
Figure 5
Localization of APP-N2C50 heterodimers was modulated by OSBP1.A, Diagram showing the BiFC method. APP and
Notch constructs with a C-terminal deletion (N2C50) were fused with the amino or carboxy domains of YFP. Binding of target
constructs brings the two halves of YFP together and produces fluorescence. B, Cells were transfected with APPYC and
N2C50YN with or without OSBP1 and, after 24 h, treated with 1 μM 25OH or vehicle for another 24 h. Detection of the BiFC
fluorescence was carried out as described and cells were examined under 63× magnification. C, Panels (a) and (b) show fluo-
rescence images of cells expressing P450 2C2/CFP (ER CFP) and 1,4-galactosyltransferase/DsRed2 (Golgi DsRed) markers,
acquired 24 h after transfection; panels (c) and (d) show localization of OSBP1 in controls and cells treated for 24 h with 1 μM
25OH, respectively.

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endocytosis [41] and many other family members includ-
ing OSBP1 are known to be involved in vesicular transport
[29,46,47]. In addition, OSBP1 itself can traffic between
the ER/cytoplasm and the Golgi depending on whether it
is bound to cholesterol or 25OH. OSBP1 overexpression
did not appear to produce changes in APP subcellular
localization. However, by using bimolecular fluorescence
complementation to study the movement of APP dimers,
we found that OSBP1 overexpression caused sequestra-
tion of APP-Notch2 dimers in the Golgi. Addition of
25OH, which induces translocation of OSBP1 from the
ER/cytoplasm to the Golgi, caused APP-Notch2 dimers to
translocate from the Golgi to the ER. When OSBP1 was
not overexpressed, 25OH had no effect on the localiza-
tion of the APP-Notch2 dimers, perhaps because basal lev-
els of OSBP1 were not high enough to counteract the
effects of 25OH binding to other ORPs or LXRs. These
results suggest that OSBP1 might act to exclude a specific
population of APP heterodimers from organelles contain-
ing high levels of OSBP1, which is likely governed by
changes in subcellular cholesterol distribution.
It is of interest that while OSBP1 overexpression produced
no obvious changes in the localization of APP, an altered
localization of APP-Notch2 dimers was detected. Interac-
tion between APP and Notch2 was recently described and
shown to occur in the ER, Golgi and at the plasma mem-
brane [34,35]. However, the importance of this interac-
tion is currently unclear. In addition to binding to
Notch2, APP is known to bind to many other proteins and
therefore other types of APP heterodimers can potentially
exist. For example, SorL1, a ~250 kDa receptor that has
recently been shown to have genetic association with AD
[48], is a potential trafficking partner to APP. Overexpres-
sion of SorL1 has similar effects on APP processing to that
of OSBP1, including decreased in β-site cleavage and shift
in subcellular APP localization from ER and plasma mem-
brane to Golgi and early endosomes [49]. These effects
were shown to occur via direct interaction between SorL1
and APP. SorL1 has also been shown to interact with
BACE1 and it appears to compete with APP for interaction
with BACE1 in the Golgi [50]. It is possible that a similar
mechanism may exist whereby APP and OSBP1 both com-
pete for binding to SorL1 or other similar trafficking pro-
teins.
In this particular study our investigation was limited to
APP-Notch2 heterodimers; however, this is likely only a
small proportion of the total population of APP het-
erodimers, and it is possible that OSBP1 could also affect
the intracellular trafficking of other types of APP het-
erodimers. The homodimerization of APP has been previ-
ously suggested to positively regulate Aβ production
[51,52]. Our present results indicate that the localization
of APP-Notch2 dimers could also influence the processing
of APP, and therefore suggests that APP heterodimers may
play an important role in the regulation of APP trafficking
and Aβ generation.
Conclusion
The present findings provide the first evidence for an
involvement of oxysterol binding proteins in the regula-
tion of APP processing/trafficking. OSBP1 binds oxyster-
ols as well as cholesterol, and therefore this work also
provides further insight into the relationship between
cholesterol and APP metabolism. We hypothesize that
OSBP1 plays a pivotal role in APP metabolism when cel-
lular cholesterol levels are increased. In this model, an
increase in cellular cholesterol causes OSBP1 to shift to
cytoplasm/ER while APP-Notch dimers localize to the
Golgi, and Aβ production remains low. However, in cells
expressing low levels of OSBP1, an increase in cholesterol
levels could not be accommodated by such mechanism
allowing for Aβ levels to increase, as it has been shown in
previous studies examining the effects of cholesterol on
Aβ production [53,54]. Many questions regarding these
relationships still remain to be answered and more work
will be required to determine whether high levels of
OSBP1 do indeed protect against the detrimental effects of
increased cellular cholesterol. If this were the case, OSBP1
and/or other ORPs may prove to be promising targets in
the search for novel AD therapeutic.
Methods
Generation of constructs
The pcDNA3.1 plasmid containing myc-His tagged rabbit
OSBP1 was provided by Dr. R.G. Anderson (University of
Texas, TX). The plasmid encoding the ER marker (P450
2C2 CFP) was kindly provided by Dr. Elzbieta Skorupa
(UIUC, Urbana, IL) [55]. The plasmid encoding the Golgi
marker (81 N-terminal amino acids of human 1,4-galac-
tosyltransferase/DsRed2) was kindly provided by Dr.
Nikolaj Klocker (University of Freiburg, Germany) [56].
Human OSBP1 cDNA was obtained from Invitrogen and
cloned into 6.2 cLumio-DEST vector (Invitrogen,
Carlsbad, CA).
Cell culture
H4 human neuroglioma cells stably overexpressing APP
(H4-APP) were provided by Dr. T. Golde (Mayo Clinic,
Jacksonville, FL). Cells were cultured in OptiMEM con-
taining FBS (10%), non-essential amino acids, penicillin
(50 U/ml), streptomycin (50 μg/ml) and zeocin (100 μg/
ml). Transient transfections were carried out when cells
were 60–70% confluent using FuGENE (Roche, Indianap-
olis, IN) according to the manufacturer's instructions.
Treatment with 25OH (1 μM) was carried out for 24 h,
beginning 24 h after transient transfection. HEK293T sta-
bly overexpressing APPNFEV cells were generated as pre-
viously described [33]. APPNFEV encodes human APP

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Page 10 of 12
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isoform 1–695, modified at amino acid positions
595–598 by substitution of the amino acid sequence
KMDA for the NFEV sequence. Cells were maintained in
DMEM containing 10% FBS, penicillin (100 U/ml), strep-
tomycin (100 μg/ml) and puromycin (2 μg/ml). HEK-
APPNFEV cells (40–50% confluence) were transfected
with the OSBP1 SiRNA pool including the following
sequences: GAUAGAUCAGUCUGGCGAATT; CUUU-
GAGCUGGACCGAUUATT;
GUAATT; or non-targeting
(Dharmacon, Lafayette, CO) at a final concentration of
300 nM siRNA using Oligofectamine (Invitrogen,
Carlsbad, CA) according to the manufacturer's instruc-
tions. For overexpression experiments, the human OSBP1
cDNA was transfected into HEK-APPNFEV cells (70–80%
confluence) using Lipofectamine 2000 (Invitrogen,
Carlsbad, CA) according to the manufacturer's instruc-
tions. After overnight incubation, transfection mixture
was removed from each well and replaced with 100 μl of
DMEM containing 10% FBS. Fresh medium was condi-
tioned for 24 h before collection for analysis of APP
metabolites by ELISA. Cell viability was assessed at the
end of each experiment using the AlamarBlue reagent
(Biosource, Carlsbad, CA).
GAGAAUACUGGGAGU-
SiRNA control pool
Aβ ELISA
Cell-secreted Aβ levels were detected in the conditioned
media by sandwich ELISA. 50 μl of 20-fold diluted media
plus 50 μl of alkaline phosphatase (AP)-conjugated G210
(for Aβ40 detection), 50 μl of 2-fold diluted media plus
50 μl of 12F4 (for Aβ42 detection) or 50 μl of 100-fold
diluted media plus 50 μl of P21 (for sAPPα detection)
were incubated overnight on ELISA plates coated with
6E10. CPD-star (Tropix, Bedford, MA) was used as AP sub-
strate for detection using the Analyst AD microplate
reader (Molecular Devices, Sunnyvale, CA).
SDS-PAGE and Western blot analysis
H4-APP cells were lysed 48 h after transfection in 20 mM
Tris containing 1% Triton X-100 and a protease inhibitor
cocktail (Sigma, St. Louis, MO). Proteins were separated
by SDS-PAGE and then immunoblotted using antibodies
to the C-terminus of APP (Calbiochem, San Diego, CA), c-
myc (Sigma, St. Louis, MO) and actin (Sigma, St. Louis,
MO). Cells for sAPPα analysis were transfected with
OSBP1 and after 48 h, the medium was removed and cells
were treated with 1 μM phorbol 12-myristate 13-acetate
(PMA) for 30 min in fresh serum-free medium. The media
were collected and concentrated using a Vivaspin column
(Vivascience, Hannover, Germany). Media samples were
immunoblotted with the 6E10 antibody to detect sAPPα
(Signet Labs, Cambridge, MA), or the 192 wt antibody to
sAPPβ, which was provided by Dr. Peter Seubert (Elan
Pharmaceuticals, CA). Densitometric analysis of blots was
carried out using the Un-Scan-It automated digitizing sys-
tem. For evaluation of OSBP1, APP, and APP-CTFs expres-
sion levels in HEK-APPNFEV, cell monolayers were
washed twice with ice-cold PBS and lysed in PBS contain-
ing 1% Triton X-100, 1 mM PMSF and Complete protease
inhibitor cocktail (Roche, Indianapolis, IN) for 30 min on
ice. Cell lysates were cleared of cell debris by centrifuga-
tion at 14,000 rpm for 3 min at 4°C. Supernatants were
analyzed for protein assay and equal amounts of total cell
protein were separated by PAGE. Samples were separated
in 4–15% or 7.5% Tris-glycine gels for OSBP1 and APP or
16% Tris-tricine gels for APP-CTFs analysis. Proteins were
transferred to PVDF membranes and probed with anti-
OSBP1 goat polyclonal (ABCAM, Cambridge, MA), anti-
β-APP rabbit polyclonal (Zymed, Carlsbad, CA) or anti-
actin mouse monoclonal (Sigma, St. Louis, MO) antibod-
ies. Blots were developed using ECL Plus (Amersham Bio-
sciences, Piscataway, NJ) and scanned with the Typhoon
variable mode imager (Amersham Biosciences, Piscata-
way, NJ).
Cell surface biotinylation
Cell surface biotinylation experiments were performed
using the Cell Surface Protein Isolation kit from Pierce
(Pierce, Rockford, IL). Briefly, HEK-APPNFEV cells were
plated in 10 cm dishes and transfected with OSBP1 cDNA
as described above. 48 h following transfection, cell mon-
olayers were quickly rinsed twice with ice-cold PBS and
biotinylation of cell surface proteins was carried our
according to manufacturer instructions. After quenching
of unbound biotin, cells were scrapped and incubated on
ice for 30 min in 500 μl of lysis buffer. An aliquot (20 μl)
of clarified supernatant was reserved for running total cell
protein gels and the remaining sample was applied to
immobilized NeutrAvidin™ gel columns for separation of
biotin-labeled cell surface proteins eluted in 200 μl sam-
ple buffer. Western blots were carried out as above.
Sucrose gradient fractionation
H4-APP cells were homogenized 48 h after transfection in
10 mM Tris (pH 7.4) containing 0.25 M sucrose, 1 mM
MgAc2 and a protease inhibitor cocktail. The homogenate
was loaded on top of a step gradient consisting of 1 ml of
2 M sucrose, 4 ml of 1.3 M sucrose, 3.5 ml of 1.16 M
sucrose and 2 ml of 0.8 M sucrose (all in 10 mM Tris, pH
7.4 containing 1 mM MgAc2). The gradient was centri-
fuged at 100,000 g for 2.5 h. Twelve 1 ml fractions were
then collected from the top of the gradients and analyzed
by SDS-PAGE and Western blotting using antibodies to
the C-terminus of APP, OSBP1 or GRP78 (Santa Cruz Bio-
technology, Santa Cruz, CA).
Bimolecular fluorescence complementation (BiFC)
The preparation of the APPYC and N2C50YN constructs
was described previously [34]. Wild type H4 cells were
grown on a Lab-Tek II 8-chambered coverglass (Nalge

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Page 11 of 12
(page number not for citation purposes)
Nunc, Rochester, NY) and co-transfected with the expres-
sion vectors indicated in each experiment using FuGENE
reagent (Roche, Indianapolis, IN). 24 to 48 h after trans-
fection, cells were rinsed twice with PBS and resultant flu-
orescence was observed in living cells using a Zeiss
Axiovert 200 M microscope with YFP, CFP and rhodamine
filters. OSBP1 was detected using anti c-myc antibody, fol-
lowed by the Alexa Fluor 594 goat anti-mouse IgG anti-
body (Molecular Probes, Eugene, OR) under 63×
magnification.
Abbreviations
The abbreviations used are: ADAM, a disintegrin and met-
alloprotease; TACE, TNFα-converting enzyme; BACE1, β-
site APP-cleaving enzyme; NSAIDs, nonsteroidal anti-
inflammatory drugs; PMA, phorbol 12-myristate 13-ace-
tate.
Competing interests
CVZ, SS, WJR and GRS are full-time employees of Merck
and Co., Inc. and declare financial competing interests.
JC, CC, MG, CRA and BW have no competing interests.
Authors' contributions
CVZ and JMC designed and carried out APP processing
experiments, performed statistical analysis and draft the
manuscript; CC and MG performed the bimolecular fluo-
rescence experiments; SS carried out APP processing
experiments, WJR helped conceive of the study; GRS
helped conceive of the study and draft the manuscript;
CRA participated in design and analysis of bimolecular
fluorescence experiments; BW conceived of the study,
oversaw experiments, analyzed data and edited the man-
uscript. All authors read and approved of the manuscript.
Acknowledgements
This work was supported by a grant to BW from NIA (AG/NS17485).
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