The FASEB Journal • Research Communication

Inhibition of acyl-coenzyme A: cholesterol acyltransferase modulates amyloid precursor proteintrafficking in the early secretory pathway

Henri J. Huttunen,*,†,‡,1 Camilla Peach,*,†,‡ Raja Bhattacharyya,*,†,‡ Cory Barren,*,†,‡

Warren Pettingell,*,†,‡ Birgit Hutter-Paier,‡,§ Manfred Windisch,‡,§ Oksana Berezovska,†,‡

and Dora M. Kovacs*,†,‡,2

*Neurobiology of Disease Laboratory, Genetics and Aging Research Unit, †MassGeneral Institute forNeurodegenerative Disease, and ‡Department of Neurology, Massachusetts General Hospital,Harvard Medical School, Charlestown, MA, USA; and §JSW-Research Forschungslabor, Instituteof Experimental Pharmacology, Grambach/Graz, Austria

ABSTRACT Amyloid �-peptide (A�) has a centralrole in the pathogenesis of Alzheimer’s disease (AD).Cellular cholesterol homeostasis regulates endoproteo-lytic generation of A� from the amyloid precursorprotein (APP). Previous studies have identified acyl-coenzyme A: cholesterol acyltransferase (ACAT), anenzyme that regulates subcellular cholesterol distribu-tion, as a potential therapeutic target for AD. Inhibitionof ACAT activity decreases A� generation in cell- andanimal-based models of AD through an unknown mech-anism. Here we show that ACAT inhibition retains afraction of APP molecules in the early secretory path-way, limiting the availability of APP for secretase-mediated proteolytic processing. ACAT inhibitors de-layed the trafficking of immature APP molecules fromthe endoplasmic reticulum (ER) as shown by metaboliclabeling and live-cell imaging. This resulted in partialER retention of APP and enhanced ER-associated deg-radation of APP by the proteasome, without activationof the unfolded protein response pathway. The ratio ofmature APP to immature APP was reduced in brains ofmice treated with ACAT inhibitors, and strongly corre-lated with reduced brain APP-C99 and cerebrospinalfluid A� levels in individual animals. Our results iden-tify a novel ACAT-dependent mechanism that regulatessecretory trafficking of APP, likely contributing todecreased A� generation in vivo.—Huttunen, H. J.,Peach, C., Bhattacharyya, R., Barren, C., Pettingell, W.,Hutter-Paier, B., Windisch, M., Berezovska, O., Kovacs,D. M. Inhibition of acyl-coenzyme A: cholesterol acyltransferase modulates amyloid precursor protein traf-ficking in the early secretory pathway. FASEB J. 23,000–000 (2009). www.fasebj.org

Key Words: Alzheimer’s disease � cholesterol � endoplasmic re-ticulum � lipid � protein maturation

Amyloid precursor protein (APP) is a ubiquitouslyexpressed type 1 glycoprotein involved in cell adhesion,migration, and synaptogenesis (1–3). Even modest in-

creases in APP expression levels, due to APP geneduplication or promoter mutations, have profoundeffects on Alzheimer’s disease (AD) pathogenesis (4–7). Like most plasma membrane proteins, APP is syn-thesized and N-glycosylated in the endoplasmic reticu-lum (ER), from where the folded, immature APP movesto the Golgi complex for maturation (O-glycosylation)before transport to the cell surface. The two forms of APPholoprotein, immature N-glycosylated APP (APP-im) andmature APP (APP-m) carrying terminally modified N- andO-linked glycans, can be distinguished by their differentelectrophoretic mobilities (8) (see also (Fig. 1A). For A�biogenesis, APP is first cleaved by �-site APP cleavingenzyme 1 (BACE1; �-cleavage), producing a 99-aaC-terminal fragment (APP-C99). APP can also becleaved in the middle of the A� sequence (nonamyloi-dogenic �-cleavage by cell-surface metalloproteasesADAM10 and ADAM17), generating an 83-aa C-termi-nal fragment of APP (APP-C83). Both APP-C99 andAPP-83 can be cleaved by presenilin-dependent �-secre-tase, releasing the APP intracellular domain and eitherA� peptide (from C99) or p3 peptide (from C83) (9,10). Most A� generation is thought to occur in post-Golgi compartments of the cell, on the plasma mem-brane, or within the endocytic machinery (11–14).Consequently, altered trafficking of APP along thesecretory pathway directly affects the availability ofmature APP for A� generation (15–18).

The role of cholesterol as a risk factor for AD issupported by a long line of studies (reviewed in refs.19–21). Our previous studies have shown that inhibi-tion of ACAT activity potently reduces A� generation

1 Current address: Neuroscience Center, P.O. Box 56 (Vi-ikinkaari 4), FI-00014, University of Helsinki, Finland. E-mail:henri.huttunen@helsinki.fi

2 Correspondence: Neurobiology of Disease Laboratory,Massachusetts General Hospital, Harvard Medical School,114 16th Street, Charlestown, MA 02129, USA. E-mail:dora_kovacs@hms.harvard.edu

doi: 10.1096/fj.09-134999

10892-6638/09/0023-0001 © FASEB

The FASEB Journal article fj.09-134999. Published online July 28, 2009.

while reducing the levels of both �- and �-secretase-generated C-terminal fragments, APP-C83 and APP-C99(22–24). Notably, ACAT inhibitor CP-113,818 stronglyprotected from development of amyloid pathologycorrelating with improved cognitive capacity in a trans-genic mouse model of AD (22). Moreover, an ACATinhibitor suitable for human use, CI-1011, was able toreduce diffuse amyloid pathology also in the brains ofaged mice that displayed strong AD-like pathologybefore the treatment was started (unpublished results).Because ACAT inhibitors comprise a promising thera-peutic modality for lowering brain A� levels, under-standing the molecular events linking ACAT inhibition toreduced A� generation is essential for further clinicaldevelopment of ACAT inhibitors for AD. Furthermore,mechanistic insights into the complex relationship ofcholesterol, APP, and A� (19–21, 25) may eventuallyprovide entirely novel therapeutic approaches for treat-ment of AD.

Here we present evidence suggesting that inhibitionof ACAT activity targets nascent APP molecules in theearly secretory pathway. This leads to delayed matura-tion of newly synthesized APP, limiting the availabilityof APP for A� generation at or near the cell surface.Consistent with our cell-based data, we found correla-tions between reduced maturation of APP and �-cleav-age products of APP in the brains and CSF of ACAT-inhibitor-treated hAPP transgenic mice. We concludethat ACAT activity regulates APP trafficking in the earlysecretory pathway and consequently the availability ofAPP for A� generation.

MATERIALS AND METHODS

Cell culture and transfection

Parental CHO, H4, and mouse embryonic fibroblasts (MEFs)were grown in standard conditions (26). Parental CHO cellswere transfected with a construct encoding APP695-paGFP(18) using Effectene transfection reagent (Qiagen, Valencia,CA, USA) according to the manufacturer’s instructions. Sta-ble cell lines (CHO/APP751 and CHO/APP695-paGFP) wereselected and maintained in G418 (Calbiochem, San Diego,CA, USA).

Antibodies and Western blots

The following antibodies were used: APP (A8717, C-terminal)and �-tubulin were from Sigma (St Louis, MO, USA); V5 fromInvitrogen (Carlsbad, CA, USA); BiP/GRP78, transferrin re-ceptor, and CHOP/GADD153 from Affinity BioReagents(Golden, CO, USA); Nicastrin and GAPDH from Chemicon/Millipore (Temecula, CA, USA); and GM130 from BD Bio-sciences (San Jose, CA, USA). Cell extraction and Westernblotting was performed as described previously (26). Bandintensities on Western blot images were quantitated by usingQuantity One software (Bio-Rad, Hercules, CA, USA) andnormalized as indicated in figure legends.

Pulse-chase and cycloheximide (CHX) block assay

Semiconfluent cells treated with 10 �M CP-113,818 for 4 d on100-mm plates were first preincubated in methionine/cys-

teine-free medium for 1 h. Then 100 �Ci of [35S]-methio-nine/cysteine (MP Biomedicals, Solon, OH, USA) was addedper plate for 15 min (pulse). Cells were incubated in thepresence of excess amounts of cold methionine/cysteine (MPBiomedicals) and harvested at 40-min intervals (chase). Thecells were then washed with cold PBS and lysed in Triton-Nonidet P-40 buffer. For immunoprecipitation, 250–500 �gof total protein was immunoprecipitated with excess amountsof either APP (A8717) or transferrin receptor antibodies.Immunocomplexes were captured with protein G agarosebeads (Pierce, Rockford, IL, USA), washed 4 times with theextraction buffer, and heated for 10 min at 70°C in 1� LDSgel-loading buffer (Invitrogen) containing �-mercaptoetha-nol. Next, 4–12% gradient Bis-Tris gels (Novex/Invitrogen)used to resolve the samples were fixed, dried, and exposedto a phosphorimaging screen (Bio-Rad). Images were readand quantitated using a Personal Molecular Imager FX andQuantity One software (Bio-Rad).

For CHX block assay, CHO/APP cells were pretreated for4 d with 10 �M CP-113,818. At the end of the treatment, 10�g/ml CHX was added to block protein synthesis and allowdegradation of APP in the cells (APP t1/2 is �60 min in mostcells). After 6 h, CHX was removed to allow synthesis of newproteins. CP-113,818 or vehicle was present at all times. Cellswere harvested at 15-min intervals to analyze APP content.Immature and mature forms of APP were detected in Westernblots with A8717 antibody (Sigma).

Subcellular fractionation and retrotranslocation assay

Postnuclear supernatants (1500 g) of parental CHO cellstreated with 10 �M CP-113,818 for 4 d were fractionated on a7.5–30% continuous iodixanol gradients (OptiPrep; Axis-Shield, Norton, MA, USA) as described previously (24, 26).

Retrotranslocation of proteins to the cytosol was analyzedas described previously (26, 27). Where indicated, cells werepretreated with 1 �M epoxomicin (Biomol, Plymouth Meet-ing, PA, USA) for 5 h.

Live-cell imaging

Stably transfected CHO/APP-paGFP cells plated on 35-mmglass-bottom dishes (MatTek Cultureware; Mat Tek Corp.,Ashland, MA, USA) were pretreated with 10 �M CP-113,818or vehicle for 3 d. On the third day, the cells were transfectedwith a plasmid encoding DsRed2-ER (Clontech, Palo Alto,CA, USA), a red fluorescent protein DsRed2 fused to a signalpeptide at the N terminus and an ER retention signal KDELat the C-terminus. The cells were incubated in the presence ofCP-113,818 or vehicle for an additional 20 h. Before imaging,cells were washed once and transferred to Opti-MEM mediumlacking Phenol Red (Life Technologies/Invitrogen) withCP-113,818 or vehicle. Live-cell imaging studies were per-formed in a heated 37°C, 5% CO2 live-cell chamber attachedto a Zeiss LSM510 inverted confocal microscope equippedwith a krypton-argon laser (488 nm, 514 nm, and 543 nmlines) for confocal microscopy (Carl Zeiss, Oberkochen,Germany), and a 720- to 950-nm tunable, pulsed Ti:sapphire laser (Chameleon; Coherent Inc., Santa Clara,CA, USA) for 2-photon live-cell imaging. ER within trans-fected cells was identified by DsRed2-ER signal, and se-lected ER regions were photoactivated by a 750-nm laserpulse (5% transmission) for 5 s. Stacks of z sections ofpaGFP (excitation 488 nm, emission 500 –550 nm BP) andDsRed2 (excitation 543 nm, emission 560 nm LP) weresequentially acquired before photoactivation and at 2-minintervals after photoactivation for up to 20 min. Projectionsof z stacks were analyzed for mean fluorescence intensity

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within the selected, photoactivated regions using the his-togram and analysis tools of Adobe Photoshop CS (AdobeSystems, San Jose, CA, USA). Signal in the paGFP channelbefore photoactivation was used as a background andsubtracted from final values. Sixteen cells for both CP-113,818 and vehicle groups were imaged and quantitatedin 4 independent experiments.

Mice, treatments, and tissue sampling

hAPP transgenic mice overexpress human APP751 with theLondon (V717I) and Swedish (K670M/N671L) mutationsunder the regulatory control of the neuron-specific murineThy-1 promoter (mThy-1-hAPP751; heterozygous with respectto the transgene, on a C57BL/6 F3 background) (28). Micewere handled and treated as described previously (ref. 22 andunpublished results). CSF was obtained from anesthesizedmice during and after the treatment. Animals were sacrificedon d 56 of treatment. Brains were collected and divided alongthe sagittal plane, and hemispheres were frozen in liquidnitrogen.

A� determinations

CSF A�1-40 and A�1-42 were analyzed using commerciallyavailable ELISA kits (Genetics Company, Zurich, Switzer-land). Total CSF A� values (the sum of A�1-40 and A�1-42)were used in correlation analysis.

Statistical analysis

Statistical analyses were performed using Student’s t test. Forcorrelation analysis, Pearson coefficients were calculated. Allstatistical analyses were performed using Microsoft Excel(Microsoft, Redmond, WA, USA) and GraphPad Prism soft-ware (GraphPad, San Diego, CA, USA). Significance wasplaced at P � 0.05.

RESULTS

Effects of ACAT inhibition on maturation of APPholoprotein

To begin characterization of the molecular mecha-nisms by which ACAT inhibition modulates APP metab-olism, we have treated various cell lines expressinghuman APP with two structurally different ACAT inhib-itors, CP-113,818 and CI-1011 (29). Cells were treatedwith micromolar concentrations of ACAT inhibitors forup to 4 d to allow subcellular cholesterol distribution toreach an equilibrium (24). As shown in Fig. 1A, treat-ment of CHO/APP cells with the ACAT inhibitorCI-1011 dose-dependently reduced the steady-statelevel of mature APP (APP-m) while reducing the levelsof APP-C99 and APP-C83. Because the immature APP(APP-im) levels were affected to a much lesser extent,the ratio of APP-m/APP-im was reduced by up to 53%in this APP overexpressing cell line. We also testednaive cell lines to study the effect of ACAT inhibitionon proteolytic processing of endogenous APP. In hu-man H4 neuroglioma cells and MEFs, similar effectswere observed, suggesting that ACAT inhibitors modu-late the maturation process of both endogenous andoverexpressed APP holoprotein in various cell types(Fig. 1B).

APP holoprotein has a short turnover rate in mostcell types (t1/2�60 min) (8). Next, we used a CHXblock-release assay to study the effect of ACAT inhib-itor CP-113,818 on APP maturation kinetics. First, wetreated CHO/APP cells with CP-113,818 for 4 d, andfor the last 6 h with CHX to block the synthesis ofnew proteins, allowing nearly complete degradationof short-lived proteins such as APP. After 6 h, CHXwas removed, and maturation of APP was followed at15-min intervals for up to 60 min in the presence of

Figure 1. ACAT inhibition slows maturation of APP holoprotein. A) CHO cells expressing humanAPP751 (CHO/APP) were treated with increasing concentrations of ACAT inhibitor CI-1011 for4 d. Cell extracts were resolved on SDS-PAGE gel and probed with C-terminal APP antibodies inWestern blots to detect APP holoprotein and C-terminal fragments (APP-CTF). Immature(APP-im) and mature (APP-m) forms of APP holoprotein were quantitated. Graph shows adose-dependent decrease of the APP-m/APP-im ratio in CI-1011-treated cells. B) Parental humanH4 neuroglioma and mouse embryonic fibroblasts (MEFs) were treated with ACAT inhibitorCP-113,818 for 4 d. Endogenous APP holoprotein and APP-CTFs were detected as in A. The part

of the Western blot containing APP C-terminal fragments was overexposed. C) Maturation of newly synthesized APPholoprotein was analyzed in a cycloheximide (CHX) block/release assay. CHO/APP cells were pretreated with CP-113,818for 4 d before addition of 10 �g/ml CHX to block protein synthesis. After 6 h, CHX block was released, and maturationof APP holoprotein was followed at 15-min intervals. APP-m level at later time points (45 and 60 min) is decreased ascompared to vehicle-treated control cells at the same time points. D) Quantitation of APP maturation in 3 independentrounds of the CHX block assay in C. APP-m values were normalized to GAPDH values.

3ACAT INHIBITION MODULATES TRAFFICKING OF APP

vehicle or CP-113,818. As shown in Fig. 1C, appear-ance of mature APP is delayed in CP-113,818-treatedcells (e.g., compare APP-m levels between vehicle-and CP-113,818-treated cells at 45- and 60-min timepoints in Fig. 1C). Quantitation of these data showedthat at 60 min after CHX removal, there is 30% (n�3,P�0.0103) less mature APP present in ACAT inhibi-tor-treated cells. These data suggest that ACAT inhi-bition may target newly synthesized APP moleculesearly in the secretory pathway, likely before they getaccess to the medial-Golgi complex, where enzymesresponsible for O-glycosylation are located (30). Thisis consistent with the observed reduction of both �-and �-cleavage products in ACAT-inhibitor treatedcells, because most �-secretase cleavage occurs in thetrans-Golgi network and plasma membrane, whereas�-secretase cleavage occurs mostly in the endosomes(31–35).

ACAT inhibition slows trafficking of APP in the earlysecretory pathway

To characterize the effects of ACAT inhibition on earlysecretory pathway trafficking of APP in more detail, weused metabolic labeling and live-cell imaging. CHO/APP cells treated with CP-113,818 were pulse-labeled with[35S]-methionine/cysteine for 15 min, then chased at40-min intervals for up to 160 min. Labeled APP wasthen immunoprecipitated from cell lysates and de-tected by fluorography. In vehicle-treated cells, thelevel of mature APP was highest at 40 min, whereas inCP-113,818-treated cells the appearance of APP-m wasdelayed, peaking at 80 min (Fig. 2A). The total amountof APP-im converted to APP-m throughout the 160-minchase period was also reduced by CP-113,818 treat-ment, consistent with decreased steady-state levels ofAPP-m (Fig. 1). Notably, when transferrin receptor

(TfR), a cell surface protein with a single transmem-brane domain, was isolated in a secondary round ofimmunoprecipitation, CP-113,818 had little effect onTfR levels or maturation (Fig. 2A). The effect on APPmaturation is quantitated in a plot showing the rate andamount of APP maturation in vehicle- and CP-113,818-treated cells as percentage change of 0-min chasecontrol samples (Fig. 2B). Interestingly, the delayedand decreased maturation of APP was accompanied bystabilization of APP-im, suggesting retention of APP-imin the early secretory pathway (Fig. 2A). Indeed, deter-mination of the half-life of APP-im in vehicle- andCP-113,818-treated cells by logarithmic fitting showedthat ACAT inhibition prolonged t1/2 of APP-im from 44to 77 min (Fig. 2C).

The prolonged half-life of immature APP and thedelayed appearance of mature APP in ACAT inhibitor-treated cells suggests that APP may be retained in theearly compartments of the secretory pathway. Next, weinvestigated the subcellular distribution of APP usingOptiPrep fractionation. Naive CHO cells were treatedwith CP-113,818 for 4 d and then subjected fraction-ation in 7.5–30% OptiPrep gradients. Mature APPcodistributed with GM130 to fractions 2–4, suggestingthat most of the Golgi-complex proteins are found inthese fractions (Fig. 3A). Immature APP was localizedto fractions 5–10 together with GRP78/BiP, a typical ERprotein. CP-113,818 had no effect on the distribution ofmature APP (Fig. 3A, B). On the contrary, the distribu-tion of immature APP was significantly altered in CP-113,818-treated cells (Fig. 3A, B). In particular, fraction5 accumulated a high amount of immature APP. Inter-estingly, the GRP78/BiP-containing ER membranesfrom CP-113,818-treated cells distributed in a morecompact manner across the gradient and had a lighterbuoyant density as compared to control cells (Fig. 3A).These data suggest that ACAT inhibition specifically

Figure 2. APP trafficking is altered in the early secretory pathway by ACAT inhibition. A) APP maturation was analyzed bymetabolic labeling in CHO/APP cells. Cells were pretreated with CP-113,818 or vehicle for 4 d, followed by a 15-min pulselabeling with [35S]-methionine/cysteine. Cells were chased at 40-min intervals for up to 160 min in the presence ofCP-113,818 or vehicle. Equal amounts of total cell lysates were immunoprecipitated with APP C-terminal antibodies(A8717). SDS-PAGE gels were exposed to phosphoimaging screen for visualization and quantitation. As a control, a secondround of immunoprecipitation from unbound cell lysates was performed with transferrin receptor antibodies. B) Kineticsof APP maturation is altered by CP-113,818 treatment. Graph shows the amount of labeled mature APP (APP-m) atdifferent time points relative to 0-min time point from 3 independent metabolic labeling experiments. CP-113,818 delaysand decreases maturation of APP as compared to vehicle-treated cells. C) Half-life (t1/2) of immature APP (APP-im) wasdetermined from 5 independent metabolic labeling experiments similar to Fig. 1A. Stabilization of APP-im coincides withdelayed maturation of APP holoprotein in cells treated with CP-113,818.

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targets the immature APP molecules in the early secre-tory pathway.

To analyze the effect of ACAT inhibition on thedynamics of early secretory pathway trafficking of APP,we used a live-cell-imaging technique utilizing a photo-activatable variant of green fluorescent protein (paGFP)(36). CHO cells stably expressing a photoactivatableAPP-paGFP fusion protein (18) were first treated withCP-113,818 for 4 d. Twenty hours before imaging, thecells were transiently transfected with an ER marker,DsRed2-ER, a constitutively fluorescent protein local-ized to the ER. Using the DsRed2-ER signal, ER waslocalized within the cells, followed by photoactivationof selected areas of the ER. Trafficking of photoacti-vated APP-paGFP out of the ER was then followed forup to 20 min (Fig. 4A). Because the activated state ofpaGFP remains stable for days (36), reduced paGFPfluorescence within the activation area directly reflectstrafficking of APP-paGFP out of this area. In vehicle-treated cells, the overall signal from photoactivatedAPP-paGFP within the activation area was reduced by46% (n�16, P�0.0001 vs. 0 min) after 20 min. In

contrast, the APP-paGFP signal was reduced by only24% (n�16, P�0.0001 vs. 0 min; P�0.0001 vs. vehicleat 20 min) in cells treated with CP-113,818 (Fig. 4A, B).Intracellular retention half-times can be used to de-scribe kinetic aspects of protein trafficking (30). Weused normalized fluorescence values to calculate ERretention half-time for APP-paGFP. In vehicle-treatedcells, APP-paGFP exited the ER with a retention time of24 min, whereas in CP-113,818-treated cells, the ERretention time was significantly longer, 55 min (Fig.4C). The change caused by CP-113,818 treatment iscomparable to the turnover rate of APP-im determinedby metabolic labeling (Fig. 2C). These data also suggestthat, under normal conditions, roughly half (55%) ofthe time (24 min, Fig. 4C) it takes to convert a newlysynthesized, immature APP molecule to a mature APPmolecule (44 min, Fig. 2C) is spent in the ER. In compar-ison, CP-113,818 treatment increases the time APP spendsin the ER to �71% of the total half-life of APP-im. Thetime between ER exit and conversion of APP-im to APP-mwas not significantly different in vehicle- vs. CP-113,818-treated cells (19 vs. 22 min). Thus, together with the

Figure 3. Effect of ACAT inhibition of the distribution of APP in OptiPrep gradients. A) Naive CHO cells were treated for 4 dwith 10 �M CP-113,818. Postnuclear homogenates were fractionated in 7.5–30% OptiPrep gradients. Fractions were analyzed forAPP content as well as for Golgi marker GM130 and ER marker GRP78/BiP. B) Amount of APP species per fraction wasquantitated from optical densities of the APP Western blots and is expressed as percentage of total activity across the gradientfractions.

Figure 4. ACAT inhibition slows ER exit of APP in living cells. A) CHO cells expressing photoactivatable APP-paGFP fusionprotein and DsRed2-ER (fluorescent ER marker protein) were treated with CP-113,818 for 4 d. APP-paGFP was activated by ashort laser pulse in selected regions of the ER (marked by white boxes). Reduction of fluorescent signal due to exit ofAPP-paGFP molecules from the ER was recorded at 2-min intervals. Representative images before photoactivation and at 0, 10,and 20 min after photoactivation are shown. B) Quantitation of normalized fluorescence intensity in the photoactivated areasat indicated time points from 4 independent experiments (n�16). Green solid line, vehicle-treated cells; orange striped line,CP-113,818 cells. Values represent means se. C) ER retention half-time for APP-paGFP was determined from the live-cellimaging data in B. APP was transported out of ER with a longer retention time in cells treated with CP-113,818.

5ACAT INHIBITION MODULATES TRAFFICKING OF APP

metabolic labeling data, these results suggest that inhi-bition of ACAT activity in cells promotes retention ofAPP in the ER.

ACAT inhibition enhances retrotranslocation of APPwithout inducing ER stress

Several studies have suggested that the ubiquitin-pro-teasome system (UPS) is involved in the turnover ofAPP holoprotein, especially in the ER-associated degra-dation pathway of the nascent, immature APP mole-cules (26, 37). Cytosolic chaperones Hsc73 and theC-terminal Hsp70 interacting protein (CHIP) regulateproteasomal degradation of APP holoprotein (38, 39).Also, a signaling adaptor protein known as MOCA, alsoknown as DOCK3, reduces A� generation and APPmaturation by directing nascent APP molecules toproteasomal degradation (16). Thus, it is possible thatthe ER-associated degradation pathway (ERAD; re-viewed in ref. 40) could participate in disposal ofER-retained APP molecules in ACAT inhibitor-treatedcells.

To examine the accumulation of APP holoprotein inthe cytosol, we used a previously described retrotrans-location assay (26, 27). In this assay, cells are treatedwith proteasome inhibitor, such as epoxomicin, toallow accumulation of UPS substrates, followed byisolation of cytosolic fraction from cells semipermeabi-lized with 0.04% digitonin. To test whether ER reten-tion of APP is coupled to retrotranslocation and deg-radation by the cytosolic UPS, we used CHO cells

expressing an APP construct carrying a dilysine ERretention signal at the C terminus (APP751-V5-KKAA).C-terminal dilysine motif has been previously shown toeffectively retain APP in pre-Golgi compartments, re-sulting in reduced A� secretion (41, 42). As shown inFig. 5A, ER retention of APP751-V5-KKAA results inenhanced retrotranslocation to the cytosol as com-pared to cells expressing wild-type APP751-V5. Thus, ERretention of APP is coupled to its dislocation from theER membrane to the cytosol followed by degradationby the proteasome.

Next, we treated CHO cells and B104 neuroblastomacells expressing wild-type human APP with CP-113,818and, for the last 5 h of the treatment, with epoxomicin.CP-113,818 treatment increased the amount of cytoso-lic APP forms in both cell lines in a proteasomeinhibitor-dependent manner (Fig. 5B). We have previ-ously reported the presence of two forms of APP in thecytosol of proteasome inhibitor-treated cells: APP-imand an N-terminally truncated form of APP-im (26).Both forms seem to be increased in CP-113,818-treatedCHO/APP and B104/APP cells. To confirm that theAPP species seen in the cytosols of proteasome inhibi-tor-treated cells are newly synthesized, we combinedmetabolic labeling with the retrotranslocation assay.CHO/APP cells were treated with for 4 d with CP-113,818, and for the last 5 h, 1 �M epoxomicin wasadded. The cells were labeled with [35S]-methionine/cysteine for 15 min, then chased for 60 min (CP-113,818 and epoxomicin remained present throughoutthese steps). After isolation of the cytosolic fractions

Figure 5. ACAT inhibition enhances ERAD of APP but does not induce ERstress. A) CHO cells expressing either wild-type APP751–V5 or an ER-retention signal containing APP751-V5-KKAA were treated for 4 d with 10�M CP-113,818 or vehicle. For the last 5 h, cells were treated with theproteasome inhibitor epoxomicin (1 �M). At the end of treatment, cellswere semipermeabilized with 0.04% digitonin to allow isolation of cytosolicfractions. Cytosolic and membrane fractions were resolved in Western blotsand stained with V5, Sec61�, and �-actin antibodies. ER retention of APPenhances retrotranslocation of APP to the cytosol, which is detectable inproteasome inhibitor-treated cells only. B) Retrotranslocation of wild-type

APP from the ER to the cytosol was analyzed in semipermeabilized cells. CHO and B104 cells expressing APP were treatedas in A. Cytosolic fractions were resolved in Western blots and stained with APP (A8718) and GAPDH antibodies.Proteasome inhibition reveals retrotranslocation of APP that is enhanced in cells treated with CP-113,818. C) CHO/APPcells pretreated for 4 d with 10 �M CP-113,818 and for the last 5 h with epoxomicin (1 �M) were pulse labeled with[35S]-methionine/cysteine for 15 min. Cells were chased for 60 min in the presence of epoxomicin and either CP-113,818or vehicle. At the end of the chase, cells were semipermeabilized as in A, B. Total detergent lysates or cytosolic fractions wereimmunoprecipitated with APP antibodies (C-terminal) to analyze metabolically labeled APP. D) Accumulation of newlysynthesized APP molecules in the cytosols was quantitated from pulse-chase/retrotranslocation assays in C. Values aremeans sd from 3 replicate experiments. *P � 0.05. E) H4 neuroglioma cells were treated with CP-113,818 or vehicle for4 d. An additional control plate was treated with 2.5 �g/ml tunicamycin for 6 h to induce ER stress. Induction of ER stresswas analyzed in Western blots of total cell lysates by BiP/GRP78 and CHOP/GADD153 staining. CP-113,818 does not induceBiP or CHOP expression.

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from semipermeabilized cells, APP was immunoprecipi-tated from the fractions. Total detergent lysates wereused as controls (Fig. 5C). In the total lysates at the60-min time point, the mature APP is already decreas-ing in control-treated cells but peaking in the CP-113,818-treated cells. As shown in Fig. 5C, epoxomicintreatment reveals labeled APP accumulating in thecytosol after 60 min chase. The major species of labeledAPP migrates slightly below the immature APP (ascompared to APP in the total lysates) at �95–97 kDa,similarly to that shown in Fig. 5B. Notably, CP-113,818treatment increased the level of cytosolic APP by 58.3 17.6% (P�0.0146; Fig. 5D), suggesting that ACATinhibition enhances retrotranslocation of APP from theER for degradation by the cytosolic proteasomes.

Quality control mechanisms of the secretory pathwayare essential for cellular homeostasis (reviewed in refs.43, 44). Specific stress-sensor systems will activate theunfolded protein response (UPR) if the folding ma-chinery becomes overloaded or misfolded proteinsbegin to accumulate in the ER (reviewed in ref. 45).The cellular response to ER stress is to transcriptionallyactivate genes that encode protein chaperones, such asBiP/GRP78, as well as various transcription factors,such as CHOP [CCAAT/enhancer-binding protein (C/EBP)]-homologous protein). Increased BiP/GRP78 ex-pression appears to be an early response to ER stressors,whereas CHOP is a UPR-induced cell-death mediatorlinking prolonged ER stress to apoptosis (45). Toevaluate whether ACAT inhibition induces UPR orchronic ER stress, we treated H4 neuroglioma cellsoverexpressing APP for 4 d with CP-113,818. As apositive control, we used a prototypical ER stress in-ducer, tunicamycin (for 16 h). As shown in Fig. 5E,CP-113,818-treated cells did not contain detectablelevels of CHOP, whereas in tunicamycin-treated cells, aprominent induction of CHOP expression was de-tected. Moreover, BiP/GRP78 levels in CP-113,818-treated cells were comparable to vehicle-treated controlcells, whereas tunicamycin-treated cells displayed astrong up-regulation of BiP/GRP78 (Fig. 5E). Alto-gether, these data show that inhibition of ACAT activityin cells results in enhanced ERAD of nascent APP

molecules, and that this occurs independently of UPRactivation.

Correlation of reduced brain levels of mature APPholoprotein with APP-C99 and A� in ACATinhibitor-treated mice

Finally, we tested whether ACAT inhibition also re-duces APP maturation in animal models. Our previousstudies have shown that ACAT inhibition efficientlyreduces A� generation and amyloid pathology in atransgenic mouse model of AD (ref. 22 and unpub-lished results). A 2-mo treatment of hAPP mice withCP-113,818 resulted in markedly reduced brain levels ofA� and APP-CTFs without affecting other �-secretasesubstrates, ApoE, BACE1, or �-secretase componentlevels. Moreover, CP-113,818 had no effect on BACE1or �-secretase activity or on A� aggregation in vitro,suggesting that �- and �-secretase-mediated proteolyticprocessing of APP is specifically modulated by CP-113,818. We homogenized and extracted brain hemi-spheres from wild-type mice treated for 2 mo with 7.2mg/kg/d CP-113,818 (from 4.5 to 6.5 mo of age) (22)and analyzed the levels of endogenous APP holopro-tein in brain extracts. As compared to placebo-treatedcontrol mice, animals that received CP-113,818 showeda significant reduction in mature endogenous APP(Fig. 6A). In fact, the ratio of mature APP to immatureAPP was reduced by 50.9% (n�5, P�0.0063) in CP-113,818-treated mice (Fig. 6B). Nicastrin, one of theproteins comprising the �-secretase complex, is aheavily glycosylated type 1 transmembrane protein thatundergoes similar maturation processes along the se-cretory pathway as APP (9, 46). The ratio of mature toimmature nicastrin was not significantly altered inCP-113,818-treated mice as compared to the placebocohort (Fig. 6C), suggesting that CP-113,818 does notalter the maturation of all secretory proteins.

We have recently tested the efficacy of CI-1011, aclinically relevant ACAT inhibitor, in an hAPP trans-genic mouse model of AD. Here hAPP mice overex-pressing human APP751 carrying the Swedish and Lon-

Figure 6. Treatment with ACAT inhibitor CP-113,818 modulates maturation of endogenous APP in brains of wild-type mice.A) Wild-type mice (4.5 mo old; C57BL/6) were treated with 7.2.mg/kg/d CP-113,818 for 57 d. Dissected brain hemispheres wereextracted in 1% Triton X-100/0.2% SDS buffer and analyzed for APP and nicastrin content on Western blots (low exposuresshown). Representative Western blots are shown. B) Quantitation of APP holoprotein content in brains from placebo andCP-113,818-treated mice. Results represent mean se ratio of mature APP to immature APP (APP-m/APP-im); n � 5.C) Quantitation of nicastrin content in brains from placebo and CP-113,818-treated mice. Results represent mean se ratio ofmature nicastrin to immature nicastrin (NCT-m/NCT-im); n � 5.

7ACAT INHIBITION MODULATES TRAFFICKING OF APP

don mutations (that increase A� production) weretreated for 2 mo with 14.4 mg/kg/d CI-1011, starting at4.5 mo of age. Triton X-100 extracts of brain hemi-spheres were analyzed on Western blots for APP holo-protein content. As shown in Fig. 7A, B, the ratio ofmature APP to immature APP was reduced by 24.6%(n�9, P�0.0014) in CI-1011-treated animals. Themilder effect of CI-1011 as compared to CP-113,818may be explained by weaker efficacy of CI-1011 onACAT and/or increased presence of APP in the trans-genic mice as opposed to wild-type littermates in theCP-113,818 study. Notably, when a regression analysis wasperformed to evaluate relationship between mature APPand APP-C99 levels in individual animals, we noticed astrong positive correlation between these two forms ofAPP (Fig. 7C). Lower mature APP levels were associatedwith lower APP-C99 levels. Also, both treatment groupsseparated in different parts of the scatter plot with onlypartial overlap. Finally, we performed regression analysisbetween mature APP and CSF A� levels in individualanimals. As shown in Fig. 7D, mature APP levels displayeda significant correlation with the amount of A� in CSF.These data strongly suggest that ACAT inhibitors limit theavailability of mature APP molecules to A�-generatingenzymes, BACE1 and �-secretase, by modulating traffick-ing of APP in the early secretory pathway.

DISCUSSION

We have previously identified ACAT as a target mole-cule that modulates A� generation in cell- and animal-based models of AD (refs. 22–24). Here we haveidentified a molecular event connecting ACAT inhibi-tion with altered APP metabolism. ACAT itself is local-ized to the ER, and reduced ACAT activity slightlyincreases the level of free cholesterol in the ER mem-

brane while reducing cholesteryl ester production (23,24). Our present results suggest that ACAT inhibitionmodulates APP trafficking in the early secretory path-way, likely at the ER-Golgi interface. Consequently, thisdelays and reduces maturation of APP, limiting theavailability of APP holoprotein for A�-generating ma-chinery, mostly localized to the post-Golgi compart-ments of the cell (31, 33–35). Retention of APP in theearly secretory pathway was recently shown to reduceA� generation in cells (17). Notably, we found areduced ratio of mature APP to immature APP in thebrains of ACAT inhibitor-treated mice, correlating withdecreased brain APP-C99 and CSF A� levels. Here wehave presented evidence showing that APP trafficking isregulated in a sterol-dependent manner. AlthoughACAT inhibition may also directly regulate APP pro-cessing, our results suggest that decreased maturationof APP contributes to reduced A� generation.

What is the mechanism connecting reduced ACATactivity to enhanced ERAD of APP? One possibility isthat ACAT activity, by contributing to formation of lipiddroplets, could participate in extraction of misfoldedproteins from the ER membrane. Cytoplasmic lipiddroplets are composed mostly of neutral lipids such ascholesteryl esters that are generated by ACAT and formby budding from the ER membrane. It was recentlyhypothesized that cells could utilize lipid droplet for-mation in the ER membrane as an escape hatch tofacilitate dislocation of ER proteins for cytosolic degra-dation (47). In another study, proteasomal and auto-phagic degradation pathways were suggested to con-verge on lipid droplet surfaces, particularly for disposalof poorly lipidated ApoB (48). Thus, ACAT activitycould hypothetically be directly linked to the proteaso-mal and autophagic ERAD pathways. However, we havebeen unable to detect colocalization of APP with lipiddroplet surface markers (unpublished results). Our

Figure 7. Reduced maturation of APP correlates with decreased APP-C99 and cerebrospinal fluid A� levels in hAPP mice treatedwith CI-1011. A) hAPP transgenic mice (4.5 mo old) were treated with 14.4 mg/kg/d CI-1011 for 56 d. Dissected brainhemispheres were homogenized and extracted according to 4-step A� isolation protocol (TBS3 1% TX-1003 2% SDS3 70%formic acid). 1% TX-100 extracts (step 2) containing the majority of membrane proteins was analyzed in Western blots for APPcontent. Representative Western blots show APP holoprotein, APP-CTF, and �-tubulin levels in placebo- and CI-1011-treatedhAPP mice. B) APP holoprotein levels were quantitated from Western blots in A and normalized to �-tubulin levels. Resultsrepresent mean se ratio of mature APP to immature APP (APP-m/APP-im); n � 9. C) Correlation analysis of mature APP andAPP-C99 levels in brain extracts from placebo- and CI-1011-treated hAPP mice. Scatter plot shows mature APP content (y axis)vs. APP-C99 content (x axis). R2 and P values were calculated by regression analysis and show significant positive correlationbetween mature APP and APP-C99 levels in both treatment groups; n � 9. D) Cerebrospinal fluid was collected at the end oftreatment (at 56 d) and analyzed for A� content. Scatter plot shows mature APP content (y axis) vs. CSF A�total content (x axis).R2 and P values were calculated by regression analysis and show significant positive correlation between mature APP and CSFA� levels in both placebo- and CI-1011-treated mice; n � 9.

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data also show that ACAT inhibitors do not activate theUPR in cells with normal cholesterol levels. A recentreport suggested that when macrophages were over-loaded with acetylated LDL, ACAT inhibition inducedCHOP expression and ER stress, leading to ER dysfunc-tion and apoptosis (49). However, neither BiP/GRP78,an early UPR-induced gene, nor CHOP, a chronic ERstress marker, was induced in our cells treated withACAT inhibitors for 4 d. Moreover, we have not ob-served any indications of ER stress, such as increasedCHOP induction, in brain tissue from mice treated withACAT inhibitors for 2 mo (unpublished results).

One explanation for APP retention could be alteredAPP protein interactions in the early secretory pathway.In fact, we have recently identified several ER-associ-ated proteins, such as the chaperone GRP94, whosebinding to APP is enhanced on ACAT inhibition (un-published results). Although the causal relationshipbetween the binding of these proteins to APP and ERretention of APP needs to be established in futurestudies, increased chaperone binding of immature APPon ACAT inhibition may indicate APP misfolding.Thus, based on our current data, we hypothesize thatincreased ERAD of APP is rather a consequence of theER retention than an actual mechanism responsible forreduced ER exit and maturation of APP. Regardless ofthe exact mechanism responsible for sterol-responsiveER retention and ERAD of APP, our current data addanother layer of complexity to the elaborate, bidirec-tional relationship between APP/A� and cellular lipidhomeostasis.

At a subcellular level, we found that in cells withreduced ACAT activity additional mechanisms affectAPP metabolism, possibly later in the life cycle of APP.Indeed, in AC29 cells, a cholesterol mutant cell linedefective in ACAT activity, an alternative endosomalproteolytic processing event replaces normal �-, �-, and�-secretase-mediated processing of APP, leading to adramatic reduction in A� secretion (50). However, it isplausible that this alternative cleavage occurs only whentotal cellular cholesterol levels are increased (as seen inAC29 cells due to a mutation in the SREBP pathway)because we have been unable to detect these events innormocholesterolemic cells.

Treatments aiming at lowering brain A� content areexpected to be the most effective disease-modifyingtherapies for AD. Moreover, understanding molecularmechanisms is an important factor in the overall suc-cess of clinical drug development, particularly withcomplex diseases such as AD. Our current data suggesta mechanism that may be responsible for the antiamy-loidogenic effects of ACAT inhibitors and, thus, pavethe way for clinical testing of ACAT inhibitors forprevention and/or treatment of AD.

This study was supported by grants from the Cure Alzheimer’sFund (D.M.K.), the U.S. National Institutes of Health (R01NS45860; D.M.K.), the Helsingin Sanomat Centennial Foundation(H.J.H.), and the Maud Kuistila Foundation (H.J.H.). CI-1011 andCP-113,818 were kind gifts from Lit-Fui Lau and James Harwood(Pfizer, Groton, CT, USA), respectively.

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Received for publication May 5, 2009.Accepted for publication June 25, 2009.

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