Internalized antibodies to the Aβ domain of APP reduce neuronal Aβ and protect against synaptic alterations

Cláudia G. Almeida and Gunnar K. GourasH. Takahashi, Feng Li, Michael T. Lin, Davide Tampellini, Jordi Magrané, Reisuke  against Synaptic Alterations

and Protectβof APP Reduce Neuronal A DomainβInternalized Antibodies to the A

RNA:RNA-Mediated Regulation and Noncoding

doi: 10.1074/jbc.M700373200 originally published online April 27, 20072007, 282:18895-18906.J. Biol. Chem. 

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Internalized Antibodies to the A� Domain of APP ReduceNeuronal A� and Protect against Synaptic Alterations*□S �

Received for publication, January 12, 2007, and in revised form, March 30, 2007 Published, JBC Papers in Press, April 27, 2007, DOI 10.1074/jbc.M700373200

Davide Tampellini, Jordi Magrane, Reisuke H. Takahashi, Feng Li, Michael T. Lin, Claudia G. Almeida,and Gunnar K. Gouras1

From the Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York 10021

Immunotherapy against �-amyloid peptide (A�) is a leadingtherapeutic direction for Alzheimer disease (AD). Experimentalstudies in transgenic mouse models of AD have demonstratedthat A� immunization reduces A� plaque pathology andimproves cognitive function. However, the biological mecha-nisms by which A� antibodies reduce amyloid accumulation inthebrain remainunclear.Weprovide evidence that treatment ofAD mutant neuroblastoma cells or primary neurons with A�antibodies decreases levels of intracellular A�. Antibody-medi-ated reduction in cellular A� appears to require that the anti-body binds to the extracellular A� domain of the amyloid pre-cursor protein (APP) andbe internalized. In addition, treatmentwith A� antibodies protects against synaptic alterations thatoccur in APP mutant neurons.

Active immunization for �-amyloid peptide (A�)2 has beendemonstrated to reduce A� plaques and improve cognitivefunction in transgenic mouse models of Alzheimer disease(AD) (1–4). In human AD patients actively immunized withA�, subjects with high antibody titers appeared to have slowedcognitive deterioration and reduced plaque burden, although6% of subjects developed meningoencephalitis (5–7). Passiveimmunotherapy in mouse models of AD has provided similar

benefits to those seen with active immunization (4). Themech-anisms by which A� antibodies reduce A� plaque pathology inthe brain remain unclear (8). Data suggest roles for antibody-mediated microglial activation and A� efflux from the brain inthe reduction of A� (9, 10). Interestingly, intracerebral injec-tion of A� antibody reduced levels of both extracellular andintracellular A� in a triple transgenic (3�Tg) mouse carryingmutations in amyloid precursor protein (APP), presenilin 1 andtau (11), and reduction of intraneuronal A� was the better cor-relate with cognitive improvement (12). How A� antibodiesreduce intracellular A� is not known. Increasing evidence sup-ports that intraneuronal A� accumulation is important in thepathogenesis of AD. Intraneuronal A� accumulation has beenreported in transgenic mouse models of cerebral amyloidosis(13–19), human AD (20–22) and Down syndrome (21, 23–25).Moreover, cultured neurons fromAPPmutant transgenicmicedevelop subcellular A� accumulation and synaptic alterationsthat parallel those observed in vivo in the brain with �-amyloi-dosis (26, 27).We now report that A� antibodies decrease levels of intra-

cellular A� in culture and provide evidence that antibody bind-ing to the A� domain of APP and internalization of the anti-body/APP complex appear to be required to reduceintracellular A�. Moreover, A� antibody treatment protectsagainst synaptic alterations that occur in APP mutant neuronsin culture.

EXPERIMENTAL PROCEDURES

cDNA Constructs—Wild-type (wt) and mutant (K44E)dynamin-1 cDNAcontainingGFPwere previously described (28).Cells were transfected overnight using Lipofectamine 2000(Invitrogen), according to the manufacturer’s instructions. Anti-GFP antibody was obtained fromUpstate Biotechnology.Cell Culture and Treatments—Primary neuronal cultures

from Tg2576 mice (29), and littermates were prepared asdescribed (26). Primary neurons were used at 19 days in vitro(DIV). Mouse N2a neuroblastoma cells either untransfected(N2a) or stably transfected with the 670/671 Swedish mutationhumanAPP (Sw-N2a) were grown as described previously (30).Mouse N2a neuroblastoma cells stably transfected with theC-terminal fragment of human APP C99 (C99-N2a) were pre-viously described (31). Chloroquine (Sigma, 100 �M) andNH4Cl (Sigma, 50 mM) were added to cells 1 h prior to treat-ment and kept in culture during 3 h antibody incubation. The�-secretase inhibitor N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT; Calbiochem) was dilutedin culture media to 250 nM and then added to cells for 3 h.

* This work was supported in part by the Dana Foundation, the Alzheimer’sAssociation, the American Health Assistance Foundation, National Insti-tutes of Health Grants NS045677 and AG028174 (to G. K. G.), and by adoctoral fellowship from Fundacao para a Ciencia e a Tecnologia, Portugal(to C. G. A.). The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

� This article was selected as a Paper of the Week.□S The on-line version of this article (available at http://www.jbc.org) contains

supplemental Figs. S1–S12 and movie S1.1 To whom correspondence should be addressed: Dept. of Neurology & Neu-

roscience, Weill Medical College of Cornell University, 525 East 68th St.New York, NY 10021. Tel.: 212-746-6598; Fax: 212-746-8741; E-mail:[email protected].

2 The abbreviations used are: A�, �-amyloid peptide; APP, amyloid precursorprotein; AD, Alzheimer disease; N2a, mouse N2a neuroblastoma cellsuntransfected; Sw-N2a, mouse N2a neuroblastoma cells stably transfectedwith the 670/671 Swedish mutation human APP; C99-N2a, mouse N2aneuroblastoma cells stably transfected with the C-terminal fragment ofhuman APP C99; �CTFs, � C-terminal fragments; GFP, green fluorescentprotein; wtDyn, wild-type GFP-dynamin; DynK44E, dominant negativeK44E mutant GFP-dynamin; LDH, lactate dehydrogenase; DIV, days in vitro;DAPT, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butylester; TUNEL, transferase dUTP nick end labeling; PBS, phosphate-bufferedsaline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ELISA,enzyme-linked immunosorbent assay; YFP, yellow fluorescent protein;BACE, �-site amyloid cleaving enzyme; CTF, C-terminal fragment.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 26, pp. 18895–18906, June 29, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Antibody Treatment—Several well characterized A� anti-bodies were used: monoclonal 6E10 (human A� residues 5–10;IgG1) and 4G8 (A� residues 17–24; IgG2B) (Signet Laborato-ries), G2-11 (A�42 C terminus; IgG1; Genetics Co.), anti-A�42(A�42 C terminus; Chemicon), polyclonal anti-A�40 (A�40 Cterminus; Chemicon), and polyclonal APP-ab2 (humanA� res-idues 1–10; Labvision NeoMarkers). Other antibodies usedwere: monoclonal P2-1 (specific for the N terminus of humanAPP; IgG1; Affinity BioReagents) and mouse anti-human IgG(Jackson ImmunoResearch). We added fresh medium to cellsjust prior to each treatment. Final antibody concentration in alltreatments was 2 �g/ml. Antibodies were added to the culturefor different time points, as indicated. Treatment for 24 h withA� antibody 6E10 did not induce neuronal death, evaluatedusing the TUNEL labeling method and lactate dehydrogenase(LDH) assay (see below). For internalization studies, neuronswere kept at 4 °C for 45 min with the indicated antibody. Cellswere then either immediately fixed (0 min) or incubated at37 °C for 10, 30, 60, or 180 min and then fixed in 4% paraform-aldehyde in phosphate buffer (PB).Cell Death Assay—19 DIV Tg2576 neurons grown on poly-

D-lysine precoated coverslips were incubated 24 hwith or with-out 6E10 antibody. Neurons were washed twice with PB saline(PBS) and fixed in 4% paraformaldehyde. The TUNEL stainingkit (Roche Applied Science) was used to stain apoptotic neu-rons according to the manufacturer’s instructions. Nuclei werestained with the Hoechst stain. Counts of TUNEL positivenuclei and total nuclei were performed with Metamorph (Uni-versal Imaging Co.) on 6–10 fields per coverslip at 20� magni-fication. The ratio between TUNEL-positive nuclei andHoechst-positive nuclei was calculated. LDH kit (Sigma) wasused to evaluate whether antibody treatment was inducing celldamage after incubation for 24 hwith orwithoutA� antibodies.Levels of LDH were measured in the media according to themanufacturer’s instructions.A� Immunoprecipitation and Detection—Primary neurons

and Sw-N2a cells were washed twice, harvested in ice-cold PBS,and centrifuged. Cell lysates were treated with 6% SDS contain-ing 10 �l/ml of �-mercaptoethanol, sonicated, and then heatedat 95 °C for 6 min. After centrifugation, supernatants wereeither loaded directly (neuron lysates) into 10–20%Tricine gels(Invitrogen) for A� detection or immunoprecipitated (N2acells) overnight at 4 °C with 4G8 antibody (in 190 mM NaCl, 50mMTris-HCl pH 8.3, 6mM EDTA and 2.5% Triton X-100). Thelatter were then incubated with rabbit anti-mouse secondaryantibody (Cappell) together with protein A-Sepharose beads(GEHealthcare) for 2 h at 4 °C. Samples were subjected to elec-trophoresis and transferred to polyvinylidine difluoride mem-branes (Millipore). Membranes were boiled in PBS for 5 minand immunoblotted as described (26). The immunoreactionwas visualized by a chemiluminescence system (Pierce). Bandintensities were quantified using Scion Image software. Thearea under the bandpeak and above the baselinewas quantified.To determine secreted APP� levels, media were centrifuged 5min at 1,000 � g to pellet cellular debris. 1 ml of supernatantwas collected and incubated with an antibody against secretedAPP� (Signet) overnight at 4 °C (in 190 mM NaCl, 50 mM Tris-HCl, pH 8.3, 6 mM EDTA, and 2.5% Triton X-100). Samples

were incubated with rabbit anti-mouse secondary antibodytogether with protein A-Sepharose beads for 2 h at 4 °C. Thesame media were further used to immunoprecipitate secretedAPP� fragments with antibody 6E10 in the same conditionsdescribed for secreted APP�. Western blot analyses were per-formed as described above using 22C11 (Roche Applied Sci-ence) as primary antibody.ELISA Analyses—19 DIV Tg2576 primary neurons or

Sw-N2a cells were incubated for 24 h in the presence or absenceof antibody 6E10 or 4G8 and collected as described previously.Concentrations of A�1–40 and A�1–42 were measured byusing the respective ELISA kits (BIOSOURCE) according tomanufacturer’s instructions.BiochemicalMeasurements of Surface APP—19DIV primary

neurons or Sw-N2a cells were incubated for 3 h with antibody6E10. After two washes with PBS containing 1 mM CaCl2 and0.5 mM MgCl2 (PBS-Ca-Mg), cells were placed on ice to blockendocytosis and incubated with PBS-Ca-Mg containing 1mg/ml Sulfo-NHS-LC-Biotin (Pierce) for 20 min. Cultureswere rinsed in ice-cold culture medium to quench the biotinreaction. Cultures were lysed in 200 �l of 3% SDS. The homo-genates were centrifuged at 14,000 � g for 15 min at 4 °C. Fif-teen microliters of the supernatant were removed to measuretotal protein levels; the remaining supernatant was incubatedwith 100 �l of Neutravidin agarose (Pierce) overnight at 4 °C.Sampleswere thenwashed three timeswith a buffer containing:150 mM NaCl, 10 mM Tris-HCl, pH 8.3, 5 mM EDTA, 0.1%Triton X-100, 0.01% bovine serum albumin, and proteaseinhibitor mixture (Roche Applied Science). Bound proteinswere resuspended in 30 �l of SDS sample buffer and boiled.Quantitative Western blots were performed on both total andbiotinylated (surface) proteins using APP N-terminal antibody22C11 and tubulin antibody (Sigma). Immunoreactive bandswere visualized by enhanced chemiluminescence (ECL, Amer-sham Biosciences) and captured on autoradiography film(Amersham Biosciences Hyperfilm ECL). Digital images, pro-duced by densitometric scans of autoradiographs on a Scan-Maker 8700 (Microtek) were quantified using NIH Image 1.63software. The surface/total APP ratio was calculated for eachculture.Degradation Assay for APP and C99—Sw-N2a or C99-N2a

cells were treated with biotin as described above. After cellswere rinsed in ice-cold culture medium to quench the biotinreaction, fresh ice-cold medium containing 6E10 antibody (2�g/ml) was added. Cells were kept on ice for 5 min to allow theantibody recognition of surface APP or C99. Cells were thenplaced at 37 °C for 45min. After treatment, cells were collectedand lysed as described for the assay of surface APP, above.Western blot analyses of full-length APPwere performed using22C11 antibody; Western blot analyses of C99 peptides wereperformed using 6E10 antibody.Immunofluorescence—Cells were grown on poly-D-lysine-

coated coverslips (Fisher). After antibody treatment, cells werewashed in ice-cold PBS and fixed for immunofluorescence, asdescribed previously (27). The following antibodies were used:A�42, synapsin I, and PSD-95 (Chemicon), APP 369 (anti-Cterminus of APP, (30), EEA1 (BD Transduction), Tsg101(Genetex), and Lamp2 (Zymed Laboratories Inc.). Fluores-

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cent secondary antibodies were either Alexa-488 or Alexa-546 (Molecular Probes) or Cy2- or Cy3-conjugated (JacksonImmunoResearch). To determine antibody uptake, cells wereincubated with secondary antibodies with or without prior per-meabilization with 0.1% saponin. Cells were viewed with anOlympus Optical IX-70 microscope equipped with anORCA-ER CCD camera (Hamamatsu Photonics) and a 60�,1.4 NA plan apochromat objective. Metamorph software wasused for quantitative analysis. To quantify A� immunofluores-cence, 5–10 neurons or Sw-N2a cells were randomly pickedfrom each of three independent experiments, and averageintensities were measured in selected areas. To quantify A�staining in Sw-N2a cells after transfection with the dynamin-1cDNA containing GFP, we only considered cells that weretransfected (GFP-positive). For quantification of 6E10 antibodyinternalization and A�42 staining, intensity threshold was setusing Metamorph 6.1 so that fluorescence of neurons wasabove background fluorescence. Total fluorescence per 30 �mof a thresholded neurite was automatically quantified. PSD-95punctawere quantified as described previously (27).One or twocoverslips from each culture were analyzed, 5–10 neurons percoverslip. From each neuron, 3–5 neuritic segments 30 �m inlength were selected from areas where single puncta could beoutlined. Images were thresholded so that only the brightestpuncta, with intensity at least twice that of the neuritic shaft,were outlined. Using the integrated morphometric analysisfeature in Metamorph, puncta density was automaticallymeasured.Confocal Microscopy—Immunofluorescence A� antibody

internalization and intracellular A� reduction were examinedby confocal microscopy using an Axiovert 100 M invertedmicroscope equipped with an LSM 510 laser scanning unit anda 63 � 1.4 NA plan apochromat objective (Carl Zeiss, Inc.),Ar488, HeNe1543 nm lasers, and LP560 and BP505–530. Opti-cal sections were acquired at 0.7 �m thickness.Live CellMicroscopy of Antibody Uptake—Cells were imaged

using an Olympus Optical IX-70 microscope equipped with anORCA-ERCCDcamera, a 60�, 1.4NAplan apochromat objec-tive and a 37 °C heated chamber. Images were obtained using aHamamatsuOrca ER digital camera. N2a cells were transfectedwith human APP-YFP for 3–4 h. Cells were incubated for 10min on ice with Alexa-555-conjugated 6E10 antibody (6E10was labeled using an Alexa Fluor 555 Monoclonal AntibodyLabeling Kit, Invitrogen) in live imaging solution (120 mMNaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10mM Hepes). Cells were washed twice in ice cold live imagingsolution before imaging. Frames were automatically andsequentially acquired every 20 s with the FITC and Rhodaminefilters using Metamorph.Metabolic Labeling—Primary neurons were plated in 10-cm

dishes. 7-Day-old cultures after 30-min starvation in cysteine/methionine-freemedium (Invitrogen) were pulsed in fresh cys-teine/methionine free medium with 1 mCi [35S]methionine/cysteine (PerkinElmer Life Sciences) in the presence or absenceof 2�g/ml A� antibody 6E10 for 20–30min and then chased inNeurobasal medium (Invitrogen) for 15 min, 45 min, or formedia studies, 90 min. Media were centrifuged for 10 mintransferred to clean tubes and immunoprecipitated with A�

antibody 4G8. Cells were collected in ice-cold PBS and lysed.Samples were immunoprecipitated with A� antibody 4G8. 35Ssignal was visualized using a phosphoimager system (HewlettPackard Cyclone).Statistical Analysis—Statistical comparisons were made

using unpaired t tests with significance placed at p� 0.05. A setof cultures prepared fromonemouse embryowas considered asone independent experiment (n � 1). One section preparedfrom one Tg2576 mouse was considered as one independentexperiment (n � 1). Data were expressed as mean � S.E. Sta-tistical analysis was performed using GraphPad Prism 3.0 soft-ware (GraphPad Software).

RESULTS

Treatment with monoclonal A� antibodies (6E10, 4G8; Fig.1A) reduced levels of A� in primary neurons at 19 DIV derivedfrom Tg2576 mice harboring the Swedish mutant human APPand in N2a neuroblastoma cells stably transfected with ADSwedish mutant human APP (Sw-N2a) (Fig. 1, B–G). Specifi-cally, treatment of culturedTg2576neuronswith 2�g/ml ofA�antibodies for 24 h resulted in a 36 � 12% (6E10) and 30 � 11%(4G8) decrease in levels of A� as quantified by Western blot(Fig. 1B, E). In contrast to these N-terminal to mid A� domain/APP antibodies, treatment of neurons with G2-11, a C-termi-nal-specific A�42 antibody (Fig. 1A), did not induce changes inintracellular levels of A� (Fig. 1E), suggesting that binding tothe exposed extracellular domain of A� was required for A�antibody-mediated reduction in intracellular A�. Similarreductions in levels of A� after treatment with A� antibodieswere evident by ELISA analyses, where reduction of both A�40andA�42was observedwith either 6E10 or 4G8 treatment (Fig.1D). To further confirm these biochemical results, we also eval-uated intracellular A� immunofluorescence (32) following A�antibody treatment, which revealed a reduction in intracellularA�42 immunofluorescence in cultured Tg2576 neurons asshown by confocal imaging (Fig. 1C). Treatment for 24 h withA� antibody 6E10 did not alter neuronalmorphology or induceneuronal death as evaluated using the TUNEL labeling methodor the LDH assay (supplemental Fig. S1).Western blot analysesdemonstrated that incubation of Sw-N2a cells with A� anti-bodies had similar reductions in levels of A�. Specifically, 24 hof treatment with A� antibodies 6E10 or 4G8 reduced levels ofintracellular A� by 51 � 10% and 54 � 11%, respectively, com-paredwith untreated controls (Fig. 1F). Since theseA� antibod-ies bind A� peptides and the A� domain within APP, we alsotreated Sw-N2a cells with a monoclonal antibody directedagainst the N-terminal ectodomain of human APP (antibodyP2-1, Fig. 1A); this treatment did not reduce levels of intracel-lular A� (Fig. 1F). ELISA analyses confirmed the reductions ofboth A�40 and A�42 after 24 h treatment with A� antibodies6E10 or 4G8 in Sw-N2a cells (Fig. 1G).To investigate the mechanism whereby A� antibodies

reduced intracellular A�, we examined whether the binding ofcell surfaceAPPbyA� antibodiesmight be involved. Sw-N2a oruntransfected N2a cells were first incubated with 6E10 or 4G8for 24 h and then fixed, rinsed and stained with a fluorescentsecondary antibodywithout cell permeabilization. The human-specific antibody 6E10 stained the cell surface of Sw-N2a cells

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(expressing humanAPP) but not thesurface of untransfected N2a cells(expressing only mouse APP) (Fig.2A). Remarkably, when the second-ary antibody was applied after cellpermeabilization, both A� antibod-ies 6E10 and 4G8 were pronouncedwithin Sw-N2a cells (Fig. 2B), con-sistent with the internalization ofthe antibodies. In contrast, incuba-tion for 24 h with human IgGrevealed absence of labeling insidecells (Fig. 2B), indicating that non-specific antibody uptake was notoccurring. Moreover, treatment ofN2a cells (lacking the human APPtransgene) with human specific6E10 antibody did not reveal signif-icant intracellular labeling, indicat-ing that 6E10 antibody uptake onlyoccurred in the presence of humanAPP (Fig. 2B). To evaluate whetherthis resultmight be due to less intra-cellular A� in untransfected N2acells, we treated N2a cells with A�antibody 4G8, which is not humanspecific and also recognizes themurine A� domain. Confocal Zstack images indicated a cytoplas-mic staining of 4G8 in permeabi-lized N2a cells (supplemental Fig.S2), supporting that lower intracel-lular A� levels do not preclude A�antibody uptake.Since endocytosis of surface pro-

teins does not occur at 4 °C, we alsotested for A� antibody uptake at4 °C. After 45-min incubation ofSw-N2a cellswith 6E10 at 4 °C, 6E10was now only evident at the plasmamembrane and not in intracellularcompartments with permeabiliza-tion, compared with 37 °C whereinternalization was evident (supple-mental Fig. S3). Treatment ofSw-N2a cells for 24 h with antibod-ies to the C terminus of A�40 (anti-A�40) or A�42 (G2-11), but not toAPP, did not reveal intracellularstaining after permeabilization (Fig.2B), supporting that antibody bind-ing to the extracellular A� domainof APP was important for internal-ization. Indeed, Sw-N2a cells incu-bated with antibody P2-1, directedat the ectodomain of human APP,revealed both a cell surface patternof staining when secondary anti-

FIGURE 1. A� antibody treatment reduces cellular levels of A�. A, sequences in APP/A� recognized bythe antibodies used in this study (not drawn to scale). G2-11 and anti-A�40 are specific for the C terminusof A�42 and A�40 peptides, respectively. 6E10 (human-specific), APP-ab2 (human-specific), and 4G8 arespecific for the extracellular A� domain of APP. P2-1 (human specific) and 369 are specific to the N- andC-terminal regions of APP, respectively. B, treatment with A� antibodies reduces levels of intracellular A�.Incubation of Tg2576 neurons with the indicated antibodies 6E10 or 4G8 (2 �g/ml for 24 h), directed at theextracellular portion of the A� domain within APP, reduces intraneuonal A�. Levels of full-length APPwere unchanged. C, representative images of A�42 immunofluorescence. Tg2576 neurons treated for 24 hwith A� antibody 6E10 revealed reduced A�42 immunofluorescence compared with untreated controlTg2576 neurons. Scale bar: 10 �m. D, ELISA analysis revealed reduction of A�40 and A�42 in Tg2576neuron cell lysates after 24-h incubation with either antibody 6E10 or 4G8 (n � 3; *, p � 0.05; **, p � 0.01).E, Tg2576 neurons were treated with the indicated antibodies (2 �g/ml) for 24 h (n � 9 for 6E10 and 4G8;n � 3 for G2-11) and neuron cell lysates were then analyzed by Western blot. A� levels were reduced onlyby antibodies 6E10 and 4G8 but not G2-11, directed at the C terminus of A�42. Densitometric quantitationof Western blots performed on Tg2675 neuronal lysates is expressed as relative amount of A� in treatedcompared with untreated cells (*, p � 0.05; **, p � 0.01). F, immunoprecipitation followed by Western blotanalysis on Sw-N2a cells also revealed reductions in levels of A� after 24-h incubation with antibodiesagainst A� (6E10, 4G8) but not by an antibody against the APP ectodomain (P2-1). Densitometric quan-titation is expressed as relative amount of A� in treated compared with untreated cells (n � 4; **, p � 0.01).G, ELISA analysis confirmed reduction of intracellular A� (A�40 and A�42) in Sw-N2a cells after 24-hincubation with either antibody 6E10 or 4G8 (n � 4; *, p � 0.05; **, p � 0.01).

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body was applied without permeabilization (Fig. 2A) and anti-body internalization when secondary antibody was appliedafter permeabilization (Fig. 2B). To better delineate A� anti-body uptake in Sw-N2a cells and cultured Tg2576 neurons, weanalyzed Z stack confocal images that more clearly reveal theintracellular localization of antibody (supplemental Fig. S4, Aand B). These results supported that recognition of antibodiesto the extracellular exposed A� domain of cell surface APP wasrequired for antibody internalization.To investigate whether A� antibodies remain associated

with APP after internalization, live cell imaging was done onN2a cells transiently transfected with APP-YFP and treatedwith fluorescently conjugated A� antibody 6E10 (Fig. 2C andsupplemental movie S1). As indicated by the arrows, internal-ized fluorescent 6E10 co-localized with APP-YFP within thesame vesicle transported in the retrograde direction within aneurite. Internalized A� antibody 6E10 also co-localized withAPP (369) in neuronal processes in fixed neurons (supplemen-tal Fig. S5). To determine whether there was a correlationbetween antibody internalization and reduction in A�, we per-formed a time course of incubation with 6E10 (10 min, 1 h, or7 h) in Tg2576 neurons, which revealed a progressive accumu-lation of A� antibody in neurons, especially evident within neu-ronal processes (Fig. 2,D andE). Quantification ofA�42 immu-nofluorescence in neuronal processes after antibody treatmentrevealed a reduction of intraneuronal A�42 over time, whichinversely correlatedwith antibody uptake in these neurites (Fig.2, E and F). Internalized A� antibody 6E10 did not co-localizewith intracellular A�42 in neurites after 10 min, at which time6E10 would be expected to localize in early endosomes. How-ever, at 1 h some co-localization of 6E10with A�42was evident(Fig. 2E), consistent with a late endosomal localization at thistime (32).To investigate whether treatment with A� antibody pro-

moted internalization of full-length APP from the plasmamembrane, we used biotin to specifically label APP at the cellsurface of Tg2576 neurons. After 3-h incubation, there was a40 � 14% reduction in surface levels of full-length APP in anti-body 6E10-treated compared with untreated neurons (n � 5;p � 0.05), consistent with increased APP internalization uponA� antibody binding (Fig. 3A).To determine whether endocytosis of the A� antibody/APP

complex upon A� antibody treatment was required to reduceintracellular A�, Sw-N2a cells were transfected with eitherwild-type GFP-dynamin (wtDyn) or the dominant negativeK44E mutant GFP-dynamin (DynK44E) cDNA, which blocksendocytosis (28). Sw-N2a cells transfectedwith the wtDyn con-struct and incubated for 3 h with A� antibody 6E10 displayed asimilar pattern of antibody internalization to the untransfectedSw-N2a cells, while cells transfected with the dominant nega-tive DynK44E construct revealed only surface staining and nointernalization of the antibody (Fig. 3B). In Sw-N2a cells trans-fectedwithwtDyn, treatmentwith 6E10 reduced levels of intra-cellular A� by 61 � 1% compared with untreated cells (n � 3,p � 0.01). In contrast, in Sw-N2a cells transfected withDynK44E, 6E10 treatment did not reduce levels of intracellularA� (Fig. 3C). These results were confirmed by immunofluores-cence experiments, which revealed a 41 � 8% decrease in cel-

lular A�42 in wtDyn transfected cells after 6E10 treatment,whereas antibody treatment did not reduce levels of A�42 inDynK44E transfected Sw-N2a cells (Fig. 3D). The overall reduc-tion inA� levels upon transfectionwithDynK44E (Fig. 3,C andD) confirms recent work (33) and is consistent with previousstudies indicating that APP internalization is important for A�generation (34, 35). At the same time, the reduction in A� gen-eration by the dynamin mutant also limits the interpretation ofthe data obtained with regards to effects of A� antibodytreatment.We used antibody co-localization studies to examine

whether internalization of the A� antibody/APP complex fol-lowed the classical endocytic pathway for cell surface receptors.Since antibodies against subcellular markers are mostly mousemonoclonal antibodies, APP mutant neurons were incubatedwith a 6E10-like rabbit A� antibody, APP-ab2, for differenttime points (0, 10, 30, 60, 180 min) and then co-stained withmarkers for subcellular compartments. After 10-min incuba-tion, A� antibody was internalized within neurites and showeda punctate pattern that co-localized with the early endosomalmarker EEA1 (supplemental Fig. S6). In contrast, after 30-minincubation, A� antibody co-localization was more pronouncedwith the late endosomal/multivesicular body marker Tsg101.At later time points (60 and 180 min), internalized APP-ab2co-stained with the late endosomal/lysosomal marker Lamp2within processes and cell bodies (supplemental Fig. S6).It has been reported that cell surface binding by antibodies

can promote internalization and degradation of receptors inlysosomes (36). Antibody binding to the N terminus of cell sur-face APP was previously used to follow APP internalization toendosomal-lysosomal compartments (37, 38). To investigatewhether A� antibody binding targeted internalized APP to thedegradative pathway, we carried out biotin surface labeling onSw-N2a cells at 4 °C followed by incubation of cells at 37 °C inthe presence or absence of A� antibody 6E10 or the APPN-ter-minal antibody P2-1. After 15-min incubation there was nosignificant difference in levels of biotinylated APP within cellswith 6E10 or P2-1 treatment (supplemental Fig. S7). In contrastafter 45-min incubation with antibody 6E10, levels of biotiny-lated APP decreased by 45 � 10% compared with untreatedcontrol cells (Fig. 4A, n � 3; p � 0.01), suggesting eitherincreased degradation and/or increased secretase cleavage ofAPP. Although P2-1 did not reduce levels of A� (Fig. 1E), it diddecrease levels of biotinylated APP. However, themagnitude ofthe effect (28 � 9% compared with control) was less than thatfor 6E10 (Fig. 4A, n � 3, p � 0.05).

To further investigate the mechanism whereby A� antibod-ies reduce intracellularA�, we assessedA� generation at earliertime points of treatment usingmetabolic labeling. Primary neu-rons were pulsed for 20 min in [35S]methionine/cysteine con-taining media and then chased for 15 min and 45 min in thepresence or absence of 6E10. Although there was no significantchange in levels of newly generated intraneuronal A� at 15min(not shown), there was a significant 30� 9% reduction in levelsof [35S]methionine/cysteine-labeled intraneuronal A� at 45min (Fig. 4B). Another explanation for the reduction of intra-cellular A� levels after A� antibody treatment could have beenan increase of A� secretion. To investigate this hypothesis, we

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pulsed primary neurons for 30min in [35S]methionine/cysteinecontainingmedia and then chased for 90min in the presence orabsence of 6E10. In the media of 6E10-treated neurons, therewas a trend for decreased levels of secreted A� compared withuntreated cells which did not reach significance (supplementalFig. S8A) suggesting that antibody treatment either reduced

generation and/or increased degra-dation of newly generated A�.Therefore, we examined the effectof A� antibody treatment on thegeneration of A� by �- and�-secretases.

To assess whether A� antibodydecreased �-site amyloid cleavingenzyme (BACE) processing of APP(39), we measured the amount ofthe N-terminal APP fragmentsecreted after �-site cleavage of full-length APP (sAPP�) using an anti-body specific for sAPP�. Remark-ably, there was a 41 � 3.5% increasein levels of secreted sAPP� (n � 4;p � 0.05; Fig. 4C and supplementalFig. S8B, upper panel) in A� anti-body 6E10 treated (3 h) comparedwith untreated Sw-N2a cells. Wealso measured levels of N-terminalAPP secreted after�-site cleavage offull-length APP (sAPP�). There wasa trend for reduced levels of sAPP�in the media of 6E10 treated com-pared with untreated Sw-N2a cells,although this did not reach signifi-cance (supplemental Fig. S8B, lowerpanel). To further investigate theeffect of A� antibody treatment onAPP processing, wemeasured levelsof APP C-terminal fragments(CTFs) after BACE cleavage (�CTFsor C99) or �-secretase cleavage(�CTFs or C83) in Tg2576 neuronstreated for 24 h with A� antibody6E10. In 6E10 treated neurons,there was a 92 � 34% increase of�CTFs compared with untreated

neurons (supplemental Fig. S8C, middle and lower panels). Incontrast, there was a trend for decreased levels of �CTFs whichdid not reach significance (supplemental Fig. S8C, lower panel).The lack of increase in �CTFs in the presence of increased�CTFs argues against a generalized inhibition of �-secretase,

FIGURE 2. A� antibodies that bind at the cell surface are internalized into cells. A, untransfected N2a and Sw-N2a cells were treated with the indicatedantibodies for 24 h and then stained with fluorescent anti-mouse IgG without permeabilization. A� antibodies 6E10 and 4G8 revealed a cell-surface pattern ofstaining in Sw-N2a cells. The human-specific antibody 6E10 did not show such surface labeling in untransfected mouse N2a compared with the Sw-N2a cells.APP ectodomain antibody P2-1 revealed surface staining in Sw-N2a cells. B, untransfected N2a and Sw-N2a cells were treated with the indicated primaryantibodies for 24 h, fixed, and then permeabilized and stained with fluorescent secondary antibodies. Only antibodies directed against the extracellulardomain of APP (P2-1), including those to the A� domain (6E10, 4G8), were internalized. In contrast, mouse IgG, antibody G2-11 (A�42), and antibody A�40 werenot internalized, and untransfected N2a cells lacking human APP did not demonstrate uptake of the human A�-specific antibody 6E10. C, live cell imaging ofN2a cells transfected with APP-YFP and incubated with Alexa-555-conjugated 6E10 for 30 min with images acquired at 20-s intervals. Internalized fluorescentlytagged A� antibody 6E10 (red) co-localized with APP-YFP (green) in the same vesicle moving retrograde in a process toward the cell body. The images wereoffset 5 pixels horizontally to better differentiate the fluorophores. The artificially large and confluent yellow area of co-staining just to the right and under thedarkly staining nucleus in the cell body is secondary to the increased gain required to visualize fluorescence in the thinner process below (magnified in thelower set of frames). D, internalization of A� antibody 6E10 with time correlated with reduced A�42 within Tg2576 neurons. Scale bar: 10 �m. E, detail ofrepresentative processes more clearly revealed the correlation between increasing internalization of A� antibody 6E10 (green) and decreasing levels of A�42(red). Although internalized A� antibody 6E10 co-localized with APP/�CTF using the APP C-terminal antibody 369 (see supplemental Fig. S5), internalized 6E10did not co-localize with intracellular A�42 at 10 min, while at 1 h some co-localization was evident (F, arrowhead, merged panel). F, quantification of A�42 andantibody 6E10 fluorescence in neurites of Tg2576 neurons over time (n � 3; *, p � 0.05; **, p � 0.01; compared with 10-min time point; scale bar: 10 �m).

FIGURE 3. APP endocytosis is promoted by A� antibody binding and is required for reduction of cellularA�. A, Tg2576 neurons (19 DIV) were treated with A� antibody 6E10 for 3 h and then incubated on ice withbiotin to label surface APP. Western blot analysis revealed decreased levels of surface full-length APP in A�antibody 6E10-treated compared with untreated control neurons. Total levels of APP were unchanged in celllysates. B, Sw-N2a cells transiently transfected with wild-type GFP-dynamin (wtDyn) or dominant negativeGFP-dynamin (DynK44E) revealed internalization of A� antibody 6E10 after 3 h treatment in both untrans-fected control cells (untransf) and wtDyn-transfected cells but not in DynK44E-transfected cells, which revealeda surface pattern of staining. C and D, in Sw-N2a cells transfected with wtDyn, treatment with 6E10 reducedlevels of intracellular A� by 61 � 1% compared with untreated cells. In contrast, in Sw-N2a cells transfectedwith DynK44E, 6E10 treatment did not reduce levels of intracellular A�. A� was measured by Western blotting(C) and A�42 immunofluorescence (D). Corresponding quantitation was expressed as a relative amount of A�in treated compared with untreated cells (n � 3; *, p � 0.05; **, p � 0.01; scale bar: 10 �m).

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because if that had occurred both�-and �CTFs should have beenincreased. Thus, A� antibody 6E10did not inhibit BACE or �-secretaseprocessing of APP but ratherappeared to augment BACE cleav-age of APP. To further confirm thatA� antibody treatment did notdecrease �-secretase processing ofAPP, we examined N2a cells stablytransfected with human APP C99,which is cleaved only by �-secretase(31). Similar to Sw-N2a cells, whenC99-N2a cells were treated with A�antibody 6E10, there was a cell sur-face pattern of staining when sec-ondary antibody was applied with-out permeabilization and evidenceof internalization when secondaryantibody was applied after perme-abilization (data not shown). Treat-ment of APP C99-N2a cells withantibody 6E10 for 3 h did notincrease levels of C99 comparedwith untreated cells but rathershowed a trend for decrease (Fig.4D). Had 6E10 decreased �-secre-tase activity, C99 levels should haveincreased, as was seen when�-secretase was inhibited withDAPT (Fig. 4D). To examine theeffect of A� antibody treatmentwith inhibition of �-secretase activ-ity, we treated Sw-N2a cells for 3 hwith �-secretase inhibitor DAPT inthe presence or absence of 6E10.Levels of �CTFs were increased by19 � 6% in DAPT and 6E10 treatedcells compared with those treatedwith DAPT alone (supplementalFig. S9A). This result is consistentwith the data demonstrating thatA� antibody treatment increasedlevels of sAPP� and �CTF (Fig. 4Cand supplemental Fig. S8, B and C).Performing the same experiment onC99-N2a cells, which precludes�-cleavage, there was a trend for adecrease, which did not reach sig-nificance, in levels of C99 after 3 h oftreatment with DAPT and 6E10compared with DAPT alone (sup-plemental Fig. S9B). Since the A�antibody mediated reduction of A�did not appear to result fromdecreased secretase cleavage ofAPP, and since internalized A�antibodies trafficked to late endo-

FIGURE 4. A� antibody induced reduction of A� does not act via �- or �-secretase inhibition and requiresthe late endosomal/lysosomal system. A, levels of internalized biotinylated full-length APP in Sw-N2a cellstreated on ice with 6E10 or P2-1 antibody, followed by 45-min incubation at 37 °C, were reduced comparedwith untreated Sw-N2a cells (n � 3). Densitometric quantitation is expressed as a ratio of biotinylated APP tototal APP in treated compared with untreated cells. B, metabolic labeling of primary neurons pulsed for 20 minwith [35S]methionine and chased for 45 min in the presence or absence of antibody 6E10. In the presence of6E10 antibody, levels of newly generated A� were reduced compared with untreated controls (n � 4). Densi-tometric quantitation is expressed as relative amount of A� in treated compared with untreated cells. C, levelsof secreted APP� (sAPP�) were increased in conditioned media of Sw-N2a cells after 3-h incubation with A�antibody 6E10 (n � 4). Densitometric quantitation of sAPP� in treated compared with untreated cells is shown.D, A� antibody treatment of C99-N2a cells did not inhibit �-secretase cleavage. C99-N2a cells were treated for3 h with either A� antibody 6E10 or �-secretase inhibitor DAPT. There was a tendency for reduction in levels ofC99 in 6E10-treated cells compared with untreated C99-N2a cells. As expected, DAPT treatment induced anincrease in levels of C99 (n � 4). Densitometric quantitation of C99 in treated compared with untreated cells isshown. E, C99-N2a cells were incubated on ice with biotin in the presence or absence of A� antibody 6E10. After45 min of incubation with 6E10 at 37 °C, levels of internalized biotinylated C99 were reduced in C99-N2a cellscompared with untreated control cells (n � 3). Densitometric quantitation is expressed as a ratio of biotiny-lated C99 to total C99 in treated compared with untreated cells. F, lysosomal inhibition with chloroquine (100�M) prevented the A� antibody-mediated (6E10) reduction of intracellular A� (n � 4). Densitometric quanti-tation is expressed as relative amount of A� in treated compared with untreated cells (*, p � 0.05; **, p � 0.01).

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somes/lysosomes, we considered that A� antibodies promotethe late endosomal/lysosomal degradation of APP and APP-derived products, such as C99 and A�. To investigate whetherA� antibody treatment induced the degradation of C99, wecarried out biotin labeling on C99-N2a cells at 4 °C followed byincubation of cells at 37 °C in the presence or absence of A�antibody 6E10. After 45-min incubation with antibody 6E10,levels of biotinylated C99 decreased by 57 � 7% (Fig. 4E),consistent with A� antibody induced degradation of C99. Tofurther investigate whether the late endosomal/lysosomalsystem is involved in the A� antibody mediated reduction ofA�, we tested whether inhibition of late endosomal/lysoso-mal function would interfere with the ability of A� antibod-ies to reduce intracellular A�. Indeed, incubation of Sw-N2acells with 6E10 (3 h) in the presence of the lysosomal inhib-itor chloroquine (40) prevented and/or counterbalanced theA� antibody-induced reduction of intracellular A� (Fig. 4F;similar results were obtained using ammonium chloride;data not shown).Synaptic dysfunction is considered to be the earliest neu-

robiological alteration in AD (41, 42), and reduction of intra-neuronal A� was the best A�-correlate of cognitive improve-ment in an AD mouse model (12). Therefore, we examinedwhether reduction of intraneuronal A� by A� antibodiescould protect against the synaptic alterations that we previ-ously described in APP mutant neurons in culture (27). Weconfirmed that the number of PSD-95 puncta (an importantscaffold protein of the post-synaptic density) was reduced inprocesses of APP mutant compared with wild-type neuronsat 19 DIV (Fig. 5A). Immunofluorescence for synapsin-1, apresynaptic protein that remains unchanged in Tg2576 neu-rons (27), highlights the similarity of the neurites in the rep-resentative images (Fig. 5A). Remarkably, treatment ofTg2576 neurons with A� antibody 6E10 (or A� antibody4G8; supplemental Fig. S10) for 24 h restored the number ofPSD-95 puncta to 96 � 6% of wild-type levels (Fig. 5B; for anadditional representative figure, see supplemental Fig. S11).In contrast, treatment with a C terminus-specific A�42 anti-body (Chem42) was unable to restore PSD-95 puncta inTg2576 neurons (supplemental Fig. S12). Treatment of wild-type neurons with A� antibody 6E10 had no effect on levelsof PSD-95 puncta (data not shown).

DISCUSSION

A� immunotherapy remains an exciting therapeuticdirection for AD, although the biological mechanism(s)whereby A� antibodies reduce A� in brain and improve cog-nitive function in ADmouse models are incompletely under-stood. Several hypotheses have been proposed. The “sinkhypothesis” suggests that peripherally administrated A�antibodies can reduce levels of A� in the plasma and driveefflux of A� from the brain, where it is more concentrated, tothe periphery (9). Another hypothesis is based on evidencethat peripherally administrated A� antibodies can cross theblood brain barrier and enter the central nervous system (4),where A� antibodies can mediate degradation of A� aggre-gates by inflammatory cell activation (4, 43). However, evi-dence also indicates that Fc-mediated antibody-directed

microglial activation is not necessary to reduce A� plaques,since A� immunotherapy was effective in FcR-� chainknock-out/APP mutant transgenic mice (44) and F(ab�)2fragments reduced plaque pathology in Tg2576 mice (45).This suggests that other mechanism(s) may be occurring aswell. Recent evidence and our data suggest an additionalscenario where A� antibodies reduce intracellular A�.

Intraneuronal A� accumulation is increasingly being linkedwith early, preplaque electrophysiological, synaptic, and path-ological abnormalities (46). For example, A�42 accumulationand oligomerization were associated with ultrastructuralpathology within distal processes and synapses prior to, and inareas devoid of, plaques in AD transgenic mice and human ADbrain (17, 26). A� antibodieswere shown to reduce intracellularA� in triple transgenic mice (11), and this reduction in intra-cellular A� was the best correlate of cognitive improvement(12). The biological mechanism by which A� antibodiesreduced intracellular A� was unclear. Interestingly, antibodies

FIGURE 5. A� antibody treatment protects against synaptic alterations inAPP mutant neurons. A, cultured neurons from Tg2576 mice demonstrated amarked reduction in the number of PSD-95 puncta compared with neurons fromnon-transgenic mice (n � 5). PSD-95 puncta in Tg2576 neurons were restored towild-type levels after 24 h of treatment with A� antibody 6E10. B, quantificationof PSD-95 puncta density in APP mutant compared with non-transgenic neuronsafter 24 h of treatment with A� antibody 6E10 (n � 5). Synapsin-1 staining high-lights the similarity of the neurites in the representative images (**, p � 0.01relative to nontransgenic neurons; ##, p � 0.01 relative to untreated Tg2576 neu-rons; scale bar: 10 �m).

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against the cleavage site of BACE on APP (47) and intrabodiesagainst A� (48) were suggested as new cellular therapeuticstrategies for AD.We provide evidence that antibodies against A�, previously

shown by multiple groups to reduce plaque pathology in vivo(8), reduce intracellular levels of A� in cultured neurons bybinding to the extracellular A� domain and protect against syn-aptic alterations in APPmutant neurons.We demonstrate thatA� antibodies directed to the N-terminal tomid-domain of A�can be specifically endocytosed after binding at the cell surface.Our results could be important in why passive immunization isespecially effectivewhen using antibodies directed at theN-ter-minal region of A� (4, 11, 12, 49, 50).A potential mechanism by which A� antibodies reduced

intracellular A� could have been inhibition of �- or �-secretaseactivities. We did not observe such inhibition. Specifically, theincrease in sAPP� and, in C99-N2a cells, the lack of an increasein C99 with A� antibody treatment argued against �- or�-secretase inhibition, respectively. In fact, the increase of C99in cells expressing full-length APP in conjunction withincreased sAPP� secretion suggests that the internalization offull-length APP induced by A� antibody treatment actuallypromotes BACE cleavage. Another possible mechanism for theantibody mediated reduction in A� could have been fromincreased clearance of intracellular to extracellular A�,although the lack of an increase in levels of extracellular A�argues against this possibility. In fact, there was a trend fordecreased A� secretion with A� antibody treatment that didnot reach significance. Another potential mechanism for A�antibody-mediated clearance of intracellular A� is by enhanc-ing cellular degradation after antibody binding to the A�domain of cell surface APP. Since A� antibodies directed to theC terminus of A� did not reduce intracellular A�, we hypoth-esized that A� antibodies act by binding to full-length APPand/or APPCTFs and not on potentially surface-associated A�in our experiments. The binding and internalization of antibod-ies to the ectodomain of cell surface APPwas previously used tostudy APP internalization and trafficking and was employed tofollowAPP to endosomal-lysosomal compartments (37, 38, 51).Recent studies are increasingly suggesting an important role ofendosomes inAPPprocessing; for example, themajority ofAPPtransported down axons by fast axonal transport was reportedto be full-length, supporting endosomal secretase cleavage inneurons (52). Interestingly, alterations in the endosomal-lyso-somal system are among the earliest changes described in ADandDown syndrome brains (53). Our data provide evidence forinvolvement of the endosomal/lysosomal system in intraneuro-nal A� clearance after A� antibody treatment. Co-localizationof endocytosedA� antibodywithA�42was evident at 1-h incu-bation, when the internalized antibody co-stained with lateendosomal/lysosomal markers and not at 10 min when inter-nalized antibody co-localized with early endosomes. Ourresults are most consistent with A� antibody-inducedincreased internalization of APP from the cell surface to earlyendosomes, followed by increased �-cleavage (elevated C99)and then enhancement of C99 trafficking to the late endosomallysosomal pathway for degradation. We hypothesize thatenhanced trafficking of A� antibody bound C99 through late

endosomes where �-secretase components have been localized(54, 55) limits �-cleavage and thereby also increased A� secre-tion. That inhibition of late endosomes/lysosomes with chloro-quine or ammonium chloride prevented A� clearance after A�antibody treatment supports the involvement of the endoso-mal-lysosomal system in the reduction of A�. Since both APPectodomain and A� antibodies promoted surface APP reduc-tion, although the latter were more effective, only A� antibod-ies reduced levels of cellular A�, and this suggests that the dis-sociation of the APP ectodomain antibody from �CTFs afterBACE cleavage might preclude the enhanced degradation thatoccurs when A� antibody remains bound to the A� domain ofC99. Our results support the scenario of A� antibodies induc-ing the internalization of APP from the plasma membrane toearly endosomes where increased BACE cleavage appears tooccur, followed by induction of the late endosomal-lysosomal-dependent clearance of �CTFs, and potentially A�. Since A�antibody alone tends to decrease levels of C99 in C99-N2a cells,our results suggest increased degradation of C99 and A� ratherthan�-secretase inhibition. The lack of a statistically significantdecrease of�CTFs in C99-N2a cells treatedwithDAPT andA�antibody compared with DAPT alone might be due to alteredtrafficking of C99 upon �-secretase inhibition that therebyinhibits C99 degradation. In fact, altered �CTF trafficking wasreported in neurons of PS1 conditional knock-out mice, where�CTFs accumulated abnormally at synapses (56).Our data can-not fully exclude that A� antibody treatment promotes �CTFprocessing by �-secretase followed by increased degradation ofthe resultant A�-A� antibody complex rather than, or in addi-tion to, primarily promoting degradation of the �CTF-A� anti-body complex.A� antibodies have been reported to block alterations of syn-

apses induced by extracellular A� oligomers (50, 57). ThatC-terminal specific A� antibodies can also be protective in A�immunotherapy supports that antibody effects on extracellularA� are also involved (58, 59). Increasing evidence supports anas yet poorly understood dynamic relationship between extra-cellular and intracellular A�, modulation of which might beespecially important in A� antibody induced therapeuticeffects (60). High levels of extracellular A� were shown toinduce up-regulation of newly generated intracellular A�42(61). The mechanism whereby extracellular A� causes celldeath in cultured neurons appears to be related to a dynamicrelationship also between extracellular A� and cell surfaceAPP, since toxicity did not occur in APP knock-out neurons(62) or cells harboring mutations in the YENPTY motif withinthe C terminus of APP (63). Thus, neurotoxicity might addi-tionally require effects on intracellular A�.In summary, in addition to effects on inflammatory mecha-

nisms of A� clearance and on extracellular A� oligomers,among others, our data underscores that another mechanismwhereby A� antibodies may play a critical role in A� immuno-therapy is via reduction in intracellular A�. A better under-standing of themolecularmechanism(s) whereby A� immuno-therapy leads to reduced A� accumulation and improvedcognitive function may lead to novel therapeutic approachesfor AD.

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Acknowledgments—We thankGopal Thinakaran and Sangram Siso-dia (University of Chicago, Chicago, IL) for providing Sw-N2a cells,Richard B. Vallee (Columbia University, New York) for providing thedynamin constructs, andWilliamNetzer and Paul Greengard (Rock-efeller University, New York) for providing the N2a-APPC99-trans-fected cells. We appreciate the technical assistance from CharlaFisher. We are grateful to Noel Y. Calingasan for technical expertiseand helpful discussions and to Paul Szabo and Marc Weksler forhelpful dicussions.

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