Large Quantities of A� Peptide Are Constitutively Releasedduring Amyloid Precursor Protein Metabolism in Vivoand in Vitro*

Received for publication, October 4, 2010, and in revised form, March 1, 2011 Published, JBC Papers in Press, March 22, 2011, DOI 10.1074/jbc.M110.191262

Abhay Moghekar‡, Sneha Rao‡, Ming Li‡, Dawn Ruben§, Andrew Mammen‡, Xiaopei Tang‡, and Richard J. O’Brien‡1

From the Departments of ‡Neurology and §Comparative Medicine, The Johns Hopkins University School of Medicine,Baltimore, Maryland 21205

The metabolism of the amyloid precursor protein (APP) hasbeen extensively investigated because its processing generatesthe amyloid-�-peptide (A�), which is a likely cause ofAlzheimerdisease. Much prior research has focused on APP processingusing transgenic constructs andheterologous cell lines.Work todate in native neuronal cultures suggests that A� is produced invery large amounts. We sought to investigate APP metabolismand A� production simultaneously under more physiologicalconditions in vivo and in vitro using cultured rat cortical neu-rons and live pigs.We found in cultured neurons that both APPand A� are secreted rapidly and at extremely high rates into theextracellular space (2–4molecules/neuron/s forA�). LittleAPPis degraded outside of the pathway that leads to extracellularrelease. Two metabolic pools of APP are identified, one that ismetabolized extremely rapidly (t1⁄2 � 2.2 h), and another, surfacepool, composed of both synaptic and extrasynaptic elements,that turns over very slowly. A� release and accumulation in theextracellular medium can be accounted for stoichiometricallyby the extracellular release of �-cleaved forms of the APP ecto-domain. Two �-cleavages of APP occur for every �-cleavage.Consistent with the results seen in cultured neurons, anextremely high rate of A� production and secretion from thebrain was seen in juvenile pigs. In summary, our experimentsshow an enormous and rapid production and extracellularrelease of A� and the soluble APP ectodomain. A small, slowlymetabolized, surface pool of full-length APP is also identified.

Alzheimer disease (AD)2 is a progressive, neurodegenerativeprocess characterized pathologically by accumulation of the�-amyloid peptide (A�) in the form of extracellular plaques (1).On the basis of genetic, cellular, and animal studies the amyloidhypothesis postulates that increased deposition of A� in thebrain is the primary influence in the pathogenesis of AD (2). A�is part of the amyloid precursor protein (APP) that is expressed

ubiquitously by neuronal andnon-neuronal cells (3–5).APP is atype 1 transmembrane glycoprotein that undergoes sequentialsite-specific proteolytic cleavages by either �- or �-secretasefollowed by �-secretase, which cleaves APP in the transmem-brane domain to yield several secreted derivatives. Cleavage ofAPP by the �-secretase BACE1 releases a large fragment calledsAPP� leaving behind a fragment that is acted upon by �-secre-tase at different sites to release several short peptides of whichA�40 and A�42 are the major components (6). Alternatively,cleavage of APP by �-secretase occurs within the A� domain,precluding the formation of A� and resulting in the secretion ofsAPP�, a short peptide called p3, and a C-terminal domain,none of which are amyloidogenic.Both in vitro and in vivo evidence suggests that A� is secreted

from cells under normal physiological conditions (3, 4, 7–9) butthe absolute amount, an important consideration given its pro-posed roles and pharmacologic interest, has not been preciselycalculated. In addition, the relative amount of intracellular ver-sus extracellular A� production and its ultimate disposition inneurons is unclear (10).Mostmodels of A�metabolismdependon the analysis of cells transfected with human APP constructsor cells from transgenic mice. Establishing a model to obtaindetectable, consistent, and reliable levels of solubleA� andAPPectodomain directly from non-transgenic and non-transfectedneurons is important to understand the normal metabolism ofAPP and A� and to explore novel therapeutic approachesaimed at manipulating soluble A� levels. Toward this end wehave developed a series of assays for endogenous rodentA� andAPP and confirmed the key result in vivo using juvenile pigs.The picture that emerges is one of an enormous quantity andrapid turnover of APP, all leading to the extracellular secretionof sAPP and A�. A second, slowly metabolized, largely extra-synaptic, surface pool of APP is also identified, confirming andextending prior work (11).

EXPERIMENTAL PROCEDURES

Antibodies—An antibody (2B) to the first 16 amino acids ofrat� amyloid peptidewas raised in rabbits by immunizing themwith the peptide DAEFGHDSGFEVRHQKC coupled to key-hole limpet hemocyanin. The resultant serumwas affinity puri-fied. Three different APP antibodies were used in this study.Two were commercially obtained (rabbit anti-APP CT andmouse monoclonal 22C11, both from Chemicon). One wasmade in our laboratory, using a GST fusion protein coupled toamino acids 30–300 of rat APP, injected into rabbits. This anti-

* This work was supported, in whole or in part, by National Institutes of HealthGrant P50 AG05146 from the NIA and the Burroughs Wellcome Fund forTranslational Research.

1 To whom correspondence should be addressed: Mason F. Lord CenterTower, Rm. 5100, Johns Hopkins Bayview Medical Center, 5200 EasternAve., Baltimore, MD 21224. Tel.: 410-550-8703; Fax: 410-550-8715; E-mail:robrien@jhmi.edu.

2 The abbreviations used are: AD, Alzheimer disease; APP, amyloid precursorprotein; A�, amyloid-�-peptide; sAPP, soluble amyloid precursor proteinectodomain; PSD-95, postsynaptic density protein, 95 kDa; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

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body, termed N-terminal APP, was used after affinity purifica-tion. A polyclonal antibody that specifically recognizes the Cterminus of sAPP� was purchased from IBL.Rat Primary Cortical Cultures—Embryonic day 19 rat corti-

cal cultures were grown at a density of 2,000,000 cells/60-mmdish for 14–21 days as described (12) in theNeurobasal plus 2%B-27 supplement (Invitrogen). Serial counts of these culturesusing randomly selected live �40 fields (n � 4) or �60 fields offixed neurons stainedwithDAPI (n� 5) demonstrate a 31� 5%loss of cells over the 2–3 week period of growth, a numbercalculated into the A� release kinetics. When A� and APPsecretion assays were performed, a complete change of the neu-ronal growth medium to 1.5 ml of fresh Neurobasal B27medium was performed at the start of the assay. This freshmedium was preincubated with glia for 8 h prior to use.A� ELISA—96-Well ELISA plates (Kirkegaard & Perry Lab-

oratories) were coated with affinity purified 2B antibody at 1mg/well as the capture antibodywith biotinylated 4G8 antibodyas the detection antibody. The intra-assay and inter-assay coef-ficients of variation were both less than 10%; the lower limit ofdetection of rat A� was 8 pg/ml. Verification experiments wereperformed using two commercially available ELISA kits, therat/human A�40 assay purchased from Wako and the rodentA�1–40 kit purchased from Invitrogen. Both assays use anti-bodies distinct from those used in our assay including a mono-clonal antibody specific for the C-terminal portion of A�40common to both. TheWako kit recognizes full-length A�1–40as well as A� truncated prior to amino acid 11, a result we haveconfirmedwith rat and humanA�11–40 standards (purchasedfrom AnaSpec). The Invitrogen kit does not recognize A�11–40, a result we have also confirmed. For all 3 rodent ELISA weused the same synthetic rodent A�1–40 standard (Bachem)from 1 ng/ml to 8 pg/ml. In theWako A�40 ELISA, rodent andhuman A�1–40 (Bachem) standards gave O.D. results within18.3 � 6.1% of each other (rodent higher; n � 4) at a standardconcentration of 400 pg/ml, a concentration relevant for com-parison between Fig. 1 (rodent) and Table 1 (pig).Quantitation of APP Secretion—Rat cortical neurons were

switched to fresh medium. Samples of the supernatant werecollected at various times after the medium switch. Abso-lute levels of APP secretion were determined with quantitativeimmunoblotting using either a purified APP-GST fusion pro-tein (amino acids 30 to 300 of rat APP) or a His6 APP fusionprotein (purchased from R&D Systems) as standards. Bothstandards were highly purified by several criteria. Duringexperiments, multiple concentrations of the synthetic APPwere run on a SDS-polyacrylamide gel along with the culturesupernatants and probed with our APPN-terminal antibody orwith the monoclonal 22C11 antibody, which recognizes anunglycosylated epitope on human and rodent APP betweenamino acids 66 and 81 (13). Both antibodies and fusion proteinsgave quantitatively similar results. Protein band intensity forsecreted APP and the protein standards were quantitated usinga densitometer. A standard curvewas generated for each exper-iment, such that band intensity could be converted to a proteinconcentration. As an independent method to quantitate thesecreted APP ectodomain, we used an ELISA fromMeso ScaleDiscovery that detects the �-cleaved form of APP using a

sAPP� C-terminal specific antibody (which recognizes theidentical C terminus of rodent and human sAPP�) and the22C11 antibody that also detects rat and human APP. This kitwas standardized using a supplied, highly purified sAPP�fusion protein.Metabolic Labeling of APP in Primary Cortical Cultures—

Cultured neurons were labeled with 0.5 mCi/ml of [35S]Met-Cys (PerkinElmer Life Sciences) for 10 or 30 min and thenrinsed thoroughly and switched to growth medium supple-mented with unlabeledmethionine and cysteine. After baselinelabeling, two 60-mm dishes of neurons were harvested in 1 mlof RIPA lysis buffer each (total cellular APP) and supernatantsand cells from other dishes were collected at various timepoints. Labeled sAPP in the supernatant was immunoprecipi-tated with 10 �l of our N-terminal rat APP serum (a saturatingdose). Total cellular APP signal at t� 0 and at other time pointswas immunoprecipitated with saturating amounts of both theAPP CT antibody and our N-terminal APP antibody. By satu-rating, we mean that no further material could be immunopre-cipitated with a second round of immunoprecipitation. Theimmunoprecipitates were washed and fractionated on 4–20%Tris-HCl gels. The gels were dried, exposed, and radioactivebands were quantified using densitometry with appropriatesubtraction of background. A series of dilutions of the t � 0samplewere used to construct a standard curve for quantitatingthe densitometry. The radioactive signal in the immunopre-cipitates of supernatants and cells was also counted using liquidscintillation, subtracting the nonspecific signal due to immuno-precipitation with a similar amount of nonspecific IgG.Half-life Quantitation—The quantified APP data from met-

abolic labeling was fitted to a biexponential model by usingSigma Plot (SPSS Inc.). Themodel is described by the equation:y � A � exp(��t) � B � exp(��t), where � and � are thedegradation rate constants in the initial and terminal phases ofthe degradation curve. The model fitting was achieved by theleast squares criteria. For the biotinylation experiments, half-life determinations were based on previously published tech-niques (14). Protein band intensitywas quantitated using a den-sitometer. A standard curve was generated for each film usingAPP fusion protein or an internal standard, such that bandintensity of biotinylated material could be expressed as a per-centage of t � 0material. To calculate half-lives, we plotted thenatural logarithm of the percentage of protein remaining as afunction of time and found the slope of the resulting regressionline. Assuming first-order decay kinetics, the half-life is equal toln 2/slope.Immunoprecipitation and Immunoblotting of A�—Ten �l of

4G8 antibody was incubated with 1 ml of the supernatantand with unconditioned growth medium containing knownamounts of synthetic rodent A�1–40 (Bachem) overnight at4 °C. The antibody was immunoprecipitated with 50 �l of Pro-tein A. The amount of 4G8 antibody used for immunoprecipi-tation was sufficient to reduce the A� signal in the medium(determined with our ELISA assay) to levels detected in fresh,unconditioned medium. The immunoprecipitate was thenboiled in sample buffer and run on a denaturing Tris-Tricinegel, transferred to a nitrocellulose membrane, and probed withpurified rabbit anti-rat A� antibody and the signal visualized

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using chemiluminescence. We saw no oligomeric forms in ourgels using either these antibodies or the oligomer-sensitiveantibodies A11 (purchased from U. S. Biological) and 82E1(purchased from IBL).APP Surface Biotinylation—After 2–3weeks in vitro, cortical

cultures were cooled on ice for 15 min and surface biotinylatedfor 5 min using LC Biotin (Pierce) dissolved in PBS at 1 mg/mlfor saturation biotinylation and at 0.1 mg/ml for subtotal bio-tinylation. The saturating conditions have previously beenshown to result in complete biotinylation of all surface proteins(14). After washing, 1 ml of glial conditioned Neurobasalmedium containing 1% B27 supplement was added to eachdish, which was then placed back in the incubator. At differenttime intervals the cells were harvested in 0.5 ml of MPER(Pierce) with 1% protease inhibitor mixture. The cell lysatewere then precipitated with streptavidin-coated beads asdescribed earlier (14), and immunoblotted with the N-terminalor C-terminal APP antibodies as designated in the text. Quan-titative estimates of surface APP were determined by compar-ing the total streptavidin-precipitated material to various dilu-tions of the total cell material as described (12).Immunostaining and Fab Fragments—The APP N-terminal

and 2B antibodies were primarily labeled with DyLight 633 andDyLight 488, respectively (Pierce), and then digested withpapain using Pierce kit number 44985 to generate and purifyFab fragments. Fab fragments were used at a concentration of 7�g/ml for 30min of live staining of 2-week-old cultures inNeu-robasal medium. Cultures were then fixed, permeablized, andstained with mouse anti-synaptophysin or PSD-95 antibodies(Millipore). Consecutive, randomly selected neurons from 2platings were photographed (cell centered) at �100 and ana-lyzed for colocalization off-line.Neuronal Transfections—Human APP 695 was tagged mid-

way through the ectodomain by inserting a HA tag into theunique XhoI restriction site starting at base pair 909 of 2087.Two-week-old cultures were transfected with this construct asdescribed (15). Thirty-six hours after transfection, cells werestained livewith rhodamine-labeled rat anti-HAand then fixed,permeablized, and stained with FITC-labeled mouse anti-HA.Axons and dendriteswere identified by permeablizedHA stain-ing and differentiated as described previously (12).Measurement of Brain A� Production in Vivo—Juvenile

female Yorkshire pigs (90–102 pounds) were anesthetized with2.5% isofluorane andmaintained at 37 °C. The common carotidand internal jugular vessels were dissected on both sides. Anangle-independent and calibrated dual-beam Doppler ultra-sound device (FlowGuard; Cardiosonix Inc.) was used to mea-sure right and left common carotid blood flow. Fiveml of bloodwas collected from the right carotid artery and internal jugularvein in EDTA tubes twice, each separated by 15 min. Carotidblood flows were stable throughout the procedure. Each bloodassay for A�40 was done in triplicate using the Wako rat/hu-man A�40 kit at 1:6 and 1:8 dilutions using human standardsfrom Bachem (which gave very similar results to the rodentstandards) and at 1:6 using the Wako human A�1–40 ELISAusing the same human standards from Bachem. By using thesame standards across kits, the results can be more directlycompared. In one pig (pig 3) we measured right and left jugular

A�40 levels, which were identical (259 pg/ml (R) versus 265pg/ml (L)).

RESULTS

The A� Peptide Accumulates Rapidly and Linearly in theSupernatant of Cultured Neurons—To assess the normal pro-duction andmetabolism of A� in rodent neurons we developedan ELISA sensitive to both of the common forms of the A�peptide (A�1–40 and A�1–42) released during �-cleavage ofAPP. Using this assay, A� levels in supernatants from corticalneuronal cultures were quantified at 6, 12, and 24 h after acomplete change in medium using either our own standards orcommercially available standards (which gave identical results).As seen in Fig. 1A the levels of A� in the supernatant increaselinearly with time. Assuming there is no degradation or uptakeof A� (see below), and that all neurons release similar amountsin a non time-varying pattern (see “Discussion”), the amount ofA� released was quite substantial, calculated at roughly 4.7 �0.43 (S.D.) molecules/neuron/s (n� 4). Performing the assay inminimal essential medium (without protein) for periods up to5 h gave the same result (4.3� 0.49molecules/neuron/s; n� 3).Assays of supernatants from cultures derived from BACE1 orAPP knock-out mice, or from cultures composed only of gliashowed no A� in the supernatant. To further ensure the accu-racy of the ELISA, we compared the amount of A� in thesupernatant at 24 h as measured by ELISA to that measured bydenaturing immunoblot. As seen in Fig. 1B, maximal immuno-precipitation of secreted A� from culture supernatants at 24 h

FIGURE 1. A� and sAPP release by cultured neurons. A, time course of A�secretion by primary rat neuronal cultures measured by ELISA. Data are rep-resentative of four independent experiments (each assayed in duplicate) andare shown with mean � S.D. In B, an immunoblot of A� immunoprecipitatedfrom culture medium at 24 h is compared with standards immunoprecipi-tated in a similar fashion. The blot was probed with 2B antibody. The arrowsindicate the positions of the 3.8- and 8.4-kDa standards. In C, a portion of thesupernatant from cultured cortical neurons (top) was collected (in duplicate)at the indicated times and probed with our N-terminal APP antibody alongwith specified amounts of an N-terminal APP fusion protein (bottom). InD, mean APP ectodomain accumulation per ml of medium (�S.D.) over time ispresented from 4 experiments.

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produced a signal that was visually similar to a 2 ng/ml A�1–40standard, immunoprecipitated in a similar fashion. By con-structing a standard curve of the densitometer signals of vary-ing A�1–40 concentrations immunoprecipitated from culturemedium under the same conditions as the secreted material,the concentration of secreted A� at 24 h, determined by dena-turing immunoblot, was 1.8 � 0.5 ng/ml (mean � S.D. in 3experiments), similar to the A� concentration determined bythe ELISA. Finally, to ensure our assay was generalizable, weused two commercially available ELISA, a rodent/humanA�40kit (Wako) and a rodent A�1–40 kit (Invitrogen) for compari-son with our own ELISA using our own rodent standards.Results at the 6-h time point for both commercial kits (520� 51(S.D.) pg/ml of A�40 (n � 8) and 467 � 43 pg/ml of A�1–40(n � 4)) were in substantial agreement with our own assay (570pg/ml; Fig. 1A).APP Ectodomain Release Parallels the Rise of A� in

Supernatant—To measure APP ectodomain secretion andaccumulation we used two antibodies, the commercially avail-able 22C11 antibody and our own N-terminal polyclonal APPantibody generated from an APP N-terminal fusion proteincapable of distinguishing APP and its secreted ectodomainfrom other related molecules either by size or specificity (Fig.2).Quantitative immunoblotting of supernatants fromculturedneurons (n � 4, in duplicate) demonstrated that the levels ofsecreted APP ectodomain in the supernatant increased linearlywith time, similar to the increase in A�. Using an APP ectodo-main fusion protein of known concentration (and confirmedwith a second commercially purchased fusion protein), wewereable to quantitate the accumulation of the ectodomain. Asshown in Fig. 1, C and D, the amount of APP ectodomain thataccumulates in the supernatant by 24 h, on a nanogram basis, is67 times greater than A�, and equates to 12.7 � 2.9 (S.D.) mol-ecules/neuron/s. Using the 22C11 antibody to quantify APPectodomain accumulation at 24 h we obtained a very similarresult (158� 23 (S.D.) ng/ml; n� 4). On amolar basis, the ratioof APP ectodomain accumulation to A� accumulation in thesupernatant is 2.7 to 1 at 24 h.Stoichiometry and Site of APP Cleavage—To determine the

ratio of �- versus �-cleavage of APP, we immunoprecipitatedthe APP ectodomain component corresponding to cleavage ofAPP at the �-secretase site (sAPP�) from the supernatant ofneuronal cultures after a 24-h incubation. To do this we usedthe 2B antibody that recognizes sAPP� 1–16 (cleaved beforeamino acid 17 of the A� fragment) and sAPP 1–10 (cleavedbefore amino acid 11 of the A� fragment) but not sAPP�(cleaved prior to the A� fragment), so that any non-amyloido-genic cleavages of APP would be immunoprecipitated. Whenneuronal supernatants are immunoprecipitated using saturat-ing concentrations of the 2B antibody, a substantial portion ofthe total APP ectodomain (30.5 � 8.6%; n � 4) is left in thesupernatant (Fig. 3A). This residual component presumablyrepresents sAPP�, that portion of the APP ectodomain thatshould be produced in a 1:1 ratio with A�. Probing the immu-nodepleted supernatant with the 2B antibody (Fig. 3A) showsthat no residual signal attributable to residual sAPP� remains.Repeat immunoprecipitation with the 2B antibody broughtdown no further sAPP� (not shown). Performing the comple-

mentary experiment (Fig. 3B), we found that an antibody thatspecifically immunoprecipitates sAPP� left a residual sAPP sig-nal of 61 � 9.8% (n � 4) in the medium. Assuming 2 moleculesof sAPP� are generated for every molecule of sAPP�, andknowing the amount of total sAPP released by neuronal cul-tures (12.7 � 2.9 molecules/neuron/s), the amount of A�expected to be released into the supernatant if the release of A�and sAPP� is a 1:1 phenomenon would be 4.2 molecules/neu-ron/s, which is very close to that actually observed (4.7 mole-cules/neuron/s), suggesting that there is a close (possibly 1:1)relationship between �-cleavage of APP and the release of A�and sAPP�. To confirm our results of a nearly 1:1 relationshipbetween the �-cleavage of APP and the extracellular release ofA� and sAPP� we used a commercially available ELISA, whichdetects full-length rodent sAPP�. Using this assay to test cul-ture supernatants after a 24-h incubation, the sAPP� contentwas 39 � 7 ng/ml (n � 3) compared with a total A� content(done simultaneously) of 2.2 � 0.3 ng/ml, a molar ratio of 1.1molecules of A� for every molecule of sAPP� detected in thesupernatant. Of note, immunoprecipitation of the culturesupernatant with the 2B antibody prior to the sAPP� ELISA

FIGURE 2. Specificity of APP antibodies. A, cells, supernatant, and surface(Biot) material from cultured cortical neurons are shown after blotting withour N-terminal APP antibody. In B, the same antibody along with a series ofother APP family antibodies are used to blot 293 cells transfected with theindicated constructs. Twenty-five �g of total protein was added per lane.There is little signal overlap with APLP1 using the APP N-terminal and 22C11antibodies and little size overlap with APLP2. In C, cultured neuronal super-natants are probed with an APLP2-specific antibody and a sample of the samesupernatant is probed with the N-terminal APP antibody. The size of the faintAPLP2 signal in the supernatant is much larger than the APP signal. InD, material from cortical neurons was immunoprecipitated (IP) with the APPC-terminal antibody and then probed with the APP N-terminal antibody orwith an APLP1 antibody, showing little APLP1 signal is immunoprecipitatedwith the APP C-terminal antibody. The bottom band in D represents the heavychain of the immunoprecipitating antibody. In E, differing concentrations of 3different N-terminal APP-GST fusion proteins are shown probed with theaffinity purified 2B antibody. All GST fusion proteins contained the APP ecto-domain (amino acids 202–597) plus the indicated contiguous segment of theA� peptide.

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had no effect on the results (not shown), demonstratingspecificity in the immunoprecipitation experiments of Fig. 3,A and B.Metabolic Labeling of APP Shows That theMajority of APP Is

Metabolized through Extracellular Release—Metabolic label-ing with [35S]methionine allowed us to examine the percentageof APP, which, once synthesized, is secreted extracellularly, andwhether it is degraded. As shown in Fig. 4, APP is secreted at arapid rate. The majority of the cellular APP signal at t � 0 ispresent in the extracellular medium at 3 h, consistent with an

extremely fast turnover as noted previously in cultured neurons(11). Approximately 90% of the total cellular APP present attime 0 appears in the extracellular medium by 9 h (Fig. 4B)when calculated by liquid scintillation following immunopre-cipitation (91 � 11%; n � 4) or by densitometry (92 � 9%; n �4). The percentage of metabolically labeled APP released intothe medium was not dependent on the metabolic labeling timeof the cultured neurons. A 10-min metabolic labeling time (ascontrastedwith the typical 30-min labeling time) resulted in thesame release of total labeled cellular APP into the medium by9 h. The fact that nearly all the cellular APP at t � 0 is recover-able from the supernatant 9 h later at the correct molecularweight for the APP ectodomain indicates that nearly all APPcatabolism leads to extracellular release and that little degrada-tion of the secreted APP ectodomain is occurring in the super-natant over this time scale.Surface APP Represents a Small Proportion of Total Cellular

APP and Turns Over at a Much Slower Rate—Consistent withthe rapid release of soluble APP from neurons, the metabolichalf-life of radioactively labeled full-length endogenous APP isquite rapid (Fig. 5A) with themajority of the cellular signal goneby 12 h but with a small residual signal lasting past 24 h. Usingliquid scintillation, we quantitated the cellular APP C-terminal

FIGURE 3. Stoichiometry of APP cleavage. In A, neuronal supernatants wereimmunodepleted with the 2B antibody, which recognizes the 16 amino acidson the C terminus of sAPP�. The residual supernatant was then run on adenaturing gel and blotted with the 22C11 APP N-terminal antibody (top) orthe affinity purified 2B antibody (bottom). A serial dilution of non-immunode-pleted supernatant was run for quantitative purposes. As controls, themedium was immunodepleted with the rabbit N-terminal APP antisera (APP)and with control rabbit preimmune serum (IGG) at the same concentration asthe 2B antibody. No residual 2B signal was seen in the supernatant afterimmunoprecipitation (IP) with the 2B antibody. In B, neuronal supernatantswere immunodepleted with an sAPP�-specific antibody (at 4 �g/ml) or withcontrol antibodies at similar concentrations. The residual supernatant wasthen run on a denaturing gel and blotted with the 22C11 APP N-terminalantibody. A serial dilution of non-immunodepleted supernatant was run forquantitative purposes.

FIGURE 4. Metabolic labeling shows near complete release of cleavedAPP ectodomain into the extracellular medium. A, metabolic labeling ofcellular APP followed by immunoprecipitation of APP from cells (t � 0) orsupernatant (other time points). Cellular APP was immunoprecipitated with acombination of the APP CT and the N-terminal APP antibodies (APP) or withcontrol IGG. The supernatants were immunoprecipitated with only the N-ter-minal APP antibody. In B, data from a series of 8 experiments is shown (4quantitated after SDS-PAGE and 4 quantitated by immunoprecipitation fol-lowed by liquid scintillation as described under “Experimental Procedures”).The data are presented as the mean � S.D.

FIGURE 5. Metabolic labeling and surface biotinylation demonstrate fastand slow APP turnover. A, metabolic labeling of APP followed by immuno-precipitation (IP) from cells using the APP C-terminal antibody or control IGG.In B, the data (�S.E.) for the observed decay of cellular APP immunoprecipi-table material is plotted and fitted to a theoretical curve for the biexponentialdecay of cellular APP as described in the text (n � 4). In C, cultured corticalneurons were surface biotinylated using a saturation protocol and streptavi-din immunoprecipitated. 100% of the surface (IP) material was run along witha standard curve of varying percentages of the total cellular material and theblot was probed with the APP N-terminal antibody. D, 50% of the biotinylatedsurface material (Surf) from sister cultures at t � 0 along with 20% of the totalcell material (Tot) at each time point are displayed along with 7% of the totalsecreted supernatant material at the 3-h time point (Sup). E, 100% of thesurface material from sister cultures following biotinylation at t � 0 alongwith 10% of the total cell material at each time point are displayed along withan APP fusion protein standard curve run simultaneously. Both D and E wereprobed with the 22C11 APP antibody. F, the half-life curve calculated from theexperiment shown in E.

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antibody precipitable signal as a function of time, subtractingnonspecific signal immunoprecipitated by a similar amount ofnonspecific IGG. As shown in Fig. 5B, the degradation of theradioactive signal can be fitted to a biphasic decaywith an initialrapid degradation (t1⁄2�) of 2.2 h (95% confidence interval 1.9–2.8; n� 4) and a subsequent relatively slow degradation (t1⁄2�) of29 h (95% confidence interval 15–68 h). The fraction thatturned over at a slower rate amounted to 7% of the total signal.Using densitometry, rather than liquid scintillation, the half-lifefor the major portion of the decay was 2.5 h (95% confidenceinterval 2.0–3.2; n� 4) with a residual component of 5%, whichhad a t1⁄2 of 23 h (95% confidence interval 14–45 h). Similarresults were obtained when the N-terminal APP antibody wasused for immunoprecipitation.The component of total cellular APP that turns over slowly

was similar in amount and turnover rate to the fraction of APPwe found present on the surface of neurons. Using saturatingsurface biotinylation (14), the amount of the total APP presenton the surface (i.e. was subject to biotinylation using a non-permeable biotinylation reagent) was 5.3 � 1.8% (n � 5) usingthe N-terminal APP antibody to quantitate APP (Fig. 5C) and6.5 � 2.2% using the C-terminal APP antibody. Degradation ofthe surface componentwas quite slow comparedwith release ofthe ectodomain. As shown in Fig. 5D, over a short time period(3 h) and using either a saturating or subtotal biotinylationstrategy, there is almost no decrease in the surface APP com-ponent whereas, over the same period, the amount of ectodo-main recoverable from the supernatant becomes a considerablefraction of the total cellular APP pool. Half-life experimentslooking at the decay of the surface APP signal over longer timepoints (Fig. 5, E and F) show that the surface component decayswith a half-life of 19 � 4 h (mean � S.D.; n � 4), a number wellwithin the confidence intervals of the slow phase of APP deg-radation seen with metabolic labeling. Live surface immunola-beling of cultured neurons with tagged Fab fragments of theAPP N-terminal antibody or the 2B antibody showed micro-clusters of immunostaining for each of these two APP epitopes,which overlapped completely (Fig. 6A). The surface clusters ofAPP were both synaptic and non-synaptic; 336 of 1142 surfaceAPP clusters (30%) from 30 neurons showed colocalizationwith the synapticmarker synaptophysin. Approximately 28% ofPSD-95 immunopositive excitatory synapses (184 of 663PSD-95 positive synapses examined in 25 neurons) had colocal-ized surface APP immunostaining (Fig. 6B, arrows), with APPpositive and negative excitatory synapses clustered nearby onthe same neuron. Fourteen percent (38 of 254) of glutamic aciddecarboxylase-positive inhibitory synapses also had colocalizedsurface APP clusters. Neurons derived from APP knock-outmice showed no surface staining (not shown), whereas perme-ablized staining of neurons with both antibodies showed a dif-fuse pattern (not shown). Transfection studies using humanAPP 695 HA-tagged in the extracellular ectodomain indicatedthat surfaceAPPonneurons could be both axonal anddendriticin origin (Fig. 6, C and D), as all dendrites and axons of trans-fected neurons showed punctate, live, surfaceHA staining (red/yellow) in addition to a nonhomogeneous intracellular pattern(green).

Brain A� Production and Secretion in Vivo Is Similar to ThatMeasured in Cultured Neurons—To validate our experimentsin cultured neurons, we measured the amount of A� that isproduced and then transported intact from the brains of juve-nile Yorkshire pigs. We simultaneously measured bilateralcommon carotid blood flows using a Doppler ultrasound probewhile measuring blood A�40 (sensitive to both A�11–40 andA�1–40) and A�1–40 in the right carotid artery and jugularvein in 5 pigs. The common carotid blood flow was then cor-rected for the known fraction of common carotid blood flow,which supplies the cortex in pigs (58%) resulting in a calculatedcortical blood flow (16). The cortical flow values we obtainedare similar to those seen in humans (17–19) corrected for thedifferences in brain weight between human and pig. Carotid-jugular A�40 and A�1–40 differences were determined twicein each animal separated by 15 min and averaged. As shown inTable 1, brain A� production and secretion in the pig is alsosubstantial, with a mean of 13.1 � 3.4 (S.D.) billion A�40 and11.4 � 2.8 billion A�1–40 molecules released per second,assuming that release does not vary significantly over shorttime intervals (see “Discussion”). Given that the pig neocortexand basal ganglia (the part of the brain whose blood flow andvenous drainage we are measuring) is composed of 7 � 108neurons, extrapolating fromFig. 2 of Braitenberg (20), and leav-ing out the cerebellum (because we do not sample vertebralblood flow or the vertebral venous system), we come to a rough

FIGURE 6. Surface APP immunostaining. A, Fab fragments of the APP N-ter-minal (red) and 2B (green) antibodies are used to stain cultured neurons simul-taneously. Near complete overlap is shown in the bottom panel. In B, livestaining with an Fab fragment of the N-terminal APP antibody was followedby permeablized staining for the excitatory synapse-specific antigen PSD-95.Examples of synaptic colocalization are shown with white arrows. The spacebars represent 10 �m. In C and D, a dendrite and axon, respectively, trans-fected with HA-tagged APP and stained live for surface HA-APP (red/yellow)and cellular HA-APP (green) both showed surface APP staining.

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figure of 2molecules of A�40 per neuron per second (assuminga uniform release probability and that all neurons release thesame amount of A�). It should be noted that the A�40 assayused in these experiments was the same assay used to confirmour results in cultured neurons (it recognizes rodent and swineA�40 equally) and that neuronal culture medium and dilutedserum spiked with standards give readings within 22.4 � 3%(n � 3) of each other (serum being lower). Unfortunately, wewere not able to detect reliable differences in the amount ofsAPP ectodomain in arterial and venous samples from the pigbrain. Although our results demonstrate the extent of brain A�production they do not exclude the possibility of additionaltransport of A� out of brain arterioles into the brain paren-chyma and then into the cerebral venous circulation. This proc-ess would not effect our results or conclusions because it wouldhappen following arterial sampling in the carotid artery andprior to venous sampling in the jugular vein, respectively, can-celling each other out.

DISCUSSION

The Quantity and Speed of APP Processing and Its Implica-tions for Alzheimer Disease—To develop an effective strategyfor reducing A� production in pathological conditions, it isimportant to precisely understand the quantity of APP metab-olized under normal circumstances, as well as its pathways fordegradation and A� release. In our cultures, APP is processedin excess of 10 molecules/neuron/s, and the vast majority of itsmetabolism leads to the extracellular release of its cleavageproducts. The only portion of APPmetabolism that is slow is itssurface component. Although we describe APP and A� releaseusing the phrase “per neuron per second” as ameans of convey-ing the amount released, the amount of A� that individual neu-rons release may differ substantially depending on the subclassof neuron, and there may be time relevant, activity dependent,fluctuations in APP and A� secretion on a second by secondscale (21).A� is produced in 1 of 3 cleavages of APP in vitro and is

produced and released from the brain at a similarly large rate invivo. Although the rapid turnover of APP might be consistentwith a role as a tightly regulated growth factor (22) or regulatoryenzyme, the large amounts of APP and all its degradative

metabolites that are produced and secreted by neurons wouldargue against these roles for the APP ectodomain and its pep-tide metabolites. The rapid turnover and large quantities ofAPP we have seen metabolized in vivo and in vitro is consistentwith a proposed role for APP as a cargo protein in fast axoplas-mic transport (23) with a compulsory degradation and releaseafter one trip down the axon, although the significance of thisrole for APP is still in dispute (24). Our data are also consistentwith work that suggests that large amounts of the APP ectodo-main shed into the extracellular space may serve a nonspecificneuroprotective role (25). Our work is consistent with that ofothers (7, 9) who have shown large amounts of A� accumulatein the supernatant of cultured neurons. The work by Seubert etal. (7) is particularly relevant as they measured 72-h accumula-tion of A� in the supernatant of cultured human neurons at 4ng/ml, a number not inconsistent with our results (by extrapo-lation). Our work more precisely quantitates A� release, ratherthan accumulation, and quantitatively places A� release in theoverall context of APP metabolism. Moreover, our results invivo are an important extension and validation of the tissueculture work.The large quantities of solubleAPP ectodomainwe have seen

released in these cultures and presumably in vivo needs to bereconciled with the recent work suggesting sAPP� is a ligandfor DR6-mediated axonal pruning (26). In this formulation,growth factor deprivation triggers BACE1 cleavage of APPreleasing the ectodomain, which then binds to DR6 and acti-vates caspase 6 and caspase 3, causing axonal and cell bodyapoptosis, respectively. How this could become manifest in anenvironment in which sAPP� is in such plentiful supply isunclear, suggesting that quantities of sAPP� may be morerestricted in specific compartments. One further implication ofthe large quantities of APP and A� that are produced in vitroand in vivo is that clearance mechanisms (27, 28) and theirregulation by aging should assume a more important target fortranslational research efforts.Secretion of the APP Ectodomain and the A� Peptide—Our

data suggests a tight relationship between the amount of�-cleavage of APP and the extracellular release of the sAPP�ectodomain fragment and the A� peptide. In addition, most

TABLE 1Brain A� production in vivoFive juvenile Yorkshire pigs had simultaneous right carotid and right jugular A�40 and A�1–40 (parentheses) levels determined along with measures of bilateral commoncarotid blood flow (CCBF). The cortical blood flow is calculated. Carotid and jugular A�40 and A�1–40 levels were determined twice in each animal 15 min apart. Eachassay was done in triplicate (themeanCV is 7.1%). TheA�40 andA�1–40 levels are themeans of the twomeasures (C.V. betweenmeanswas less than 5.0%). Cortical bloodflow was calculated from the measurement of bilateral common carotid blood flow as described under “Experimental Procedures.”

Pig

Right carotidA�40 (A�1–40)

levels

Right jugularA�40 (A�1–40)

levelsBrain A�40 A-V

(A�1–40) gradientBilateralCCBF

Corticalblood flow

BrainA�40 (A�1–40)

production

pg/ml ml/min Molecules/s1 253 311 58 231 135 18.5 � 109

(217) (264) (47) (15.0 � 109)2 179 212 33 250 147 11.4 � 109

(246) (286) (40) (13.8 � 109)3 215 259 44 227 133 12.8 � 109

(230) (261) (31) (9.1 � 109)4 415 439 24 283 166 9.4 � 109

(299) (326) (27) (10.3 � 109)5 212 253 41 240 141 13.5 � 109

(241) (268) (27) (8.9 � 109)

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APP synthesized by these neurons appears in the extracellularspace in short order. Reports of the amount of APPmetabolismthat leads to extracellular release versus intracellular accu-mulation have been variable depending on the cell lines stud-ied and constructs used (10, 29, 30). In our neurons, process-ing endogenous APP, most A� is released into theextracellular medium. Intraneuronal A� has been implicatedin the early pathogenesis of AD (31) and studies have docu-mented the intracellular accumulation of A� and its effectson neurodegeneration (30–32). In this regard, it should beemphasized that even if only a small percentage of APPmetabolism led to intracellular A� retention, this would stillrepresent a large quantity of A�, given the overall quantitieswe have documented. Moreover, under pathological condi-tions the ratio of intracellular versus extracellular accumu-lation could certainly vary.The Slow Turnover of Surface APP Suggests New Models for

APPMetabolism—Our data shows a biexponential metabolismof APP and further suggests that the component of APP withthe slower turnover rate is the surface component. The quantityofAPPwith a slowmetabolic turnover ratematches the percentof the surface component of APP relative to the overall pool.Moreover, the slow rate of surface APP turnover is consistentwith the slow phase of metabolism from metabolic studies.These observations are consistent with prior observations byStorey et al. (11) who described two pools of APP in culturedneurons with different metabolic half-lives. This group alsopresented evidence that the slowly metabolized pool was thesurface component of APP. Our work extends these importantresults in a more quantitative fashion, accounting for nearly allAPP metabolism and release. These results are importantbecause the current model of APPmetabolism postulates a cellsurface phase for the full-length amyloid precursor proteinprior to � and � metabolism (6). If the surface component ofAPPhas amuch slower turnover rate than the total cellular poolof APP, it would exclude it from being the obligate precursor tometabolism of the total pool. However, other explanations arepossible. First, there could be a second pool of surface APP thatturns over so fast that it never amounts to a significant compo-nent of the total surface pool even though it might still be themain pathway for APP catabolism. Our results are also consis-tent with a model in which the majority of �- or �-cleavageoccurs prior to the surface, except for the small pool of slowlymetabolized, full-length, surface APP, and that reinternaliza-tion is only necessary for �-cleavage. Whether the slowlymetabolized surface pool serves a unique role or has simplyescaped normal cellular processing mechanisms is unclear.Glutamate receptors are known to be stabilized on the cell sur-face by their synaptic localization (14), but we have notobserved the same degree of synaptic localization for surfaceAPP as is seenwith glutamate receptors. Interestingly, however,the turnover rate for surfaceAPP is quite similar to the turnoverrate for amixture of synaptic and nonsynaptic glutamate recep-tors (14), potentially offering a clue to the mechanism for deg-radation of these molecules. Our finding that much surfaceAPP is non-synaptic and that themajority of synapses lack clus-tered APP is different from some previous reports (33, 34) butmore consistentwith other reports, which suggest surface func-

tions other than synaptic integrity (11). It is possible that sub-surface pools of APP aremore synaptically localized (we did notobserve this) or that rapid �- or �-cleavage at the synapseobscures the synaptic localization of APP in these cultures.Limitations of this study include the lack of quantitation of

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and Richard J. O'BrienAbhay Moghekar, Sneha Rao, Ming Li, Dawn Ruben, Andrew Mammen, Xiaopei Tang

in Vitro and in VivoPrecursor Protein Metabolism Peptide Are Constitutively Released during AmyloidβLarge Quantities of A

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