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NATURE MEDICINE • VOLUME 6 • NUMBER 2 • FEBRUARY 2000 143

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Accumulation of amyloid β-peptide (Aβ) in brain plays an up-stream pathogenic role in the decades-long pathological cascadeleading to Alzheimer disease (AD) as established by a series ofstudies analyzing the phenotypes of gene mutations causingearly-onset familial AD (refs. 1–3). Paradoxically, Aβ is also aphysiological peptide constantly secreted from cells throughoutlife and known to be trophic under certain conditions, althoughits definite in vivo function remains unclear4,5.

Upon aging, most humans start to accumulate Aβ in brain byyet unknown mechanism(s)6. It should be noted that aging isstill the major risk factor even for those carrying the familial AD-causing gene mutations. Therefore, Aβ is, at least in young andhealthy brains, fully catabolized immediately after secretion be-fore being deposited. This catabolic mechanism of determiningthe in vivo life span of Aβ in brain is important for two reasons.First, a reduction in this process would lead to pathological accu-mulation of Aβ and thus to AD development. Second, we may beable to prevent the disease onset or decelerate its progression bymanipulating this process.

However, our current knowledge on the catabolism of Aβ isquite limited as compared with the mass of information avail-able concerning the anabolism of Aβ. This can be attributed tothe technical limitations of the molecular and cellular biologicalapproaches that have proved very powerful for analyzing theproduction of Aβ from amyloid precursor protein (APP). In cul-ture, cells exist in a monolayer in the presence of a medium withmore than 100-fold the cell volume. In contrast, neuronal andglial cells in brain tissue are organized close to each otherthrough an extracellular matrix in the presence of much smallervolumes of extracellular fluids. The extracellular environmentsare so different among these systems that it is simply inappropri-ate to apply the knowledge obtained in experiments using cul-tured cells to understanding the catabolism of secreted peptidesin brain. It should also be noted that proteolytic systems are

often drastically altered when cells are transferred from an organto culture. We need to establish a physiologically relevant exper-imental paradigm in which we can capture the actual catabolicprocesses in brain.

In addition to the technical difficulties mentioned above, ex-tracellular protein catabolism is rendered quite complex by thepresence of various possible mechanisms. Clearance of a givenpeptide from brain may be achieved by (i) transport to vascularsystems, which may not depend on peptidases in brainparenchyma (ii) proteolysis conducted by extracellular pepti-dases, or (iii) receptor-mediated endocytosis followed by endoso-mal-lysosomal proteolysis. We may also need to consider thepossible existence of physiological cofactor(s) that assist or regu-late these processes. To overcome these problems, we developeda new experimental design in which we injected multiple-radio-labeled Aβ peptide into rat hippocampus and analyzed its metab-olism by tracing the labels. The peptide was internallyradiolabeled without altering its chemical structure to minimizethe risk of producing artifacts. This new approach led to novelfindings and medical implications that would otherwise have re-mained veiled. Here we describe identification and characteriza-tion of the rate-limiting step in the in vivo Aβ catabolism.

Degradation of 3H/14C-labeled Aβ1-42 in rat hippocampusThe 42-amino-acid Aβ peptide carries several possible cleavagesites that may be attacked by various peptidases (Fig. 1a). So asnot to overlook any possible proteolytic fragments derived fromthe intact Aβ during the course of catabolism, we synthesizedAβ1–42 multiple-radiolabeled with 3H and 14C at distinct aminoacid residues (Fig. 1b). The amino- and carboxyl-terminal halveswere differentially marked so that we could observe whether ornot either side of the peptide would undergo preferential prote-olysis. This peptide was injected into the CA1 sector of rat hip-pocampus and subjected to in vivo degradation (Fig. 1c and d).

Identification of the major Aβ1–42-degrading catabolic pathwayin brain parenchyma: Suppression leads to biochemical and

pathological deposition

NOBUHISA IWATA1, SATOSHI TSUBUKI1, YOSHIE TAKAKI1, KAORI WATANABE1, MISAKI SEKIGUCHI1,EMI HOSOKI1, MAHO KAWASHIMA-MORISHIMA2, HAHN-JUN LEE1, EMI HAMA1,

YOKO SEKINE-AIZAWA1 & TAKAOMI C. SAIDO1

1Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, JAPAN2Department of Neuropathology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033

Correspondence should be addressed to T.C.S.; email: [email protected]

Alzheimer amyloid β-peptide (Aβ) is a physiological peptide constantly anabolized and catabo-lized under normal conditions. We investigated the mechanism of catabolism by tracing multi-ple-radiolabeled synthetic peptide injected into rat hippocampus. The Aβ1–42 peptide underwentfull degradation through limited proteolysis conducted by neutral endopeptidase (NEP) similaror identical to neprilysin as biochemically analyzed. Consistently, NEP inhibitor infusion resultedin both biochemical and pathological deposition of endogenous Aβ42 in brain. This NEP-cat-alyzed proteolysis therefore limits the rate of Aβ42 catabolism, up-regulation of which could re-duce the risk of developing Alzheimer’s disease by preventing Aβ accumulation.

© 2000 Nature America Inc. • http://medicine.nature.com©

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Fig. 2 Profiles of in vivo 3H/14C-Aβ1-42 proteolysis. The 3H/14C-Aβ1-42

peptide (Fig. 1b) was injected into rat hippocampus and extracted at 0min (a and e), 3 min (b and f), 10 min (c and g), and 30 min (d and h)after injection. The radioactive profiles are shown in 14C mode in panelsa–d and 3H mode in panels e–h. Insets (a and e), homogenized in theabsence of sodium dodecylsulfate (SDS). Arrow, the elution volume ofthe major catabolic intermediate. A flow scintillation monitor gives rel-ative values of radioactivity rather than absolute measurements.

144 NATURE MEDICINE • VOLUME 6 • NUMBER 2 • FEBRUARY 2000

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The resultant products were extracted and analyzed by high-pressure liquid chromatography (HPLC) connected to a flowscintillation monitor. We first inspected the relevance of this ex-perimental paradigm by tracing the metabolism of a 3H-an-giotensin II peptide. The intact peptide was completelycatabolized to amino acids within 5 minutes, producing cata-bolic intermediates, angiotensin III and IV, detected at 1 minute,and co-injection of an aminopeptidase inhibitor, amastatin, si-multaneously blocked the entire degradation and the appear-ance of intermediates (data not shown). This observationindicates that aminopeptidase action is a rate-limiting process inangiotensin II catabolism7 and that the co-administration of aprotease inhibitor effectively suppresses the target peptidases inthis experimental paradigm.

The 3H/14C-Aβ1-42 peptide also underwent proteolyticdegradation in hippocampus as analyzed by HPLC al-though it proceeded more slowly, with a half-life of 15–20minutes (Fig. 2). The major peak at retention time of 42minutes represents the full-length Aβ1-42 peptide whereasthe early peak at 7–8 minutes corresponds to a void volumewith catabolites composed of small peptides and aminoacids. The possibility that transport to vascular systems maybe the primary mechanism of Aβ1-42 clearance can be dis-carded because the injected radioactivity remained insidethe hippocampus throughout the catabolic processes. Thissuggests that brain parenchyma possesses independent andpotent proteolytic systems capable of determining the lifespans of extracellular peptides.

A notable feature is the appearance of a radioactive peakat 39 minutes corresponding to a catabolic intermediate(arrows, Fig. 2b–d and f–h). This observation suggests thatAβ undergoes typical limited proteolysis in vivo first.Because there is only a single major peak and because it isobserved at all time points after injection, processes in-volved in the production of this fragment are likely to playa key rate-limiting part in the major catabolic pathway as inthe cases of other peptides including angiotensin II andopioids8. The HPLC profiles of the 3H and 14C radioactivities

Fig. 1 Experimental scheme. a, Possible cleavage sites in Aβ1–42 by vari-ous peptidases. a, aminopeptidase; a′, dipeptidyl peptidase; b, chy-motrypsin-like endopeptidase; c, trypsin-like endopeptidase; d, gelatinaseA (MMP-2), α-secretase; e, endopeptidase 24.11 (NEP, neprilysin); f,cathepsin D/E, gelatinase B (MMP-9); g, collagenase; h, carboxypepti-dase; h′, peptidyl dipeptidase; i, insulin-degrading enzyme. Most of thecleavages sites were determined using Aβ1−40 or Aβ1−42 as a substrate andthe others were assessed based on the specificities of peptidases13,34. b, Aβ1–42 peptide multiple-labeled with 3H at positions 2 (A), 4 (F), 9 (G),and 17 (L) and with 14C at 20 (F), 29 (G), 34 (L), 40 (V), and 42 (A) as in-dicated by shadowed and underlined letters. c, Experimental scheme. d, Radiolabeled peptides were injected into the brain of an anesthetizedrat as demonstrated by the localization of an injected dye marker, Evansblue, in the CA1 sector of hippocampus.

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Fig. 3 Two-dimensional HPLC separation ofthe catabolic intermediate and its aggregationproperty a, Second HPLC analysis of catabolicintermediate. The catabolic intermediateshown in Fig. 2d was further analyzed. Half ofthe eluent was used for scintillation countingand the other half was collected for furtheranalyses, peptide sequencing and mass spec-trometry, that identified the catabolic interme-diate as Aβ10-37. Inset, mass spectrometric datagiving an MH+ value of 3,092.306 correspond-ing to the molecular weight of 3091.306. b, Aggregation property of synthetic Aβ1–42

and Aβ10–37 peptides.


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are essentially identical to each other at varioustime points, indicating that there is no notablepreferential proteolysis of Aβ from either theamino- or carboxyl-terminal side. Therefore,Aβ1–42 seems to be fragmented, essentially, simul-taneously from both ends. Interestingly, weneeded a detergent, sodium dodecyl sulfate(SDS), to extract the Aβ peptide immediatelyafter administration (see the insets in Fig. 2a, e),suggesting that Aβ enters an insoluble phaseprobably representing membrane fractions be-fore the subsequent proteolytic event(s).

Structure of catabolic intermediateTo investigate whether the catabolism involvesspecific limited proteolysis and to gain insightsinto the responsible peptidase(s), we determinedthe primary structure of the catabolic intermedi-ate as follows. We subjected the correspondingfractions obtained in the initial HPLC separationto a second HPLC under different conditions(Fig. 3a). This 2-dimensional HPLC analysis indi-cated that the intermediate is composed essen-tially of a single peptide component. Chemicalsequencing of this peptide gave an amino-termi-nal sequence of Aβ starting with tyrosine atresidue 10. Furthermore, mass spectrometric analysis gave a mol-ecular mass of 3,091.3, which is identical within the range oftechnical deviation to the calculated molecular weight of3,091.55 for Aβ10–37 with the methionine residue oxidized. Thismethionine oxidization occurred during HPLC separation,which used columns heated to 50 °C under both basic and acidicconditions9. This primary structure of the catabolic intermediateindicates that it is specific endoproteolysis from both ends thatproduced the intermediate.

This intermediate peptide probably does not contribute topathological Aβ accumulation in brain for the following reasons.First, a synthetic Aβ10–37 peptide was less fibrillogenic than Aβ1–42

peptide (Fig. 3b). Second, because production of this intermedi-ate seems to constitute a rate-limiting step in Aβ1–42 catabolism(see next section), the physiological steady-state amount of thispeptide in brain is expected to be very low.

Suppression of Aβ catabolism by specific peptidase inhibitorsTo further characterize the nature of the in vivo Aβ catabolism,we examined the effect of various protease inhibitors (Table 1).First, none of the inhibitors against the endosomal-lysosomalproteolysis, chloroquine, pepstatin A, leupeptin, E-64, and Z-

LLL-al, had any appreciable influence. This suggests that recep-tor-mediated endocytosis followed by lysosomal-endosomaldegradation does not function as a primary catabolic mechanismin the time range examined. Among all the protease inhibitorstested, only phosphoramidon and thiorphan caused an almostcomplete inhibition of in vivo Aβ1–42 catabolism (Fig. 4).Phosphoramidon is an inhibitor of metalloendopeptidase with arelatively narrow inhibitory spectrum. For instance, it does notinhibit matrix metalloproteases (MMP’s) or insulin-degradingenzyme (IDE) and, in accordance, a potent MMP inhibitor,CGS27023A (ref. 10), or insulin, respectively, had no effect.There are three known major groups of phosphoramidon-sensi-tive metalloendopeptidases: neprilysin (also known as neutralendopeptidase 24.11 (NEP) and enkephalinase), angiotensin-converting enzyme (ACE), and endothelin-converting enzyme(ECE)11–15. ACE was excluded as a candidate peptidase because theACE inhibitor cocktail did not block Aβ catabolism.Furthermore, thiorphan, a selective inhibitor of neprilysin andACE, was quite effective even at a very low initial concentrationof 1 µM, which corresponds to a final in vivo concentration of ap-proximately 10 nM. Together, these observations suggest thatneutral endopeptidase (NEP), similar or identical to neprilysin, is

Table 1 Summary of protease inhibitor effects on in vivo 3H/14C-Aβ1-42 degradation

Inhibitor Initial concentration Target peptidases Effect on catabolism

chloroquine 0.2 mM lysosomal -pepstatin A 0.2 mM aspartate -leupeptin 10 mM serine

cysteineE-64 0.2 mM cysteine -Z-LLL-al 0.1 mM cysteine

proteasomeAPMSF 1 mM serine -TPCK 0.1 mM serine -aprotinin 1 mg/ml serine -chymostatin 0.1 mM chymotrypsin -insulin 10 mg/ml IDE -phosphoramidon 0.1, 1, 10 mM metallo

NEP > ECE > ACE +thiorphan 0.001, 1 mM NEP > ACE +ACE inhibitorsa 0.05 mM each ACE -CGS27023A 0.01 mM MMP -amastatin 0.5 mM aminopeptidase -aminopeptidase inhibitorsb 0.5 mM each aminopeptidases -puromycin 1 mM aminopeptidase -

-, no or little inhibition; +, potent inhibition. Z-LLL-al, benzyloxycarbonyl-Leu-Leu-leucinal; APMSF, 4-(amidinophenyl)methanesulphonyl fluoride); TPCK, L-1-choler-3-(4-tosylamido)-4-phenyl-2-butanone tosyl-L-phenylalanine chloromethyl ketone; IDE, insulin-degrading enzyme; NEP, neutral endopeptidase 24.11(neprilysin); ECE, endothelin-converting enzyme; ACE, angiotensin-converting enzyme; MMP, matrix metal-loproteases. aMixture of lisinopril, enalapril and captopril. bMixture of amastatin, leuhistin and bestatin.

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Fig. 4 Effect of protease inhibitors on 3H/14C-Aβ1–42 degra-dation. Various peptidase inhibitors listed in Table 1 were ad-ministered to hippocampus before the injection of3H/14C-Aβ1–42, the degradation of which was monitored at 30min. Only two compounds displayed almost full inhibition ofin vivo catabolism. a and b, Effect of phosphoramidon at theinitial concentrations of 10 mM and thiorphan at 1 mM, re-spectively. c and d, Effect of 0.5 mg/ml spinorphin peptidein the absence and presence of 0.5 mM amastatin, respec-tively. Compare these data to Fig. 2d.


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the most probable candidate peptidase among allthe possibilities. Accordingly, the in vivo cleavagesites of Aβ1–42 inferred by the structure of catabolicintermediate, G(9)-Y(10) and G(37)-G(38), are con-sistent with the known specificity of neprilysin16,17

and are partially identical to those determined byHowell et al. using purified neprilysin from kidneyand synthetic Aβ1–40 peptide18.

A notable feature of the phosphoramidon/thiorphan effect isthat they inhibit not only the full degradation but also the ap-pearance of the catabolic intermediate. This suggests that NEP isinvolved in the production of the catabolic intermediate andthat this process primarily limits the rate of Aβ1–42 catabolism.We also examined the effect of an endogenous neprilysin in-hibitor, spinorphin, discovered by Nishimura and Hazato19. This7-mer peptide, LVVYPWT, decelerated the degradation of Aβ1–42

(Fig. 4c). The partial effect of spinorphin is probably the result ofaminopeptidase-catalyzed proteolysis of this peptide19 becauseco-administration of amastatin enhanced the inhibitory effect(Fig. 4d). These results indicate that Aβ catabolism may possiblybe regulated by endogenous inhibitor(s) in vivo and thataminopeptidase inhibition could exert an indirect effect.

In vitro proteolysis of Aβ by isolated NEPTo examine whether brain NEP can indeed proteolyze Aβ1–42 pep-tide in vitro, we isolated NEP activity from rat brain by anion-ex-change chromatography using Z-Ala-Ala-Leu-pNA as isperformed to isolate neprilysin from kidney20. The Aβ-degradingactivity appeared in parallel with the phosphoramidon- andthiorphan-sensitive NEP activity and with anti-neprilysin im-munoreactivity in western blot analysis (data not shown).Furthermore, this brain-derived NEP was capable of proteolyzing3H/14C-Aβ1–42 peptide in a manner essentially identical to the invivo processes and its action was inhibited by thiorphan (Fig. 5)as well as by phosphoramidon (not shown). This in vitro observa-

tion, consistent with the effect of protease inhibitors on the invivo catabolism, strongly supports the direct involvement of NEPactivity in Aβ catabolism.

Accumulation of Aβ42 in brain by NEP inhibitor infusionIf NEP is involved in the physiological Aβ catabolism, inhibitionof this peptidase should also decelerate the degradation of en-dogenous Aβ in rat brain. To address this issue, we examined theeffect of thiorphan on the amount of the endogenous rat Aβ.Thiorphan was delivered to the hippocampus for 3 days using anosmotic pump. The concentrations of Aβ40 and Aβ42(43) in the sol-uble and insoluble fractions were determined by enzyme-linkedimmunosorbent assay (ELISA)21 (Table 2). Thiorphan administra-tion had no effect on the amount of APP or soluble APP frag-ments or C-terminal fragments of APP in the hippocampus asanalyzed by western blotting (data not shown), indicating thatthe synthesis or subsequent proteolytic processing of APP wasnot affected. Thiorphan treatment resulted in a more thantwofold increase in Aβ42 in the insoluble fraction of hippocam-pus. The induced increase of insoluble Aβ42 corresponds to threetimes the amount of soluble Aβ42 in the controls. Extrapolationof this observation predicts that chronic inhibition of the pepti-dase for 1 month would lead to accumulation of 30 times thesteady-state amount of soluble Aβ42. The results suggest that athiorphan-sensitive peptidase is indeed involved in Aβ42 catabo-lism and thus strongly support the assumption drawn from theexperiments using the radiolabeled Aβ42. The data also indicate

that alternative proteolytic pathway(s) or possiblesecondary catabolic processes such as lysosomalproteolysis that may be mobilized upon Aβ accu-mulation are not efficient enough to fully com-pensate for the suppression of thethiorphan-sensitive degradation even at a timespan longer than 30 minutes. Also notable is thatthe ratio of Aβ42 to Aβ40 (Aβ42/Aβ40), elevation ofwhich is the most commonly accepted phenotypeof early onset familial AD-associated gene muta-tions1–3, was also elevated in the insoluble frac-tion. This increase in the Aβ42/Aβ40 ratio iscomparable to that achieved in the transgenicmice overexpressing presenilin 1 with a familialAD mutation22.

Neither Aβ42 nor the Aβ42/Aβ40 ratio increased

Table 2 Effect of thiorphan administration on endogenous Aβ40 and Aβ42(43)

in rat hippocampus

Aβ42 (pmol/g) Aβ40 (pmol/g) Aβ42/Aβ40

Soluble Aβ (TS sup.)control 0.103 ± 0.005 0.170 ± 0.010 0.607 ± 0.009thiorphan 0.114 ± 0.009 0.219 ± 0.020* 0.528 ± 0.042

Insoluble Aβ (GuHCl sup.)control 0.271 ± 0.065 2.367 ± 0.157 0.119 ± 0.033thiorphan 0.586 ± 0.039** 2.551 ± 0.158 0.235 ±0.028***

Total Aβ (TS+GuHCl)control 0.374 ± 0.060 2.537 ± 0.161 0.152 ± 0.031thiorphan 0.700 ± 0.040** 2.770± 0.168 0.257 ±0.027***

Each value represents the average ± s.e.m. (n = 4). *P < 0.05; *P < 0.005; ***P < 0.02 as compared with con-trols (Student’s t-test). TS, Tris-buffered saline; GuHCl: Guanidine-hydrochloride.

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Fig. 5 Proteolysis of 3H/14C-Aβ1–42 by isolated brain NEP.3H/14C-Aβ1–42 was incubated with isolated rat brain NEP at 37 °Cin the absence (a–d) or presence (e, f) of 10 µM thiorphan for3 h (b), 8 h (c, e) and 16 h (d, f) and analyzed by HPLC as in Fig.2. Arrow, elution volume of the catabolic intermediate pro-duced during the in vivo catabolism (see Fig. 2). The catabolicintermediate was subjected to further analysis by 2-dimen-sional HPLC followed by mass spectrometry and identified asAβ10–37 (inset, d).


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significantly in the soluble fraction. This observation is consis-tent with the results demonstrating that 3H/14C-Aβ42 injectedinto hippocampus became rapidly insoluble before being frag-mented (Fig. 2) and with the fact that NEP is a membrane-boundenzyme9,10. Therefore, it is reasonable to assume that the majorAβ42 catabolism takes place near or on the cell surface. Our re-sults also indicate possible differences in the ways Aβ40 and Aβ42

are metabolized, because the amount of Aβ40 was much less af-fected by thiorphan administration. Clearance of Aβ40 thereforeseems not to depend on NEP-catalyzed proteolysis as that of Aβ42

does or could even be transport-dependent rather than catabo-lism-dependent (see introductory remarks). Several recent stud-ies indicated possible involvement of intracellular proteolysis23,insulin-degrading enzyme24, and unidentified serine protease in-fluenced by metalloendopeptidase 24.15 (ref. 25) in Aβ catabo-lism, using cell culture experimental paradigm. Although ourdata (Table 1) does not provide positive support for these possi-bilities, it is still possible that they may be involved in the catab-olism of Aβ40, another pathogenic agent in AD pathogenesis, orin the clearance of Aβ42 that has escaped NEP action.

Based on this biochemical observation, we examined whetherlong-term administration of thiorphan would produce patholog-ical Aβ deposition. Figure 6 shows Aβ plaque formation in rathippocampus treated with the protease inhibitor for 30 days.Immunohistochemistry using the anti-Aβ carboxyl terminal an-tibodies indicated that the plaques were composed of Aβ42, butnot Aβ40 or Aβ43, consistent with the biochemical observationshown in Table 2. The plaques did not give strong Congo red orthioflavin staining as compared to the typical cored plaques inhuman AD brain, indicating that they are essentially diffuseplaques representing an initial stage of amyloid deposition. Themorphology of these plaques also resembled that of typical dif-fuse plaques in senile human brain despite the difference in theprimary structure between human and rodent Aβ1-42

peptides at three amino acid residues. The plaqueswere distributed in hippocampus and the cortical areaoutside hippocampus.

DiscussionThe balance between anabolism and catabolism deter-mines the in vivo amount of any peptide. Physiologicalbut potentially amyloidogenic peptides such as Aβcould be turned into pathogenic agents by subtle alter-ations in this balance over a long period of time. Inmost early onset familial AD cases, Aβ accumulation iscaused by only ∼ 50% increase in Aβ42 anabolism1–3,26. Incontrast, most of the sporadic AD cases do not seem toaccompany such simple overproduction of Aβ42 (ref.26). Therefore, aging-associated reduction of catabo-lism is a strong candidate mechanism that could ac-count for the accumulation in senile brain. Suchcatabolic reduction can be achieved either by down-

regulation of the responsible peptidase(s) or by upregulation ofthe corresponding inhibitor(s). It is noteworthy that almostevery peptidase is under strict regulation by endogenous in-hibitor(s) in vivo because any uncontrolled excessive proteolyticaction would be destructive to cells and tissues. Inhibitors couldalso be taken up from outside through diet and therefore maypossibly pose an environmental risk factor.

The present study has demonstrated that a peptidase similar toor identical to neprilysin is involved in Aβ42 catabolism as a rate-limiting enzyme. As the conventional nomenclature ofneprilysin, enkephalinase, infers, opioids and other neuropep-tides are known physiological substrates for this peptidase8. Thissuggests that both Aβ42 and some neuropeptides may share, atleast in part, a common catabolic pathway. If this assumptionhappens to be true, a catabolic reduction in this pathway couldelevate opioid concentrations under normal conditions or com-pensate for a decreased opioid synthesis that could be caused byaging. Although these are speculations that need to be verified,aging-dependent accumulation of Aβ may possibly be a conse-quence of sacrificing Aβ clearance for maintaining appropriateneuropeptide concentrations. Aβ accumulation, in turn, maycause disturbed neuropeptide metabolism. It is also possiblethat, in more pathological circumstances, both the Aβ and neu-ropeptide concentrations may be seriously perturbed togetheraccelerating the disease progression.

As already mentioned, catabolic reduction may be achieved byan increase in the corresponding peptidase inhibitor. We haveshown that an endogenous inhibitor, spinorphin, can deceleratethe in vivo Aβ42 degradation. Similar in vivo inhibitory peptides, ifpresent, could regulate Aβ clearance and, therefore, Aβ concen-trations. Also, anabolic and catabolic regulation of such in-hibitory peptide(s) would influence the metabolism of Aβ andother important peptides. More importantly, we demonstrated

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Fig. 6 Immunohistochemistry of rat brain treated with thiorphan.Hippocampal sections from rat brain treated with thiorphan for 30 d wereimmunostained with mouse monoclonal antibody against Aβ, 4G8, thatrecognizes the epitope commonly shared by both human and rodent-typeAβ peptide (a–e). The plaques were also stained with anti-C42 antibody(f), but not with antibodies against C40, C43, or APP amino- and carboxyl-termini. The plaques were distributed in hippocampus (a) and the corticalarea outside hippocampus (b), where the infused inhibitor permeated asconfirmed by infusing Evans blue. Control rats infused with saline only,saline containing 0.5 mg/ml leupeptin, or saline containing 0.5 mg/mlinactivated thiorphan did not show such structures. Scale bar represents400 µm (a), 200 µm (b) and 100 µm (c–f).


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that inhibition of the catabolic process would lead to Aβ accu-mulation in brain. To our knowledge, this is the first demonstra-tion of Aβ deposition induced by peptidase inhibition. It isnotable that even the transgenic mice overexpressing extremelyhigh concentrations of APP thus far described require as long asseveral months to display apparent Aβ pathology27,28. Our obser-vation therefore suggests not only that a reduction in Aβ catabo-lism would indeed lead to pathological Aβ accumulation in vivobut also that catabolic down-regulation could be more influen-tial than anabolic up-regulation in the pathological cascadeleading to AD.

These findings have the following medical implications.Catabolic reduction may possibly be used as a diagnostic markerfor certain cases of sporadic AD. We may also be able to up-regu-late the responsible peptidase(s) or down-regulate the corre-sponding endogenous inhibitor(s) and thus facilitate Aβclearance through, for instance, gene therapy. Another possiblestrategy would be to seek peptidase inhibitors capable of pene-trating blood-brain barrier as environmental risk factors in diets.Avoiding such risk factors could preclude down-regulation of Aβcatabolism in brain. The advantage of this strategy is that dietaryapproaches would be much less costly to the society than phar-maceutical approaches.

MethodsInhibitors and general analytical procedures. Protease inhibitors, pep-statin A, leupeptin, E-64, Z-LLLal, phosphoramidon, and leuhistin, werepurchased from Peptide Institute (Osaka, Japan). Chloroquine, thiorphan,lisinopril, enalapril, captopril, amastatin, and bestatin were from Sigma.Insulin was from Wako (Osaka, Japan). APMSF, TPCK, aprotinin, and chy-mostatin were from Roche Diagnostics (Mannheim, Germany). An MMP in-hibitor, CGS27023A, was provided by M. Nakajima, Novartis-Pharma,Osaka, Japan. A spinorphin peptide was produced, purified, and qualitycontrolled as described29. A protein sequencer Model 492 (PE Biosystems,Foster City, California) was used to determine the amino acid sequences ofpeptides. Mass spectrometry was done with the matrix assisted laser des-orption ionization-time of flight mass spectrometry (MALDI TOF-MS) sys-tem using BIFLEX III (Bruker, Bremen, Germany) according to the methodsdescribed30,31. All the procedures involving radioisotopes were done in a ra-dioisotope-restricted area.

Synthesis of 3H/14C-labeled Aβ peptides. We synthesized 3H/14C-labeledAβ peptides by the method of Chang and Meienhofer32 using a double-coupling protocol with an Advanced Chemtech (Louisville, Kentucky)peptide synthesizer ACT90. All the cold Fmoc (Nα-fluorenylmethyloxy-carbonyl)-amino acids were purchased from Advanced Chemtech. For ra-diolabeling, Fmoc-amino acids containing 3H or 14C within the amino acidmoiety were used. The basic strategy was to react [3H] or [14C] Fmoc-amino acids diluted with cold Fmoc-amino acids so that the molar ratio oftotal Fmoc-amino acid to the reactive sites in the supportive resin, Wangresin, would be ∼ 1:1 during the initial coupling of each cycle. The secondcoupling used a 20-fold excess amount of cold Fmoc-amino acids to en-sure structural integrity of the product. [14C]Fmoc-L-Ala (148 Ci/mol),

[14C]Fmoc-L-Val (245 Ci/mol), [14C]Fmoc-L-Gly (56 Ci/mol), [14C]Fmoc-L-Leu (287 Ci/mol), [3H]Fmoc-L-Leu (63 Ci/mmol), and [3H]Fmoc-L-Gly (18Ci/mmol) were from Amersham-Pharmacia (Buckinghamshire, UK), and[14C]Fmoc-L-Phe (498 Ci/mol), [3H]Fmoc-L-Phe (68.5 Ci/mmol) and[3H]Fmoc-L-Ala (42Ci/mmol) were from NEN (Boston, Massachusetts).The synthesis was done sequentially from the carboxyl-terminus startingwith alanine corresponding to amino acid 42 of Aβ1–42. Following exten-sive deprotection and cleavage with trifluoroacetic acid, ethandithiol,anisol, and dimethylsulfide, two times, followed by filtration, the peptideswere washed three times by cold ether precipitation and subjected toHPLC purification on a gel filtration column (Shodex Ohpak SB-802.5HQ;Showa Denko, Tokyo, Japan) equilibrated with dimethylformamide. Afterremoval of the solvent in the presence of an azeotropic solvent under vac-uum, the purified peptides were dissolved in a minimum volume of di-methylsulfoxide and stored in aliquots at -80 °C until use. The structuralintegrity of the peptides was confirmed by reversed phase HPLC analysis,peptide mapping with lysilendopeptidase, direct chemical sequencing,and mass spectrometric analysis. The peptides were evenly labeled to aspecific activity of 1.92 × 104 dpm /µg for 3H and 6.6 × 104 dpm/µg for14C. The 3H specific activity was somewhat lower than expected probablybecause [3H] Fmoc-amino acids needed to be diluted much more than[14C] Fmoc-amino acids with cold Fmoc-amino acids during synthesiscausing reduced efficiency of the labeled amino acid coupling.

In vivo experiments. Male, 8–10-weeks-old, Sprague-Dawley rats weighing280–350 g were used for all experiments. For injection of radiolabeled pep-tides, rats anesthetized by intraperitoneal administration of 60 mg/kgsodium pentobarbital (Abbott Labs, North Chicago, Illinois) were mountedon a small-animal stereotaxic instrument (David Kopf, Tujunga, California).Based on the stereotaxic coordinates measured from bregma (–3.3 mm an-terior–posterior, 2.5 mm medial–lateral, and 2.6 mm dorsal–ventral) ac-cording to Paxinons and Watson33, a 26S-gauge needle equipped with a2-µl motorized Hamilton syringe (KD Scientific Inc., Boston, Massachusetts)was inserted into the CA1 sector of hippocampus. One minute after theneedle was inserted, 0.5 µl of 0.5 µg 3H/14C-Aβ42 peptide dissolved in phos-phate-buffered saline (pH 7.5) (PBS) was unilaterally injected at a constantflow rate of 0.25 µl/min. The exact delivery was confirmed using Evans blue,repeatedly, in separate experiments (Fig. 1d). The needle was kept in thisconfiguration to prevent reflux of the injected material along the injectiontrack until the animals were killed by decapitation at various time points.The body temperature of the rat was monitored with a microprobe ther-mometer, MK-900 (Muromachi Kikai, Tokyo, Japan), until decapitation. The

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hippocampus was immediately dissected out and stored at -80 °C until use.When we examined peptidase inhibitor effects, 0.5 µl of inhibitor in PBSwas first infused at a flow rate of 0.25 µl/min and then, after a 5-min inter-val, the 3H/14C-Aβ1–42 peptide was sequentially injected as described. Wechose not to pre-mix the inhibitor and 3H/14C-Aβ42 peptide to avoid possiblephysicochemical interactions that could lead to conformational changes inthe Aβ peptide. For HPLC analysis, the frozen hippocampus was homoge-nized in buffer containing 0.3% triethylamine, 0.1% SDS, 1 mM betaine,protease inhibitor cocktail (Roche Diagnostics), and 0.7 µg/ml pepstatin Awith 10 strokes of a Teflon glass motor-driven homogenizer, sonicated in anice bath for 2 min, and finally centrifuged at 9,000g for 20 min. Ninety-fiveto one hundred percent of the injected peptide was extracted by thismethod. The supernatant was subjected to HPLC analysis after filtration.

The injection of 3H-angiotensin II peptide was done similarly except forthe following alterations. Cold angiotensin peptide (2.5 µg) (PeptideInstitute) mixed with 1.36 × 106 dpm of [tyrosyl-3,5-3H] angiotensin pep-tide (52.8 Ci/mmol, NEN) in 0.5 µl PBS with or without 0.5 mM amastatinwas delivered to hippocampus at a flow rate of 1.0 µl/min for 30 s. The dis-sected hippocampus was, after storage at –80 °C, homogenized in 0.2% tri-fluoroacetic acid and the protease inhibitor cocktail for subsequent HPLCanalysis.

For prolonged administration of a peptidase inhibitor to rat hippocam-pus, we implanted a steel brain cannula connected by a polyethylenecatheter to a subcutaneous miniosmotic pump (Alzet 1003D and 2ML4,Alza, Palo Alto, California) fixed under the dorsal neck according to themanufacturer’s instructions. DL-thiorphan (0.5 mg/ml) in saline (pH 6.8) orsaline alone was delivered from the pump to hippocampus at an infusionrate of 1.0 µl/h for 72 h or 2.5 µl/h for 30 d. The saline for the 30-day infu-sion contained 1 mM ascorbic acid to minimize oxidation of thiorphan. Theinactivated thiorphan was produced by oxidizing thiorphan in the presenceof 0.03% H2O2 at 37 °C for 3 h, followed by purification by HPLC. The ratswere killed immediately after the end of infusion. For biochemical analysis,each set of hippocampal tissue (∼ 120 mg) from two rats treated as abovewas stored frozen at –80 °C until use. The amounts of Aβ40 and Aβ42 in thesoluble and insoluble fractions were determined as follows by sandwichELISA using monoclonal antibodies BNT77/BA27 and BNT77/BC05, respec-tively16. The frozen tissues were homogenized in four volumes of a buffercontaining 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and the protease in-hibitor cocktail with ten strokes of a Teflon-glass homogenizer and cen-trifuged at 35,6000g for 20 min at 4 °C. The supernatant was used as thesoluble fraction. The pellet was solubilized by sonication in buffer contain-ing 6M guanidine-HCl. The solubilized pellet was centrifuged at 200,000gfor 20 min at 4 °C, after which the supernatant was diluted 12 times to re-duce the concentration of guanidine-HCl and used as the insoluble fraction.

HPLC analysis. The instrumentation consisted of an LC-10AVp HPLC(Shimadzu, Kyoto, Japan) connected to Radiomatic flow scintillation ana-lyzer 525TR (Packard, Meriden, Connecticut) using Ultima-Flo M as a scin-tillation cocktail. 3H and 14C signals were detected using energy ranges of0–6 keV and 6-156 keV, respectively. The flow rate was 0.5 ml/min unlessotherwise stated. The eluents were collected by a fraction collector whennecessary. For separation of the 3H/14C-labeled Aβ peptides and their frag-ments, a reversed phase polymer column, Shodex Asahipak ODP-50 4E,heated to 50 oC and equilibrated with 0.05% triethylamine, 10 mM be-taine, and 0.1 mM EDTA was used (Figs. 2–5). The bound peptides wereeluted by a linear gradient of 0–90% acetonitrile from 20 to 50 minutes.These conditions gave the best recovery and thus were most quantitativeamong more than ten different sets of conditions that we examined. Foridentification of catabolic intermediate, the 3H/14C-Aβ1–42 peptide mixedwith 0.5 µg of cold Aβ1-42 was subjected to in vivo catabolism and subse-quent separation as in Fig. 2d. A set of two samples was separated togetherand this procedure was repeated four times. HPLC fractions correspondingto the catabolic intermediate were collected, dried under vacuum, and fur-ther separated by a second HPLC under different conditions as follows. Thesecond separation used a Capcell Pak C18 UG120 column (Shiseido, Tokyo,Japan) heated to 50oC and equilibrated with 0.1% trifluoroacetic acid witha flow rate of 1 ml/min (Fig. 6). The elution was carried out with a lineargradient of 0–70% acetonitrile from 10 to 40 minutes. The separation of 3H-angiotensin II peptide used a Shiseido Capcell Pak C18 UG120 columnequilibrated with 0.01% triethylamine at room temperature.

Aggregation assay. Aggregation property of synthetic Aβ1-42 and Aβ10-37

peptides was assayed as follows. Freshly prepared 50 µM solution of thepeptide in 20 mM Tris-HCl (pH 8) was stirred and incubated at 37 °C as in-dicated. After centrifugation at 15000g for 10 min, an aliquot of the super-natant was subjected to HPLC analysis. The peak area of the peptide at time0 was taken as 100% and the percent aggregation represents disappear-ance of the soluble peptide.

Isolation of brain NEP. Brain NEP was isolated in a manner essentially iden-tical to isolation of kidney NEP (ref. 12). Microsome fractions prepared fromwhole brain by centrifugation at 100,000g were solubilized by 1% Triton X-100 in 10 mM Tris-HCl (pH 8), 250 mM sucrose containing 10 µM leu-peptin. The supernatant was subjected to an anion-exchange HPLC usingDEAE-Toyopearl 650S (Tosoh, Tokyo, Japan) connected to Biologic system(Bio-Rad, Hercules, California). The bound proteins were eluted with a lin-ear gradient of 0–0.3 M NaCl. Phosphoramidon- and thiorphan-sensitiveendopeptidase activity appeared as a single peak at ∼ 0.1 M NaCl. Westernblot analysis using a mouse monoclonal antibody against neprilysin, 56C6(Novacastra, Tyne, UK), gave a single immunoreactive band with a relativemolecular weight of 100,000, identical to that of kidney neprilysin. The iso-lated NEP with activity of hydrolyzing 0.01 nmol of Z-Ala-Ala-Leu-pNA perminute was incubated with 0.5 µg 3H/14C-Aβ42 peptide in 20 mM Tris-HCl ina total volume of 50 µl at 37 °C for 3–16 h in the presence or absence ofprotease inhibitor (Fig. 5).

Immunohistochemistry. The rats were anesthetized and perfused intracar-dially with PBS, followed by ice-cold 4% paraformaldehyde in PBS. Brainswere then removed, postfixed in the same fixative for 4 h and cryopro-tected in sucrose solution (up to 30%) at 4 °C. Ten micrometer-thick coro-nal sections were cut on a freezing microtome, and processed forimmunohistochemistry, using the Vecstatin ELITE ABC kit (VectorLaboratories Inc., Burlingame, California). A mouse monoclonal antibodyagainst Aβ, 4G8 (Senetek, Maryland Heights, Missouri), was used to visual-ize plaques composed of rat endogenous Aβ. For immunohistochemicalstaining by the endo-specific anti-carboxyl terminal antibodies, C40 andC42 (ref. 23), the anesthetized rats were perfused intracardially with PBS,followed by ice-cold 70% ethanol in 150mM NaCl. Brains were then re-moved, postfixed in the same fixative for 24 h at 4 °C and embedded inparaffin. Six micrometer-thick coronal sections were cut, and pretreated by100% formic acid for 1 min before immunochemical staining.

AcknowledgmentsWe thank Y. Ihara for supporting and criticizing our work. We also thank H.Uchino and T. Iwatsubo for technical advice and W. Harigaya and Y. Dohzonofor secretarial assistance. We also thank M. Ito and the Brain Science PlanningOffice for support. This work was supported by research grants from RIKEN BSI,Special Coordination Funds for promoting Science and Technology of STA,CREST, Ministry of Health and Welfare, Ministry of Education, ChugaiPharmaceutical Co., Mitsubishi Chemical Co., and Takeda Chemical Industries.

RECEIVED 13 JULY; ACCEPTED 11 NOVEMBER 1999

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