the effects of amyloid precursor protein on postsynaptic

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The Effects of Amyloid Precursor Protein on Postsynaptic Composition and Activity * S Received for publication, January 8, 2009 Published, JBC Papers in Press, January 21, 2009, DOI 10.1074/jbc.M900141200 Hyang-Sook Hoe ‡1 , Zhanyan Fu §1 , Alexandra Makarova , Ji-Yun Lee , Congyi Lu § , Li Feng § , Ahdeah Pajoohesh-Ganji , Yasuji Matsuoka** 2 , Bradley T. Hyman , Michael D. Ehlers ‡‡3 , Stefano Vicini § , Daniel T. S. Pak , and G. William Rebeck ‡4 From the Departments of Neuroscience, § Physiology and Biophysics, Pharmacology, and **Neurology, Georgetown University Medical Center, Washington, D. C. 20057-1464, Harvard Medical School, Massachusetts General Hospital, MassGeneral Institute for Neurodegenerative Disorders, Charlestown, Massachusetts 02129, and ‡‡ Howard Hughes Medical Institute, Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710 The amyloid precursor protein (APP) is cleaved to produce the Alzheimer disease-associated peptide A, but the normal functions of uncleaved APP in the brain are unknown. We found that APP was present in the postsynaptic density of central exci- tatory synapses and coimmunoprecipitated with N-methyl-D- aspartate receptors (NMDARs). The presence of APP in the postsynaptic density was supported by the observation that NMDARs regulated trafficking and processing of APP; overex- pression of the NR1 subunit increased surface levels of APP, whereas activation of NMDARs decreased surface APP and pro- moted production of A. We transfected APP or APP RNA interference into primary neurons and used electrophysiologi- cal techniques to explore the effects of APP on postsynaptic function. Reduction of APP decreased (and overexpression of APP increased) NMDAR whole cell current density and peak amplitude of spontaneous miniature excitatory postsynaptic currents. The increase in NMDAR current by APP was due to specific recruitment of additional NR2B-containing receptors. Consistent with these findings, immunohistochemical experi- ments demonstrated that APP increased the surface levels and decreased internalization of NR2B subunits. These results dem- onstrate a novel physiological role of postsynaptic APP in enhancing NMDAR function. Alzheimer disease (AD) 5 is an age-related neurodegenerative disease characterized by the progressive loss of synapses and neurons and by the formation of amyloid plaques and neurofi- brillary tangles. Amyloid plaques are composed predominantly of the A peptide, a 40- or 42-amino acid cleavage product of amyloid precursor protein (APP). APP is a transmembrane pro- tein of unknown function that undergoes extracellular cleavage by one of two activities, - or -secretase, resulting in the for- mation of large N-terminal extracellular fragments of secreted APP and smaller, membrane-bound C-terminal fragments. If the initial cleavage event occurs via -secretase, then subse- quent cleavage of the C-terminal fragment by -secretase results in the production of A (1). Clues to APP function may be gleaned from studies of its different cleavage products or isoforms. Soluble forms of APP have been found to be neurotrophic, and some splice variants have proteinase inhibitor activity (2). The APP intracellular domain may alter gene transcription in conjunction with cyto- plasmic proteins (3). In addition, the A peptide has been shown to inhibit glutamate receptor activity (4, 5). However, the function of full-length APP is undefined, and it is likely that full-length APP performs distinct roles from any its cleavage products. One recent study showed that mice lacking APP have impaired development of neuromuscular junctions (6), sug- gesting an important role for APP in maintaining active syn- apses. Understanding APP function in central neurons may provide valuable information in generating interventions against the generation of A and thus AD pathogenesis and its accompanying memory loss. Further evidence for a synaptic function of APP comes from studies indicating that APP processing is regulated by synaptic activity. Neuronal activity increased production of A in hip- pocampal slices (7) and primary neurons (8). This effect on APP processing may be due to activation of the NMDA subtype of glu- tamate receptors, which inhibited -secretase cleavage of APP in primary neurons (8) and in the brain (9) and would therefore favor -secretase cleavage and A production. This mechanism of action is still controversial, since another study showed that NMDAR activation led to increased -cleavage of APP (10). NMDARs are critical for many forms of synaptic plasticity, including hippocampal CA1 long term potentiation and long * This work was supported, in whole or in part, by National Institutes of Health Grants AG14473 (to G. W. R.), NS48085 (to D. T. S. P.), NS047700 (to S. V.), AG12406 (to B. T. H.), AG032330 (to H. S. H.). This work is also supported by the Alzheimer’s Research Fund in memory of Bill and Marie Drach. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. 1 Both of these authors contributed equally to this work. 2 Present address: Neurodegeneration Research, GlaxoSmith Kline, Shanghai 201203, China. 3 Investigator of the Howard Hughes Medical Institute. 4 To whom correspondence should be addressed: Dept. of Neuroscience, Georgetown University Medical Center, 3970 Reservoir Rd. NW, Washing- ton, D. C. 20057-1464. Tel.: 202-687-1534; Fax: 202-687-0617; E-mail: [email protected]. 5 The abbreviations used are: AD, Alzheimer disease; APP, amyloid precursor protein; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; RNAi, RNA interference; PSD, postsynaptic density; mEPSC, miniature excitatory postsynaptic current; GFP, green fluorescent protein; siRNA, small interfer- ing RNA; -ME, -mercaptoethanol; PBS, phosphate-buffered saline; DIV, day(s) in vitro; CGC, cerebellar granule cell; pF, picofarads. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 13, pp. 8495–8506, March 27, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. MARCH 27, 2009 • VOLUME 284 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 8495 by guest on April 6, 2018 http://www.jbc.org/ Downloaded from

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Page 1: The Effects of Amyloid Precursor Protein on Postsynaptic

The Effects of Amyloid Precursor Protein on PostsynapticComposition and Activity*□S

Received for publication, January 8, 2009 Published, JBC Papers in Press, January 21, 2009, DOI 10.1074/jbc.M900141200

Hyang-Sook Hoe‡1, Zhanyan Fu§1, Alexandra Makarova¶, Ji-Yun Lee�, Congyi Lu§, Li Feng§,Ahdeah Pajoohesh-Ganji‡, Yasuji Matsuoka**2, Bradley T. Hyman¶, Michael D. Ehlers‡‡3, Stefano Vicini§,Daniel T. S. Pak�, and G. William Rebeck‡4

From the Departments of ‡Neuroscience, §Physiology and Biophysics, �Pharmacology, and **Neurology, Georgetown UniversityMedical Center, Washington, D. C. 20057-1464, ¶Harvard Medical School, Massachusetts General Hospital, MassGeneral Institutefor Neurodegenerative Disorders, Charlestown, Massachusetts 02129, and ‡‡Howard Hughes Medical Institute, Department ofNeurobiology, Duke University Medical Center, Durham, North Carolina 27710

The amyloid precursor protein (APP) is cleaved to producethe Alzheimer disease-associated peptide A�, but the normalfunctions of uncleavedAPP in the brain are unknown.We foundthat APPwas present in the postsynaptic density of central exci-tatory synapses and coimmunoprecipitated with N-methyl-D-aspartate receptors (NMDARs). The presence of APP in thepostsynaptic density was supported by the observation thatNMDARs regulated trafficking and processing of APP; overex-pression of the NR1 subunit increased surface levels of APP,whereas activation ofNMDARs decreased surfaceAPP and pro-moted production of A�. We transfected APP or APP RNAinterference into primary neurons and used electrophysiologi-cal techniques to explore the effects of APP on postsynapticfunction. Reduction of APP decreased (and overexpression ofAPP increased) NMDAR whole cell current density and peakamplitude of spontaneous miniature excitatory postsynapticcurrents. The increase in NMDAR current by APP was due tospecific recruitment of additional NR2B-containing receptors.Consistent with these findings, immunohistochemical experi-ments demonstrated that APP increased the surface levels anddecreased internalization of NR2B subunits. These results dem-onstrate a novel physiological role of postsynaptic APP inenhancing NMDAR function.

Alzheimer disease (AD)5 is an age-related neurodegenerativedisease characterized by the progressive loss of synapses and

neurons and by the formation of amyloid plaques and neurofi-brillary tangles. Amyloid plaques are composed predominantlyof the A� peptide, a 40- or 42-amino acid cleavage product ofamyloid precursor protein (APP). APP is a transmembrane pro-tein of unknown function that undergoes extracellular cleavageby one of two activities, �- or �-secretase, resulting in the for-mation of large N-terminal extracellular fragments of secretedAPP and smaller, membrane-bound C-terminal fragments. Ifthe initial cleavage event occurs via �-secretase, then subse-quent cleavage of the C-terminal fragment by �-secretaseresults in the production of A� (1).

Clues to APP function may be gleaned from studies of itsdifferent cleavage products or isoforms. Soluble forms of APPhave been found to be neurotrophic, and some splice variantshave proteinase inhibitor activity (2). The APP intracellulardomain may alter gene transcription in conjunction with cyto-plasmic proteins (3). In addition, the A� peptide has beenshown to inhibit glutamate receptor activity (4, 5). However,the function of full-length APP is undefined, and it is likely thatfull-length APP performs distinct roles from any its cleavageproducts. One recent study showed thatmice lacking APP haveimpaired development of neuromuscular junctions (6), sug-gesting an important role for APP in maintaining active syn-apses. Understanding APP function in central neurons mayprovide valuable information in generating interventionsagainst the generation of A� and thus AD pathogenesis and itsaccompanying memory loss.Further evidence for a synaptic function of APP comes from

studies indicating that APP processing is regulated by synapticactivity. Neuronal activity increased production of A� in hip-pocampal slices (7) and primary neurons (8). This effect onAPPprocessingmay be due to activation of theNMDA subtype of glu-tamate receptors, which inhibited �-secretase cleavage of APP inprimaryneurons (8) and in the brain (9) andwould therefore favor�-secretase cleavage and A� production. This mechanism ofaction is still controversial, since another study showed thatNMDAR activation led to increased �-cleavage of APP (10).

NMDARs are critical for many forms of synaptic plasticity,including hippocampal CA1 long term potentiation and long

* This work was supported, in whole or in part, by National Institutes of HealthGrants AG14473 (to G. W. R.), NS48085 (to D. T. S. P.), NS047700 (to S. V.),AG12406 (to B. T. H.), AG032330 (to H. S. H.). This work is also supported bythe Alzheimer’s Research Fund in memory of Bill and Marie Drach. Thecosts of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1 and S2.

1 Both of these authors contributed equally to this work.2 Present address: Neurodegeneration Research, GlaxoSmith Kline, Shanghai

201203, China.3 Investigator of the Howard Hughes Medical Institute.4 To whom correspondence should be addressed: Dept. of Neuroscience,

Georgetown University Medical Center, 3970 Reservoir Rd. NW, Washing-ton, D. C. 20057-1464. Tel.: 202-687-1534; Fax: 202-687-0617; E-mail:[email protected].

5 The abbreviations used are: AD, Alzheimer disease; APP, amyloid precursorprotein; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; RNAi, RNAinterference; PSD, postsynaptic density; mEPSC, miniature excitatory

postsynaptic current; GFP, green fluorescent protein; siRNA, small interfer-ing RNA; �-ME, �-mercaptoethanol; PBS, phosphate-buffered saline; DIV,day(s) in vitro; CGC, cerebellar granule cell; pF, picofarads.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 13, pp. 8495–8506, March 27, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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term depression, and consist of an obligate NR1 subunit andvariable NR2 or NR3 subunits (11, 12). The NR2 subunit typedetermines important pharmacological and biophysical prop-erties of NMDARs (13–16). NMDARs may also play an impor-tant role in AD, since memantine, an NMDAR antagonist, is adrug approved for moderate to serious forms of this disease.We now report a novel physical and functional interaction

betweenNMDARs andAPP. In contrast to the inhibitory effectof A� on glutamatergic function, full-length APP promotedNMDAR activity in primary hippocampal neurons, as meas-ured by increasedwhole cell current density, surface expressionof NMDAR, and analysis of spontaneous miniature excitatorypostsynaptic current (mEPSC) through NMDAR. Consistentwith this role, reduction of endogenous APP levels via RNAinterference (RNAi) decreased NMDAR current density andsurface levels. Conversely, NMDARexpression and activity lev-els regulated APP trafficking and processing in primary neu-rons. These data reveal synaptic functions of APP and demon-strate the reciprocal effects of NMDARs and APP on proteintrafficking and metabolism.

MATERIALS AND METHODS

Vector Construction—N-terminal tagged GFP for NR1,NR2A, and NR2B was used for cell surface and cell internaliza-tion assays (17).We produced full-length APP770with aN-ter-minal GFP tag or a C-terminal ofMyc tag.We also produced anM761V construct of GFP-tagged full-lengthAPP770 using site-directed mutagenesis (Stratagene) and a C-terminal Myc-tagged construct of APP C99. To generate the APP short inter-fering RNA constructs, we used pSuper vector, which expressesan siRNAunder the control of theH1promoter and can be usedfor prolonged suppression of specific gene expression (18).Using Invitrogen BLOCK-iTTM RNAi Designer to identifysequences within the cDNA for silencing, APP-siRNAsequences were targeted against rat APP open reading frame(NM_019288). The sequences for the RNAis used in this studywere as follows: number 7, GGTGCCTAGTTGGTGAGTT;number 14, GCACTAACTTGCACGACTA; number 37,GCGTGTCATCCCAAAGTTT.Animals—1-month-oldwild-type C57BL/6J andAPP knock-

out mice B6.129S7-APPtm1DBo/J were purchased from JacksonLaboratory. 1-month-old Sprague-Dawley rats were purchasedfromTaconic. All animal protocols were approved by theGeor-getownUniversity Institutional Animal Use and Care Commit-tee and were in compliance with the standards stated in Ref. 48.Synaptosomal Preparations—Synaptosome fractionation

was performed as described previously (19) with minor modi-fications. Briefly, brains from3-month-old,wild-typeC57BL/6Jand APP knock-out B6.129S7-APPtm1DBo/J 3 mice werehomogenized in 0.32 M sucrose, 4 mM HEPES-NaOH, pH 7.3,with protease inhibitors and centrifuged at 1000 � g for 10minto recover the supernatant S1 and the pellet P1. S1 fraction wascentrifuged at 12,000 � g for 15 min to obtain the pellet P2(crude synaptosome) and the supernatant S2. P2 fraction wastreated with an osmotic shock by diluting with double-distilledwater and further centrifuged at 25,000 � g for 20 min to gen-erate the pellet LP1 and the supernatant LS1. The LS1 fractionwas subjected to ultracentrifugation to obtain the synaptic ves-

icle fraction. LP1 was detergent extracted in buffer B (0.16 Msucrose, 5 mM Tris-HCl, pH 8.0, 0.5% Triton X-100, 0.5 mM�-ME, 1 mM EDTA, and protease inhibitors) and then centri-fuged at 33,000 � g for 20 min. The pellet LP1P was resus-pended and applied to a discontinuous sucrose gradient con-sisting of 1.0, 1.5, and 2.0 M sucrose layers. Afterultracentrifugation (200,000 � g for 2 h), the postsynaptic den-sity (PSD) fraction was recovered at the interface between 1.5and 2.0 M sucrose.Cell Lines and Culture Conditions—COS7 cells were main-

tained in Opti-MEM (Invitrogen) with 10% fetal bovine serum(Invitrogen) in a 5%CO2 incubator. COS7 cells were transientlytransfected with 0.5–1 �g of plasmid in FuGENE6 (RocheApplied Science) according to the manufacturer’s protocol andcultured for 24 h in DMEM containing 10% fetal bovine serum.For co-transfections, cells were similarly transfectedwith 0.5–1�g of each plasmid in Fugene 6 (Roche Applied Science) andcultured 24 h in DMEM with 10% fetal bovine serum.Antibodies—The following antibodies were used: anti-GFP

(Sigma), anti-GABA-A (Chemicon International, Temecula,CA), anti-synaptophysin (Sigma), anti-synapsin, and anti-PSD-95 (Chemicon International). For analysis of APP, we used6E10 (Signet, Dedham, MA), 22C11 (Chemicon International),and rabbit anti-APP (Sigma); Dr. Paul Mathews (Nathan S.Kline Institute, Orangeburg, NY) provided antibodies c1/6.1and M3.2; and Dr. Sam Gandy (Thomas Jefferson University,Philadelphia, PA) provided antibody 369, which all recognizethe C terminus of APP. Antibodies against NR1 (monoclonal,recognizing amino acids 656–811), NR2A (polyclonal, againstamino acids 934–1142), and NR2B (monoclonal, recognizingamino acids 934–1457) were generous gifts of Dr. Barry Wolfe(Georgetown University).APP Analysis—Cell-associated human and rodent APP was

measured in Western blots with the APP N-terminal antibody(Sigma), 22C11 (Chemicon), or C1/6.1. Rodent APP was alsomeasured inWestern blotswith antibodyM3.2. RodentA�-(1–40) in conditionedmedia was measured by ELISA, as described(20). A�-(1–42) peptidewas purchased fromAmericanPeptide(dissolved in DMSO). For immunostaining of APP, cells werefixed in cold (�20 °C) methanol for 10 min. After fixation, cellswere washed with PBS and incubated with anti-APP and anti-PSD-95 antibody overnight at 4 °C. After primary incubation,cells were washed with PBS and then incubated with AlexaFluor 488 (green color) goat anti-rabbit antibody (MolecularProbes) and Alexa Fluor 555 (red color) goat anti-mouse anti-body for 1 h at room temperature. Stained cells were viewedwith a confocal laser-scanning microscope.Co-immunoprecipitation—Adult wild-type mouse brains or

APP knock-outmouse brains were perfusedwith PBS and lysedin buffer containing 50 mM Tris-HCl (pH 8.0), 0.15 M NaCl, 1%Nonidet P-40, 0.5% sodium deoxycholate, phosphatase, andprotease inhibitors. For immunoprecipitation, lysates wereincubated for 2 h at 4 °C with the anti-APP (22C11) antibody oran anti-NR1 antibody bound to protein G-Sepharose beads(Amersham Biosciences). The precipitates were then washedthree times with buffer and resuspended in SDS sample buffer.The samples were separated by SDS-PAGE on 4–15% polyac-rylamide gels, transferred electrophoretically to nitrocellulose

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membranes, and blocked with 5% nonfat dry milk. The blotswere incubated with antibodies at room temperature for 1 h.Horseradish peroxidase-conjugated secondary antibodies werevisualized by the ECL detection system and exposed to film.Primary Neuron Culture and Transfection—Hippocampal

neurons from embryonic day 18–19 Sprague-Dawley rats werecultured at 150 cells/mm2 as described previously (21). Neu-rons were transfected at 10 or 12 days in vitro (DIV) by calciumphosphate precipitation (4–5�g ofDNA/well). 8–14 days aftertransfection, we analyzed cell surface expression levels of APPandNMDARs or the endocytosis of APP andNMDARs in vitro(DIV 16–21). Cerebellar granule cells (CGc) were culturedfrompostnatal day 5–7mouse pups and plated at 0.8–1.0� 106cells/ml, as described previously (22, 23). CGCs were trans-fected at 5 days in vitro by calcium phosphate precipitation in a4-well dish (3�g of DNA/well).Whole cell patch clamp record-ings were performed 2–3 days after transfection.Primary cultures of mouse CGCs were prepared from post-

natal day 5–7 mice and plated in a culture medium containing25mMKCl (23). AtDIV 5, themediumwas replacedwith low (5mM) KCl medium to facilitate formation of neuronal networksbetween CGCs and GABAergic interneurons in culture (24).CGCs were transfected as above.Surface Protein Assay—Immunostaining of surface APP,

NR1, and NR2B was performed as described previously (25).Briefly, live neuronal cultures were incubated with antibodiesdirected against extracellular N termini of human APP or GFP(10 �g/ml in conditioned medium) for 10 min to specificallylabel surface receptors and then lightly fixed for 5 min in 4%paraformaldehyde (nonpermeabilizing conditions). After fixa-tion, the surface-remaining antibody-labeled APP or GFP wasmeasured with Alexa Fluor 555-linked �-mouse secondaryantibodies for 1 h. Immunostaining was quantified usingMeta-morph analysis of immunostaining intensity from z-stackedimages from a Zeiss LSM510 confocal microscope (25). Surfacelocalization of staining was also confirmed visually from theseimages.Internalization Assay—Immunostaining of endocytosis of

GFP-tagged NR2 subunits was performed as described (18).Live neuronal cultures were incubated with antibodies directedagainst extracellular N termini of human GFP (10 �g/ml inconditioned medium) for 10 min and washed with prewarmedDMEM. Following this step, the coverslip was placed in theconditionedmedium and was incubated in a CO2 incubator for10 min and 30 min. After incubation, cells were fixed for 5 minin 4% paraformaldehyde, and the surface-remaining antibody-labeled NMDAR subunit was saturated with Alexa Fluor 647-linked �-rabbit secondary antibody for 2 h. After secondaryincubation, the cells were washed with PBS three times for 10min, and cells were permeabilized by incubating the coverslipsin�20 °Cmethanol for 90 s. Then coverslips were incubated inAlexa Fluor 555-linked�-mouse secondary antibodies for 1 h todecorate the internalized NMDAR subunits.Quantification and Image Analysis—Images were collected

using a Zeiss LSM510 confocal microscope (Carl Zeiss, Thorn-wood,NY). Confocal z-series image stacks encompassing entireneurons were analyzed using Metamorph software (UniversalImaging Corp., Downingtown, PA). For measures of surface or

internalized APP, dendrites from hippocampal neurons werecarefully traced, and surface fluorescence intensities weredetermined for the traced region.Biotin-labeled Cell Surface Proteins—Primary cortical neu-

rons were treated with NMDA (10 �M) and MK801 (10 �M).After 24 h, cells were washed, and surface proteins were labeledwith sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropi-onate at 500 �g/ml in PBS, including 1 mM MgCl2 and CaCl2(Pierce) under gentle shaking at 4 °C for 30 min. After quench-ing, cells were lysed, disrupted by sonication, and clarified bycentrifugation (10,000 � g, 2 min). To isolate biotin-labeledproteins, lysatewas added to immobilizedNeutrAvidinTMGeland incubated for 1 h at room temperature. Gels were washedand incubated with SDS-PAGE sample buffer, including 50mM

dithiothreitol. Eluants were analyzed by immunoblotting.Whole Cell Recordings—Coverslips with CGCs were placed

on the stage of an inverted microscope (TM2000; Nikon)equipped with fluorescent and phase-contrast optics. Allrecordings were performed at room temperature (24–26 °C)from neurons maintained for 7–8 days in vitro. Continuouslyperfused extracellular solution contained 145 mM NaCl, 5 mM

KCl, 1mMMgCl2, 1mMCaCl2, 5mMHepes, 5mM glucose, 0.25mg/liter phenol red, and 10 �M D-serine (all from Sigma)adjusted to pH 7.4 with NaOH. Osmolarity was adjusted to 325mosmol/liter with sucrose. NMDA was applied in a Ca2�- andMg2�-free extracellular solution to prevent Ca2�-mediatedinactivation of NMDAR channels. Electrodes were pulled intwo stages on a vertical pipette puller to a resistance of 5–8Mohm from borosilicate glass capillaries (Wiretrol II, Drum-mond, Broomall, PA) and filled with recording solution con-taining 145 mM potassium gluconate, 10 mM Hepes, 5 mM

MgATP, 0.2 mM NaGTP, and 10 mM 1,2-bis(2-aminophe-noxy)ethane-N,N,N�,N�-tetraacetic acid, adjusted to pH 7.2with KOH. Whole cell voltage clamp recordings from CGCswere made at �60 mV and performed at room temperatureusing an Axopatch 200 or an Axopatch-1D amplifier (AxonInstruments, Union City, CA). A transient current response toa hyperpolarizing 10-mV pulse was used to assess membraneinput resistance and capacitance throughout the recordings.Currents were filtered at 2 kHz with an 8-pole low pass Besselfilter (Frequency Devices, Haverhill, MA), digitized at 5–10kHz using an IBM-compatible microcomputer equipped withDigidata 1322 A data acquisition board and pCLAMP9.2 soft-ware (both from Axon Instruments). Off-line data analysis,curve fitting, and figure preparation were performed withClampfit 9 software and Minianalysis software (Synaptosoft,Decatur, GA).NMDA-mEPSCs were pharmacologically isolated using 25

�M bicuculline metabromide, 0.5 �M tetrodotoxin, and 2,3-di-oxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfono-mide (NBQX) (5 �M) (all from Sigma) in a Mg2�-free solution.AMPA-mEPSCs were recorded in the presence of 25 �M bicu-cullinemetabromide, 0.5�M tetrodotoxin in extracellular solu-tion withMg2� with a holding potential at �60 mV. The decayof NMDA-mEPSC was fitted using Clampfit 9 (Axon Instru-ments) fromaverages of several events selectedwithMinianaly-sis. The decay phase of currents was fitted using a simplex algo-

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rithm for least squares exponential fitting routine with a doubleexponential equation of the following form,

I�t� � I1 exp��t/�1� � I2 exp��t/�2� (Eq. 1)

where Ix represents the peak current amplitude of a decay com-ponent and x is the corresponding decay time constant. Toallow for easier comparison of decay times between experimen-tal conditions, the two decay time components were combinedinto a weighted time constant.

�W � �I1/�I1 � I2�� � �1 � �I2/�I1 � I2�� � �2 (Eq. 2)

Statistical Analyses—All data are expressed as means � S.E.Data were analyzed using analysis of variance with GraphpadPrism 4 software, using Tukey’s multiple comparison test forpost hoc analyses with significance determined as p � 0.05.Electrophysiology data were analyzed by using unpaired Stu-dent’s t test (two-tailed) with Bonferroni corrections.

RESULTS

APP Is Part of the Postsynaptic Density—Immunostaining ofendogenous APP in primary rat hippocampal neurons at DIV14 demonstrated that APP was expressed at puncta along neu-ronal processes, which co-localized with synaptic markersPSD-95 (Fig. 1A) and synaptophysin (data not shown). As acontrol, we immunostained primary hippocampal neuronsfrom wild-type and APP knock-out mice with the same anti-body. As expected, APP knock-out cultures showed dramati-cally reduced APP immunostaining compared with controlswhile still showing evidence of PSD-95 puncta (Fig. 1B). Previ-ous studies have shown that APP is presynaptic but have not

determined whether it is also postsynaptic (26–28). Using syn-aptosomal preparations from brains of wild-type and APPknock-out mice, we generated synaptic vesicle and PSD frac-tions. Synaptic vesicle fractions contained synaptophysin andsynapsin, and PSD fractions contained PSD-95 and theNMDAR subunit, NR1, as expected (Fig. 1C). As detected withtwo APP antibodies (monoclonal C1/6.1 and rabbit polyclonalanti-APP), APP was present in both the synaptic vesicle andPSD fractions (Fig. 1C, left). No APP immunoreactive bandswere present in the preparations from APP knock-out mousebrain (Fig. 1C, right), demonstrating the specificity of the anti-bodies for APP and not for other APP family members. Isola-tions of pre- and postsynaptic fractions were again demon-strated by the presence of specificmarkers, althoughwe did notattempt to determine whether absolute levels of these markerswere altered in APP knock-out mice. These data demonstrateboth a pre- and postsynaptic localization of APP.APP Associates with NMDARs—As an independent

approach to defining whether APP is present postsynaptically,we tested whether there was a physical association betweenAPP and proteins of the postsynaptic density. We performedco-immunoprecipitations from whole brain lysates, using theanti-369 and a nonspecific IgG as a negative control. Immuno-precipitation of APP resulted in the co-precipitation of PSD-95and NR1 (Fig. 2A). Neither protein was precipitated from wildtype brain lysates with the IgG (Fig. 2A). As an additional neg-ative control, immunoprecipitation with the APP antibody didnot recover NR1 from lysates from brains of mice that lackedAPP and the related family member APLP1 (data not shown).The nonsynaptic GABAA receptor did not co-precipitate withAPP in lysates fromwild-typemice (Fig. 2A), further supportingthe synaptic localization of APP. We also tested whether APPassociated with a postsynaptic marker in primary neurons. Wefound that APP co-precipitated with an antibody against NR1but not with a control antibody (Fig. 2B). Similarly, immuno-precipitation of APP resulted in co-precipitation of NR1 (Fig.2C, lane 2).NMDA Receptor Activity Regulates APP Processing—As a

third test of whether APPwas present in the PSD, we examinedwhether manipulating postsynaptic neuronal activity affectedAPP trafficking and processing. Based on a previous studyshowing that NMDA receptor activity can promote amyloido-

FIGURE 1. APP is localized to synapses. A, after 14 days in culture, primaryhippocampal neurons were fixed and immunostained for APP (with rabbitanti-APP antibody (Sigma)) (A1) and for PSD-95 (A2). Co-localization of APPand PSD-95 appears yellow in the right panels (A3). Dendritic segments athigher magnification are shown below each image. Scale bars, 10 �m (top)and 5 �m (bottom). B, primary hippocampal neurons from wild-type (wt) mice(top) and APP knock-out (ko) mice (bottom) were immunostained as above forAPP (red) and PSD-95 (green). C, immunoblot analysis of APP (rabbit poly-clonal and C1/6.1 monoclonal antibodies), NR1, synapsin, synaptophysin, andPSD-95 in brain homogenate fractions of presynaptic vesicles (SV) and PSDsfrom wild-type mice (left) and APP knock-out mice (right).

FIGURE 2. NMDA receptors interact with APP in primary cortical neuronsand brain lysates. A, deoxycholate extracts of mouse brain lysates (300 �g)were immunoprecipitated (IP) with antibody (Ab) 369 (lane 3) or control anti-body (lane 2) and probed for APP (c1/6.1), PSD-95, NMDA R1, or GABAA recep-tor. Input (lane 1) represents 5% of the lysate used for immunoprecipitation.B and C, cell lysates of primary cortical neurons (3 mg) were immunoprecipi-tated with anti-NR1 antibodies or irrelevant control antibody (�-P-JNK) andimmunoblotted for APP (B) or immunoprecipitated with anti-APP antibodiesor control antibody and probed for NR1 (C). Immunoblots below each immu-noprecipitation show total levels of the indicated protein in cell lysates. Anasterisk marks IgG heavy chain.

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genic processing of APP (7), we tested whether NMDA treat-ment could regulate proteolysis of endogenous APP in our cul-tures of primary cortical neurons. NMDA treatment (10 �M,24 h) of cultures at DIV 14 significantly decreased secretedAPPby over 80% without changing total cellular APP (representa-tive blot in Fig. 3A and quantification of blots in Fig. 3B). Thesedata are consistent with previous findings that NMDA receptoractivation inhibited �-cleavage of APP (8). Control treatmentsof primary neurons with NMDAR antagonists MK-801 (Fig. 3,A and B) and AP-5 (data not shown) alone had no effect, butMK-801 treatment blocked the effects of NMDA on APP proc-essing (Fig. 3,A and B). NMDAR activation and inhibition werenot toxic in these cultures, as measured by LDH release (datanot shown). To test whether NMDAR activity affected produc-tion of endogenous A�, we treated primary cortical neuronswith NMDA, MK801, or both NMDA and MK801 for 24 h.

NMDA greatly increased A�40 lev-els (189% increase versus untreatedcontrol, p 0.02), and this effectwas largely blocked byMK-801 (Fig.3C). These findings support a rolefor NMDAR activity in the regula-tion of APP processing that shiftsthe balance from �- to �-secretaseprocessing.We hypothesized that NMDARs

affect APP processing by alteringAPP trafficking in neurons. To testthis hypothesis, we measured cellsurface levels of endogenousAPP bybiotin-labeling surface proteins oncultured hippocampal neurons, iso-lating these proteins with avidinbeads, and immunoblotting forAPP. Levels of cell surface APPweresignificantly decreased (by 87%, p �0.01) by NMDA compared withcontrol or MK-801 treatments (Fig.3, D and E). MK-801 again blockedthe effects of NMDA on APP traf-ficking (resulting in an 11%, nonsig-nificant, decrease; Fig. 3, D and E).We also took an independentapproach to measure the effect ofNMDAR activity on APP traffick-ing, using live cell staining. Pri-mary hippocampal neurons wereco-transfected with NR1 and APP,and we measured cell surface levelsof APP using live cell staining undernonpermeabilizing conditions. NR1overexpression alone increased sur-face levels of APP compared withcontrol vector (data not shown),allowing for more reliable assays ofsurface APP. Transfected cells wereidentified with GFP (Fig. 3F, left)and surface levels of APP with anti-

bodies (right).We tested the effect of NMDAR activation over abrief period (20min) by treating these cells with vehicle control(DMSO), NMDA (200 �M), or MK801 (10 �M) (Fig. 3F).NMDAR activation reduced cell surface levels of APP, whereasMK-801 treatment had no effect (Fig. 3F). Quantification ofstaining in neuronal processes showed a significant decrease insurface APP after NMDA (87% decrease versus untreated cells;n 3; p � 0.01), whereas MK-801 treatment had no effect.These data show not only that APP is found in the PSD but alsothat synaptic activity alters APP processing and trafficking.Knockdown of APP Reduces NMDAR-mediated Current

Responses—To examine the physiological function of APP, wedecreased endogenous APP expression levels in primary neu-rons with RNAi and examined the effect on synapses usingwhole cell patch clamp recordings. This approach allowed us tospecifically examine the effects of APP in the postsynaptic cell

FIGURE 3. NMDARs affect APP trafficking and processing. A, cultured hippocampal neurons were treatedwith DMSO (vehicle) control, NMDA (10 �M), MK801 (10 �M), or NMDA and MK-801 for 24 h. Secreted APP wasmeasured in conditioned media (15 �l) with antibody 22C11, and cell APP was detected in lysates (20 �g) withC1/6.1. B, quantification of four experiments in A (control, 100 � 21%; NMDA, 19 � 9%; *, p � 0.01; MK-801, 96 �9%; NMDA plus MK-801, 94 � 9%). C, primary cortical neurons were similarly treated and A�40 levels weremeasured by ELISA. NMDA treatment significantly increased A� (289 � 22% versus control; n 4; *, p 0.02),and this effect was largely blocked by MK801 (123 � 31% versus control; n 4; p 0.05). D, cultured corticalneurons were similarly treated, and cell surface proteins were labeled with biotin, isolated with avidin beads,and immunoblotted with antibody 22C11 for APP (top). Total APP in cell lysates was measured in the lowerpanel. E, quantification of four experiments in D (control, 100 � 8%; NMDA, 13 � 7%; *, p � 0.01; MK-801, 98 �8%; NMDA plus MK-801, 89 � 10%). F, cultured hippocampal neurons were transfected at DIV 12 with GFP-NR1and APP. The left panels show transfected neurons expressing GFP, and the right panels show levels of surfaceAPP identified with polyclonal APP antibody. At DIV 18, cells were treated with DMSO control, NMDA (200 �M),or MK801 (10 �M) for 20 min, and surface APP was measured by live cell staining.

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without altering presynaptic APP. To determine the effective-ness of various APP RNAi, we transfected COS7 cells withrodent APP and RNAi constructs. Two APP RNAi constructs(numbers 7 and 14) in the pSuper vector (18) reduced trans-fected APP expression 90% (Fig. 4A); a third APP RNAi con-struct (number 37) had no effect. To test whether the RNAiwaseffective against endogenous APP in neurons, we co-trans-fected cultured primary hippocampal neurons with GFP (toidentify transfected neurons) and either empty pSuper vectoror APP RNAi-14 (Fig. 4B, left). After 3 days of expression, cul-tures were immunostained to detect endogenous APP. APPimmunostaining was substantially reduced by APP RNAi-14,

particularly in the neuronal processes, compared with controlcells (Fig. 4B; 95% reduction versus pSuper control, p� 0.01) orcells transfected with the ineffective APP RNAi-37 (data notshown). Using transfected COS7 cells and primary neuronalcultures, we found that the APP siRNA also reduced levels ofAPP proteolytic fragments, secreted APP, and APP C-terminalfragment (data not shown). Thus, as expected, changes to APPalso result in changes to APP fragments, such as secreted formsof APP, A�, and intracellular fragments.

To analyze changes in neuronal electrophysiology caused byaltered APP levels, we initially examined mouse CGCs. Thesecultures provide a homogeneous neuronal population fromwhich the total and synaptic receptor pools of receptors can bemeasured. With its small cell body size (only 3–4 �m in diam-eter) and simple dendritic arborization, the CGC allows deter-mination of whole cell current response and synaptic currentwith much higher recording resolution compared with othertypes of cultured neurons. We transfected CGCs with APPRNAi-14 and GFP using calcium phosphate, as described (29).Fewer than 5% of neurons were transfected under these condi-tions, allowing us to focus on the effects of reducing APP inindividual neurons, rather than neuronal networks. Weobtained whole cell patch clamp recordings 24–48 h post-transfection from pairs of nearby APP RNAi-transfected andnontransfected control neurons, using a focal application ofsaturating doses of NMDA (200 �M). A second control groupconsisted of CGCs transfected with only GFP. The peak ampli-tude of current responses to this NMDA application representsthe contribution of all functional surface NMDARs, includingreceptors localized to synaptic sites and those at extrasynapticsites. These currents were subsequently normalized to cellcapacitance (which is proportional to cell surface area) to gen-erate current density values (the cultured CGCs had generallyhomogenous capacitances (5 � 0.42 pF)). Neurons withdecreased levels of APP exhibited reduced NMDA-evoked cur-rent densities compared with neurons transfected only withGFP (Fig. 4C). Quantification of results from neurons trans-fected with APP RNAi (n 15) demonstrated a significantreduction in NMDA evoked current densities compared withGFP (n 31) or nontransfected neighboring cells (n 16) (70%reduction versus GFP control, p � 0.01) (Fig. 4D). These datademonstrate that endogenous APP affects normal neuronalNMDAR expression or function.APP Overexpression Increases NMDAR Currents—Reducing

APP levels decreasedNMDAR current density in cultured neu-rons; we next examined whether transiently increasing APPlevels had the opposite effect. Using the same protocol as above,we transfected CGCs with APP tagged on its C terminus withGFP. Neurons overexpressing APP exhibited significantlyhigher NMDA-evoked current densities than control untrans-fected neighboring cells or GFP-transfected cells (42% increaseversusGFP control, p� 0.01) (Fig. 5,A and B). As in Fig. 4, APPsiRNA reduced NMDA current density (Fig. 5, A and B). Toexamine the specificity of these effects, we analyzed inhibitoryGABAA receptors present in these CGCs. We found that therewas no significant difference in response to a saturating dose ofGABA (1 mM) observed between CGCs expressing APP-GFPand control cells transfected only with GFP (Fig. S1). Thus, the

FIGURE 4. Knockdown of APP decreases current density of NMDARs.A, COS7 cells were co-transfected with rat APP and 1) pSuper-APP siRNA 7, 2)pSuper-APP siRNA 14; 3) pSuper vector; 4) pSuper-APP siRNA 37; and 5)pSuper-APP siRNA 7 and 14. Rat APP in cell lysates was measured with anti-body M3.2 (recognizing rat APP). B, hippocampal neurons were co-trans-fected with GFP and pSuper (top) or GFP and APP siRNA 14 (bottom). Trans-fected cells (GFP fluorescence; left) were analyzed for APP expression byimmunofluorescence with APP antibody (Ab) M3.2 (right). C, whole cell cur-rents were elicited in mouse CGCs transfected with APP siRNA 14 and GFP orpSuper and GFP at DIV 9 by application of a saturating concentration ofNMDA (200 �M) in the absence of Mg2� and Ca2�. Application duration isindicated by solid bars above the traces. D, quantification of the results from C(GFP, 120 � 8 pA/pF, n 31; siRNA, 38 � 7 pA/pF, n 15); *, p � 0.05 versuscontrol, Student’s t test. Data are shown as mean � S.E.

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effect ofAPPonNMDARswas not due to nonspecific effects onall neurotransmitter receptors.Because the hippocampus is one of themost vulnerable brain

regions affected in AD, we tested whether the effects of APP onNMDARs were also observed in hippocampal neurons. Con-sistent with the results in CGCs, overexpression of APP causeda significant increase in NMDAR current density at DIV 11–13hippocampal neurons (41% increase versus GFP control, p �0.05) (Fig. 5, C and D). Knockdown of APP levels with RNAi

again caused a reduction in NMDAR current density (22%decrease versus control), although the decrease did not reachsignificance in these experiments (p 0.18).

The results from overexpression of APP were surprisingbecause of reports demonstrating that the APP proteolyticproduct A� has a negative effect on glutamate receptors (4, 5,7). To test whether A� produced in our cells could be having aconfounding effect in our system, we compared A� levels incultured medium from control CGCs and neurons overex-pressingAPP (from the low efficiency calciumphosphate trans-fections). We observed no significant difference in A� levelsbetween these two groups (data not shown). We also trans-fected cells with APP-MV, a version of APP mutated at its�-cleavage site to prevent the production of A� (7). As in ourexperimentswithwild-typeAPP, the�-sitemutantAPP causedincreased NMDAR current density in both CGCs (32%increase, p � 0.01; Fig. 5, A and B) and hippocampal neurons(53% increase, p � 0.01; Fig. 5, C and D).We also tested whether the direct or indirect addition of A�

to the cultures had a similar effect as adding full-length APP.First, we transfected cells with a construct expressing the APPfragment C99, which can generate A� and which contains theintracellular domain ofAPP. In contrast to full-lengthAPP,C99significantly decreasedNMDARcurrent density (36%,p� 0.05;Fig. 5, A and B). Second, we treated neurons with exogenousA�.We appliedA�42 (2�M) to culturedCGCs for 24–48 h andobserved the expected significant decrease in whole cellNMDAR current densities (52% decrease versus control, p �0.01) (Fig. S2). Therefore, we attribute the effect of APP-en-hancing NMDAR whole cell current responses to full-lengthAPP and not to any effects of the APP C-terminal fragment orthe A� peptide.APP Increases the Incorporation of the NR2B Subunit into

Receptor Complexes—Given our findings that APP increasedthe current density of NMDARs, we considered the possibilitythat APP affected subunit composition of NMDARs, sinceNR2B subunit passes larger current than NR2A (13). To testthis idea, we used the highly selective NR1/NR2B antagonist,CP101,606. Control GFP-transfected CGCs showed a modestbut significant inhibition ofNMDARwhole cell current densityby CP101,606 (28% decrease versus vehicle, p � 0.01) (Fig. 6A).Consistent with the data in Fig. 5, APP-GFP-transfected cellsshowed an increase in NMDAR current density compared withGFP control. This enhanced current density wasmore sensitiveto CP101,606 than control cells (46% reduction, p� 0.05 versusvehicle) (Fig. 6, A and B). Importantly, the whole cell currentsfromGFP- and fromAPP-GFP-transfected cells in the presenceof CP101,606were nearly identical, suggesting that the increasein NMDAR current seen with APP was due largely to theincrease in CP101,606-sensitive receptors (i.e.NR1/NR2B het-eromers). We performed similar experiments using anotherselective NR2B antagonist, ifenprodil (10 �M), and again foundthat APP selectively promoted functional expression of NR1/NR2B receptor complexes (data not shown). Thus, we concludethat APP specifically enhances the surface levels of NR2B-con-taining NMDARs.To test these electrophysiological findings using an immuno-

cytochemical assay, we conducted cell surface immunostaining

FIGURE 5. APP overexpression increases NMDAR currents. A, representa-tive traces from whole cell recordings of currents evoked by 200 �M NMDAfrom cells transfected with GFP, untransfected neighboring control cells,APP-GFP, APP M671V-GFP, APP siRNA, or APP C99 in CGC cells at DIV 5.B, quantification of the data in A, showing that the NMDA-evoked whole cellcurrents in APP or APP MV cells were significantly higher than in control cells(GFP, 120 � 8 pA/pF; untransfected neighboring cells, 99 � 12 pA/pF; APP-GFP, 156 � 11 pA/pF; APP M671V-GFP, 161 � 12 pA/pF; siRNA, 38 � 7 pA/pF);n 17–19 cells from three different batches of cultures. *, p � 0.01 versuscontrol, analysis of variance; data are shown as mean � S.E. Independentexperiments demonstrated the significant decrease in NMDA-evoked currentdensity by APP-C99 (GFP, 104 � 10 pA/pF; APP-C99, 67 � 12 pA/pF); n 10cells from two different batches of cultures. *, p � 0.05 versus control. C, rep-resentative traces from whole cell recordings of currents evoked by 200 �M

NMDA in hippocampal neurons transfected with GFP, APP-GFP, APP M671V-GFP, or APP siRNA at DIV 12–13. D, quantification of the data shown inC, showing that APP overexpression significantly enhanced whole cell cur-rent density (GFP, 22 � 2 pA/pF; APP, 33 � 3 pA/pF; siRNA, 17 � 2 pA/pF). n 12 cells, from at least three different batches of cultures. *, p � 0.01 versuscontrol. Independent experiments demonstrated the significant increase inNMDA-evoked current density by APP-MV (GFP, 21 � 3 pA/pF; APP-MV, 32 �3 pA/pF); n 10 cells from two different batches of cultures. *, p � 0.01 versuscontrol.

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ofNMDARs in primary hippocampal neurons. To allow immu-nostaining of surface NMDAR subunits, we transfected neu-rons with NR1, NR2A, or NR2B tagged on their N-terminal(extracellular) domains with GFP. The presence of the GFP tagallowed imaging of surface receptorswith aGFP antibody, sincewe were unable to identify reliable receptor subunit-specificantibodies for immunocytochemistry assays of endogenousNMDA receptor subunits. These GFP-tagged NMDAR sub-units are known to express and traffic indistinguishably fromtheir endogenous counterparts (17, 30, 31). The left panelsdefine transfected cells; themiddle panels show surfaceNMDAreceptor subunits; and the right panels show amagnification ofselected neuronal processes (Fig. 6, C–E). Compared with cellstransfected with just the NMDAR subunit alone (defined as100%), co-transfectionwith APP significantly increased surfacelevels of both NR1 (176% versus NR1 alone, p � 0.01; Fig. 6, Cand F) and NR2B (147% versus NR2B alone, p 0.02; Fig. 6, Eand H) in dendritic processes. Overexpression of APP did notaffect surface levels of NR2A (118% versus NR2A alone, p 0.05; Fig. 6,D andG). In contrast, reduction of endogenousAPPusing RNAi significantly decreased dendritic surface staining ofboth NR1 (57% versusNR1 alone, p � 0.01; Fig. 6, C and F) andNR2B (60% versusNR2B alone, p� 0.01; Fig. 6,E andH) but notNR2A (93% versus NR2A alone, p 0.05; Fig. 6, D and G).Because C99 decreased NMDAR current density, we testedwhether it decreased surface levels of NR2B. We found thathippocampal neurons co-transfected with NR2B and APPagain showed a significant increase in surface NR2B (135% ofcontrol; n 7; p � 0.01) but that C99 showed a significantdecrease in surface NR2B (76% of control; n 7; p � 0.05).These data are consistent with our observation that C99reduced NMDAR current density (Fig. 5).Because a change in NMDAR surface expression could

reflect changes in trafficking, we tested the effect of APP oninternalization of NR2A or NR2B in hippocampal neurons.Weco-transfected neurons with GFP-tagged NR2A or NR2Btogether with either empty vector, APP expression vector, orAPP RNAi (Fig. 7, A and B, left). We then used an antibodyuptake assay to measure internalization of NMDAR subunits(Fig. 7, A and B, right, at 30 min after labeling of surface NR2Aor NR2B). Using this assay, co-transfection with APP signifi-cantly decreased endocytosis of NR2B (58% versusNR2B alone,p 0.02) (Fig. 7, B and D). APP overexpression did not affectendocytosis of NR2A (90% versusNR2A alone, p 0.05) (Fig. 7,A and C). In contrast, reduction of endogenous APP usingRNAi significantly increased internalization of NR2B (139%versus NR2B alone, p � 0.05) (Fig. 7, B and D) but not NR2A(91% versus NR2A alone, p 0.05) (Fig. 7, A and C). Thesefindings are consistent with the electrophysiology data indicat-

ing that APP promoted surface levels of NR2B-containingreceptor complexes preferentially.APP Increases NMDAR-mediated Synaptic Currents—Thus

far, we have reported corresponding changes in NMDAR-me-diated whole cell currents (composed of both synaptic andextrasynaptic receptors) and surface levels ofNMDARsubunitsin cells with altered APP levels. We next tested whether APPaffected NMDA receptors specifically within synapses byrecording spontaneous miniature NMDAR-mediated excita-tory postsynaptic currents (NMDA-mEPSCs) (22, 23, 29). Sam-ple traces of NMDA-mEPSCs recorded in cultured CGCs at7–8 days in vitro in the absence of Mg2� under various condi-tions are illustrated (Fig. 8A). The peak amplitude of NMDA-mEPSCs was significantly increased by APP-GFP (129% versusGFP control, p � 0.05) and the APP-MV mutant (165% versusGFP control, p � 0.05) (Fig. 8C, left). These data indicate thatAPP causes an increased number of synaptic NMDA receptorsin the postsynaptic densities or a change of biophysical channelproperties, such as single-channel conductance ormean single-channel open time.To address the possibility that the observed effects of APP

might be due to changes in NMDAR single-channel properties,we took advantage of the fact that recordings at negative poten-tials are characterized by very low background noise in CGCs.This property allows for the observation and measurement ofthe amplitude of single-channel currents in the tails of synapticresponses (29). Using this methodology we observed no changein the single-channel NMDAR current in neurons with alteredAPP levels (Fig. 8B) (3.2 � 0.6 pA (mean � S.D.) for controlGFP-transfected cells; 3.5 � 0.7 pA for APP-transfected cells;3.3 � 0.4 pA for APP siRNA-transfected cells). Thus, APP didnot affect the single-channel conductance of postsynapticNMDARs.Finally, we observed that theweighted time constant of decay

in APP-GFP-transfected cells (�w; Fig. 8C, middle) was signifi-cantly lengthened (for APP, 138% versusGFP control, p� 0.05;for APP-MV, 133% versus GFP control, p � 0.05), suggestingincreased incorporation of NMDARs with slower deactivationkinetics. This property is consistent with a higher number ofNR2B-containing receptors in the synapse. The frequency ofNMDA-mEPSCs was not significantly affected by overexpres-sion ofAPPorAPP-MV (Fig. 8C, right). In cells transfectedwithAPP siRNA, there was a significant reduction in NMDA-mEPSC peak amplitude (29% decrease versusGFP control, p �0.05) and frequency (75% decrease versus GFP control, p �0.05) (Fig. 8C, right). The decay time decreased in APP siRNA-transfected cells compared with GFP-transfected cells,although this did not reach significance (p 0.11). Together,these data support a strong effect of APP on NMDAR function

FIGURE 6. APP promotes the surface expression of NR1/NR2B receptors. A, representative traces from whole cell recordings of currents evoked by 200 �M

NMDA from CGCs transfected with GFP (left) or APP-GFP (right) at DIV 5 in the presence or absence of CP101,606 (10 �M). B, quantification of the data shownin A, showing inhibition of NMDA-evoked whole cell current by CP101,606 in control (28%; *, p � 0.01, Student’s t test) and APP-overexpressing cells (46%;*, p � 0.05); n 17–19 cells from three different cultures. C–E, effect of APP on surface staining of NMDAR subunits. Cultured hippocampal neurons weretransfected at DIV 12 with constructs as indicated, and surface NMDAR subunits were measured at DIV 18 by immunofluorescence of live cells. Left, total GFP;right, surface NR1 (C), NR2A (D), or NR2B (E). Higher magnification at the far right shows individual dendritic processes. F–H, quantification of surface NMDARsubunits in neuronal processes. F, APP significantly increased cell surface NR1 (176 � 5% versus control; n 8; *, p � 0.01) and APP siRNA significantly decreasedcell surface NR1 (57 � 4% versus control; n 10 –15; *, p � 0.01). G, cell surface NR2A was not affected by overexpressing APP (118 � 25% versus control; n 7; p 0.05) or by APP siRNA (107 � 19% versus control; n 10 –15; p 0.05). H, APP significantly increased cell surface NR2B (147 � 5% versus control; n 10 –15; *, p 0.02), and APP siRNA significantly decreased cell surface NR2B (60 � 3% versus control; n 10 –15; *, p � 0.01). All data are shown as mean � S.E.

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within the synapse, increasing NMDAR numbers and favoringNMDAR with longer decay times.

DISCUSSION

Our data have defined a novel physiological function of APPin promotingNMDARaccumulation at postsynaptic sites. Thisconclusion is supported by several lines of evidence. APP co-localizes and co-fractionateswith postsynapticmarkers and co-precipitates with PSD proteins in the brain. Knockdown ofendogenous APP decreased (and overexpression of exogenousAPP increased) the NMDAR whole cell current density as wellas NMDA-mEPSC peak amplitudes. These electrophysiologi-cal findings were consistent with our immunocytochemicalstudies showing that APP promoted the presence of NMDARson the neuronal cell surface by inhibition of receptor endocy-tosis. The effect of APP overexpression was observed in both

CGCs and hippocampal neurons,suggesting that this functionmay bea general feature of NMDAR-ex-pressing cells. We observed theopposite effects when we added theC-terminal portion of APP (C99).These effectsmay be due to the localgeneration of the synaptotoxic A�fragment or to the competition ofthe transfected C99 with intracellu-lar adaptor proteins that normallymediate the functions of endoge-nous APP. We conclude that theextracellular domain of APP is nec-essary for its effects on synapticfunctions.We confirmed and extended the

earlier findings of Kamenetz et al.(7) that stimulating neurons withNMDA favored cleavage of APP by�-secretase over �-secretase. Weobserved decreased surface APP,decreased sAPP, and increased A�after NMDA treatment, each ofwhich was blocked by MK-801.These observations are consistentwith the hypothesis that synapticactivity promotes the endocytosis ofsurface APP, leading to its �-cleav-age (32).We and others (26, 27) have

found that APP is also presynaptic.APP may form homodimers reach-ing across the synaptic cleft (33),thus allowing presynaptic APP toaffect the function of postsynapticAPP. The experiments describedhere were aimed at studyingchanges only in postsynaptic APP.In other preliminary experiments,we used neurons from APP knock-outmice to test the effects of chang-

ing both pre- and postsynapticAPP (data not shown).However,we did not observe changes in NMDAR current density in APPknock-out CGCs. We hypothesize that this result could bebecause eliminating APP both pre- and postsynaptically resultsin a more complex phenotype. Alternatively, functional redun-dancy with APP family members or developmental compensa-tionmay limit the usefulness of knock-out cultures. PresynapticAPP may have independent functions on synaptic function byaltering presynaptic transporters (34) or vesicle release (35).Overexpression of APP caused an enhancement of NMDA-

mEPSC amplitudes in CGCs, indicative of increased spontane-ous NMDAR activity in the cultures, and APP siRNA had theopposite effect. APP siRNA also caused a dramatic reduction inNMDA-mEPSC frequency. Itmay be thatwhenAPP function islost acutely, NMDAR insertion at the synapse is compromisedto the extent that many excitatory synapses become function-

FIGURE 7. APP inhibits NR2B receptor internalization in cultured hippocampal neurons. A and B, culturedhippocampal neurons were transfected at DIV 12 with constructs as indicated, and internalization of NR2subunits was measured at DIV 18 by immunofluorescence of live cells. Left, total GFP; right, internalization ofNR2A (A) or NR2B (B). C and D, quantification of NR2 internalization in neuronal processes. C, overexpression ofAPP or knockdown of APP did not alter internalization of NR2A (90 � 19 or 91 � 32%, respectively, versuscontrol; n 7; p 0.05). D, APP significantly decreased NR2B internalization (58 � 16% versus control; n 14;*, p 0.02), and APP siRNA significantly increased NR2B internalization (139 � 27% versus control; n 14; *, p �0.05). All data are shown as mean � S.E.

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ally “NMDAR-less.” Alternatively, APPmay regulate excitatorysynapse number and play a role in synapse formation and/ormaintenance. The role of APP in promoting development ordelaying maturation of synapses is an interesting question forfuture studies.A role for APP in synaptic plasticity is also suggested by its

specific recruitment of NR2B-containing NMDARs. APPincreased the decay time ofNMDA-mEPSCs, enhanced surfaceexpression, and suppressed internalization specifically ofNR2Bversus NR2A. In addition, APP increased sensitivity of NMDAwhole cell currents to specific antagonists of NR2B-containingreceptors. NR2B-containing receptors have several distinctproperties. NR2B is expressed earlier in development thanNR2A (14, 36, 37) and has a longer mean open time (13).Although there is some debate regarding the precise roles ofNR2A versusNR2B in synaptic plasticity, bothNR2AandNR2Bappear to be able to participate in long term potentiation andlong term depression (38–40). However, NR2B contacts dis-tinct sets of signaling proteins (41–43). It is possible that APPpreferentially affects NR2B-containing receptors because theslower deactivation kinetics conferred by NR2B allows forgreater calcium influx, which could favor heightened synapticactivity in response to neuronal damage or during learning.Wedo not know the mechanism for how APP would preferentiallyaffect NR2B, and we did not observe a specific interactionbetween APP and NR2B in immunoprecipitation experiments

(data not shown). Our data demonstrate that full-length APPincreases surface NR2B and decreases its internalization.The role of APP in promoting synaptic recruitment of

NMDARs is in contrast to the effects of A� on synapses. Anumber of recent studies (4, 5, 7) have found that A� (andspecifically soluble, oligomeric A�) is synaptotoxic, resulting indecreased levels of AMPARs and NMDARs at synapses in pri-mary neurons. A� also results in decreased long term potenti-ation (4) and decreased spine number in transgenic modelsof AD (44). We confirmed in our system that A�-(1–42)decreased NMDA current densities, whereas full-length APPincreased NMDAR currents in the same cell preparations. Thisdifferential effect is likely to reflect distinct roles of full-lengthAPP and A� as well as the different levels of A� in these twoconditions. For analysis of cells with overexpressed APP, weused calcium phosphate transfections (with low transfectionefficiency) to limit the amount of excess A� that could be pro-duced. Indeed, we observed no changes in synaptic transmissionin neighboring neurons near APP-transfected cells, suggestingthat low levels of A� secreted into the conditioned media fromtransfected cells were not sufficient to affect normal synaptictransmission or counteract the positive effect of APP onNMDARrecruitment. Furthermore, we showed that APP mutated at the�-secretase site showed enhancement of NMDARwhole cell cur-rent densities comparable with that seen with wild-type APP,demonstrating unequivocally that neither �-cleavage of APP northe generation of A�was responsible for this effect. Thus, we pro-pose that full-length APP promotes synaptic transmission, but itsproteolytic product A� is synaptotoxic.Taken together, our data suggest a model that ties together

the functions of APP in physiological and pathophysiologicalconditions. APP is transported in both axonal and dendriticcompartments (45) and localized to both pre- and postsynapticregions (26, 27). Following many types of neuronal damage,levels of APP are increased (46, 47). Our data suggest that thisincrease may act as a mechanism to stabilize or strengthen syn-apses by recruitment of NMDARs or enhance plasticity by spe-cifically increasing NR2B content. A� is also produced inresponse to damage; this production may be a result of thesynaptic APP undergoing endocytosis and proteolysis in con-junction with heightened NMDAR activity. In turn, the loss ofthe stabilizing effect of full-length APP coupled with the synap-totoxic effect of A� could act as a physiological brake mecha-nism to down-regulate glutamatergic transmission (e.g. to com-bat excitotoxicity if synaptic activity levels become excessive). Thesynaptotoxic effects of A� in AD could be an aberrant or exagger-ated form of this protective negative feedback response, thusexplaining the loss ofNMDARsduringADaswell as thebeneficialeffects of the NMDAR antagonist memantine for AD patients.Further elucidation of APP function and trafficking in relation toNMDARs may contribute to our understanding of how the A�peptide is generated in normal and pathological states and informideas about how to help prevent its production in AD.

Acknowledgments—We thank Ulrike Muller (University of Heidel-berg) for the APP/APLP1 DKO tissue. We thank Drs. SamGandy andPaul Mathews for APP antibodies.

FIGURE 8. Increase in peak amplitude of NMDA-mEPSCs by APP. A, repre-sentative recordings of pharmacologically isolated NMDA-mEPSCs from cellstransfected as indicated. NMDA-mEPSCs were recorded at �60 mV in thepresence of bicuculline metabromide (25 �M; GABAA receptor antagonist),tetrodotoxin (0.5 �M), and NBQX (5 �M; AMPA receptor antagonist) in a Mg2�-free solution. B, representative single-channel traces from the tails of sponta-neous miniature synaptic responses as shown in A. There was no change inthe single channel current of NMDA receptors between neurons transfectedwith APP (3.2 � 0.6 pA), APP siRNA (3.3 � 0.4 pA), and GFP (3.5 � 0.7 pA) (p 0.5). Data are shown as mean � S.D. (n 5, from two different cultures).C, summarizing bar graphs show mean � S.E. for mEPSC peak amplitude (GFP,18.5 � 0.7 pA; APP, 28.7 � 2.2 pA; APP-MV, 30.54 � 2.57 pA; siRNA, 14.1 � 1.8pA), decay time constant (�w; GFP, 178 � 9 ms; APP, 247.1 � 28 ms; APP-MV,236.76 � 16.28 ms; siRNA, 131.5 � 40.5 ms), and frequency (GFP, 0.07 � 0.01Hz; APP, 0.08 � 0.01 Hz; APP-MV, 0.086 � 0.016 Hz; siRNA, 0.015 � 0.01Hz).The number of cells recorded (GFP, n 32; APP, n 19; APP-MV, n 9; APPsiRNA, n 10) were from at least three different culture preparations (*, p �0.05 versus GFP).

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APP and NMDA Receptors

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Stefano Vicini, Daniel T. S. Pak and G. William RebeckAhdeah Pajoohesh-Ganji, Yasuji Matsuoka, Bradley T. Hyman, Michael D. Ehlers,

Hyang-Sook Hoe, Zhanyan Fu, Alexandra Makarova, Ji-Yun Lee, Congyi Lu, Li Feng,Activity

The Effects of Amyloid Precursor Protein on Postsynaptic Composition and

doi: 10.1074/jbc.M900141200 originally published online January 21, 20092009, 284:8495-8506.J. Biol. Chem. 

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