Identification of Amino-Terminally and Phosphotyrosine-Modified Carboxy-Terminal Fragments of the Amyloid Precursor Protein in Alzheimer's Disease and Down's Syndrome Brain

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Neurobiology of Disease 8, 173–180 (2001)doi:10.1006/nbdi.2000.0357, available online at http://www.idealibrary.com on

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Identification of Amino-Terminally andPhosphotyrosine-Modified Carboxy-TerminalFragments of the Amyloid Precursor Protein inAlzheimer’s Disease and Down’s Syndrome Brain

Claudio Russo,* ,† Serena Salis,* Virginia Dolcini,*Valentina Venezia,* Xiang-Hong Song,† Jan K. Teller,†,1,2,3

and Gennaro Schettini* ,1,2

*Section of Pharmacology and Neuroscience, National Cancer Institute, AdvancedBiotechnology Center, and Department of Oncology, Biology and Genetics,University of Genova, Genova, Italy; and †Neuropathology Department,Case Western Reserve University, Cleveland, Ohio

Received June 20, 2000; accepted September 25, 2000; published online January 20, 2001

The carboxy-terminal fragments (CTFs) of the amyloid precursor protein (APP) are considered b-amyloid (Ab)precursors as well as molecular species that are both amyloidogenic and neurotoxic by themselves in vitro orin animal models. However the CTFs’ role in the pathogenesis of Alzheimer’s disease (AD) is however relativelyunexplored in human brain. In this study, we analyzed CTFs extracted from brains of subjects with AD, non-ADcontrol, and Down’s syndrome (DS) cases. Our data indicate that: (i) In fetal DS brains CTFs levels are increasedin comparison to age-matched control, suggesting that the enhanced CTFs formation is important for the earlyoccurrence of plaque deposition in DS. There is no significant difference in CTFs level is present between ADand age-matched control cases. (ii) CTFs modified at their N-terminus appear to be the direct precursors oflikewise N-terminally modified Ab peptides, which constitute the most abundant species in AD and DSplaques. This observation suggests that N-truncated Ab peptides are rather formed directly at b-secretaselevel and not through a progressive proteolysis of full-length Ab1-40/42. (iii) Among the differently cleavedCTFs, only the 22- and 12.5-kDa polypeptides are tyrosine phosphorylated in both AD and control brains whilethe full-length APP and the CTFs migrating below the 12.5-kDa marker are not phosphorylated, suggesting thatsome APP and CTFs are processed through regulated pathways. This study provides further evidence that inhuman brain CTFs constitute a molecular species directly involved in AD pathogenesis and in the developmentof the AD-like pathology in DS subjects. © 2001 Academic Press

INTRODUCTION

The pathogenesis of Alzheimer’s disease (AD)4 in-olves a progressive neuronal degeneration of cere-

1 To whom correspondence should be addressed. E-mail:[email protected] or [email protected].

2 Joint last authors.3 Present address: Department of Physiology, The Brody School of

Medicine, East Carolina University, Greenville, North Carolina.4 Abbreviations used: APP, amyloid precursor protein; CTFs,

APP’s carboxy-terminal fragments; Ab, amyloid-b peptide; AD,Alzheimer’s disease; DS, Down’s syndrome; DSf, fetal and postnatalDown’s syndrome cases; DSa, adult DS with plaques; APPfl, full-length APP.

0969-9961/01 $35.00Copyright © 2001 by Academic Press

ll rights of reproduction in any form reserved. 173

bral and limbic cortices (Terry and Katzman, 1983).Pathological hallmarks of the disease are the presenceof extracellular amyloid plaques and neurofibrillarytangles accompanied by neuronal loss and gliosis(Terry and Katzman, 1983). The amyloid present inplaques and meningeal vessels is predominantly com-posed of 40/42 amino acid peptides defined as amy-loid b-peptides (Ab) proteolytically derived from theamyloid precursor protein (APP) through the action oftwo enzymes called b- and g-secretases (Glenner andWong, 1984; Selkoe, 1998). The b-secretase cleavageproduces a number of transmembrane polypeptidespreviously referred to as C22 kDa, C18 kDa, C100,C99, or generally carboxy-terminal fragments of APP

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(CTFs). Depending on their length and sequence,CTFs may contain the entire Ab sequence and areconsidered amyloidogenic (Haass et al., 1992; Estuset al., 1992; Kammesheidt et al., 1992; Simons et al.,1996). Also, another protease the a-secretase is re-sponsible for the formation of a CTF C83, which iscleaved at residue 17 of Ab, and therefore is consid-ered nonamyloidogenic (Haass et al., 1992; Estus etal., 1992; Kammesheidt et al., 1992; Simons et al.,1996). The recently identified b-secretases (BACE1and 2) seem to be responsible only for the cleavagesat residues 1 and 11 of the Ab sequence (Vassar etal., 1999; Yan et al., 1999; Sinha et al., 1999; Acquatiet al., 2000) leaving open the question of the forma-tion of other CTFs with different N-termini (Haass etal., 1992; Simons et al., 1996). The g-secretase cleav-age at the C-terminus of CTFs is responsible for theformation of the Ab40 or Ab42, with the latter beingmore amyloidogenic and more toxic. IncreasedAb42 in the brain is correlated with familial AD andDown’s syndrome (DS) (Scheuner et al., 1996;

orchelt et al., 1996; Teller et al., 1996; Mann andIwatsubo, 1996; Citron et al., 1997). Recent studieshave demonstrated that the majority of Ab peptidespresent in AD are truncated at their N-terminus andoften contain pyroglutamate at positions 3 and 11(AbN3(pE)-40/42 and AbN11(pE)-40/42) (Saido etal., 1995; Russo et al., 1997; Kuo et al., 1997). More-over, an increased presence of N-truncated Ab iscorrelated with the early-onset familial AD due tomutations in the APP or in the presenilin 1 (PS1)genes (Ancolio et al., 1999; Russo et al., 2000). Therole of truncated Ab peptides in AD pathogenesis ispoorly explored and their formation is a matter ofdebate (Saido, 1998; Tekirian et al., 1999; He and

arrow, 1999). In this report we show that N-termi-ally truncated and pyroglutamate modified Ab

peptides beginning with pyroglutamate may origi-nate at the b-secretase level, rather than being gen-rated through a degradation process of the full-ength Ab1-40/42. We also show that different CTFsolypeptides, corresponding to different N-terminal

b-secretase cleavage sites, are present in the mem-brane-bound brain fraction and that their level isincreased in Down’s syndrome subjects long beforethe formation of amyloid deposits or plaques. Wealso demonstrate that some CTFs, in contrast, full-length APP are tyrosine-phosphorylated. The rele-vance of our findings to the AD pathogenesis isdiscussed.

Copyright © 2001 by Academic Pressll rights of reproduction in any form reserved.

METHODS

Source of Tissue

Cerebral cortex was obtained at autopsy from clin-ically and neuropathologically verified (CERAD) (Ga-lasko et al., 1995) cases of sporadic AD (N 5 8, age73 6 8 years) and control subjects (N 5 5, age 60 6 5

ears; N 5 4, postnatal age 14 days to 13 months) inhich AD had been excluded by clinical and autopsy

xamination, including immunohistochemical analy-is and Down’s syndrome cases (N 5 4, fetal age 14–24eeks; N 5 5, postnatal age 4 days to 14 months; N 5

, 51 years old with AD pathology) in which therisomy of the chromosome 21 was confirmed byaryotyping. The fetal Down’s syndrome cases wererovided by Dr. P. Gambetti, Case Western Re-erve University and The Brain and Tissue Bank Forevelopmental Disorders of the University of Miami,lorida.

ntibodies

The antibody R3659 recognizes the NH2-terminus ofAb. The aN3(pE) and aAbL-Asp antibodies (kindlyprovided by Dr. T. Saido, Riken Brain ScienceInstitute, Saitama, Japan), specifically recognize theN-terminal pyroglutamate residue at position 3, andL-asparate at position 1 of Ab sequence, respectively.Monoclonals 4G8 and 6E10, specific for residues 17–21and 6–10 of Ab, were purchased from Senetek. Thepolyclonal antibody F25608 (provided by Dr. Gam-betti) and the monoclonal antibody 643/695 Jonas(Roche) are specific for the C-terminus of APP andCTFs. The monoclonal antibody to phosphotyrosinewas purchased from Santa Cruz Biotechnology. Allthe chemicals were from Sigma unless otherwise spec-ified.

Isolation and Extraction of CTFs

Brain cortical samples were homogenized in 10 mMTris buffer supplemented with 1% of Triton X-100 andspun down at 100,000g for one hour. The supernatantswere then analyzed by immunoprecipitation with spe-cific antibodies for CTFs or Ab, followed by Tris–tricine electrophoresis, Western blotting, immunode-tection, and densitometry measurement as previouslydescribed (Teller et al., 1996; Russo et al., 1997). ECLAmersham-Pharmacia) films were scanned and ana-yzed using the GelDoc instrument (Bio-Rad) and the

IH imaging software. All the samples were analyzedt least in triplicate in separate experiments.

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175CTFs of the APP in Alzheimer’s Disease and Down’s Syndrome

RESULTS

To analyze the APP CTFs in the detergent extractsobtained from human brain with a variety of patho-logical and nonpathological conditions, we used awell-established protocol in which immunoprecipita-tion followed by SDS–PAGE and Western blottingwith specific antibodies allows for the identification ofAPP fragments. The samples available in our study(see Methods) include subjects with sporadic AD, age-matched non-AD control subjects (CO) as well as fetaland postnatal plaque-free (DSf) and adult withplaques (DSa) Down’s syndrome cases. The goal ofthis study was to qualitatively and quantitativelycharacterize the CTFs in AD and DS cases in compar-ison to non-AD control subjects and further analyzethem with specific antibodies in order to reveal theirN-terminal modifications as well as possible tyrosinephosphorylation of the polypeptides.

CTFs Characterization

The CTF polypeptides that are extracted from brainsamples can be detected with the antibody 4G8 afterbeing immunoprecipitated with the antibody F2568that is specific for the C-terminus of APP (Fig. 1). Theelectrophoretic pattern shows a single band that mi-

FIG. 1. Electrophoretic separation of brain CTFs in AD, control (CO),and DS subjects. CTFs were detergent-extracted from brain, immuno-precipitated with the antibody F2568, specific for the C-terminus ofAPP, and immunodetected, after Western blotting, with the monoclo-nal antibody 4G8 which is specific for a region comprising residues17–21 of the Ab sequence. The electrophoretic profile is similar in allsubjects analyzed, with five major bands migrating between 8 and 14kDa (B1–B5) and a single band detectable at 22 kDa. Longer exposuresvisualized additional bands migrating at 17–18 kDa (Simons et al.,1996). Note an increased presence of B1–B5 CTFs in DS fetal subjects(DSf) as compared to AD, control, or the DS adult (DSa) subject.

grates at 22 kDa, together with five bands (B1–B5) thatare detected between 8 and 14 kDa as previouslydescribed (Haass et al., 1992; Estus et al., 1992; Kamme-sheidt et al., 1992). The electrophoretic profile is similarin AD, control, and DS subjects, although in DSf anincreased level of CTFs migrating between 8 and 14kDa is evident. The DSa sample shows an electro-phoretic profile similar to the other groups; the CTFslevels comparable to AD or control cases, although arelower than those for DSf subjects (Fig. 3). The appear-ance of the 22-kDa band is similar among the differentsubjects analyzed. The immunoprecipitation with theantibody 6E10 specific for residues 6–10 of Ab (Fig. 2),followed by immunoblotting with the antibody 643/695 Jonas for the C-terminus of APP, recognized the22-kDa band and only the first two bands migrating at12.5 kDa, of the five that were previously recognizedby the antibody 4G8. This observation suggests thatthe polypeptides migrating in bands 3, 4, and 5 havesequences starting after the 10th residue of Ab. It isalso of note that DSf subjects show an increasedamount of CTFs in comparison to the other groupsanalyzed.

Densitometric analysis of CTFs migrating between 8and 14 kDa analyzed with the antibody 4G8 revealedthat these heterogeneous polypeptides are present insignificantly higher amount in DSf subjects than inAD, DSa, or control cases (Fig. 3). The total amount ofCTFs present in AD cases does not significantly differfrom control subjects. The DSa subject (with the his-tochemical hallmarks of AD—data not shown) has alower amount of membrane-bound CTFs in compari-son to the DSf group.

FIG. 2. The B1 and B2 CTFs have sequences starting before theresidue 10 of Ab, while B3 to B5 have sequences starting after thisesidue. CTFs are immunoprecipitated with the antibody 6E10 spe-ific for the Ab sequence 6–10 and immunodetected, after Western

blotting, with the monoclonal antibody 643/695 Jonas which isspecific for the C-terminus of APP. The electrophoretic profile issimilar in all subjects analyzed, with only two major bands migrat-ing between 8 and 14 kDa (B1, B2) and a single band detectable at22 kDa.

Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

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Pyroglutamate-containing CTFs

Immunoprecipitation CTFs with the antibody F2568from Triton X-100 extracted brain samples followed bydetection with the antibody aN3(pE), revealed a bandwith approximate molecular weight of 12.5 kDa de-fined as band 2. This band is present in all subjectsanalyzed and apparently corresponds to a pyroglu-tamylated 3–100 CTF sequence (Fig. 4a). Similarly,after being immunoprecipitated with the antibodyF2568, Triton X-100-extracted CTFs were analyzedwith the antibody specific for the residue L-aspartatein position 1 of Ab sequence (Fig. 4a). The immuno-blotting identified a band at 12.5 kDa (B2) that corre-spond to the same band identified with the aN3(pE)ntibody (Fig. 4a). This analysis therefore identifieshe CTF polypeptide with an initial l-Asp residue at

the N-terminus (C99) that comigrates with the N3(pE)-100 polypeptide. The N3(pE)-100 polypeptide ispresent in significantly higher amount in DSf subjectsthan in AD patients, control, or DSa cases. The anti-bodies against L-aspartate1 and for the N3(pE) resi-dues recognize Ab peptides in AD and DSa brains aswell (Fig. 4b). When control, AD, DSa, and DSf brainextracts were immunoprecipitated with the antibodyR3659 and analyzed by immunoblotting with theantibodies 4G8, aN3(pE), and aAbl-Asp, the antibody4G8 detected 3 major Ab bands in AD and DSa sub-jects. 4G8 detects three bands in the DSa subject, whileonly the top band, which comigrates with Ab1-40/42Saido et al., 1995; Russo et al., 1997) was detected in

DSf cases. No Ab was detected in control subjectsusing this methodology. In AD and DSa cases theantibodies aAbL-Asp and aN3(pE) detect the first andecond bands, as Ab1-40/42 and AbN3(pE)-40/42,

FIG. 3. Densitometric analysis of 4G8 detected B1–B5 CTFs incontrol (CO), AD, DS fetal, or early-postnatal (DSf) and adult DS(DSa). DSf subjects (N 5 9) have twice as much CTFs in comparisonto AD (N 5 8), control cases (N 5 9), or the only DS adult subjectavailable in this study (P , 0.003). No significant differences inCTFs levels are detected between young control subjects (N 5 4)and adult (N 5 5) (data not shown, see Methods).


respectively (data not shown) (Saido et al., 1995; Russoet al., 1997).

Tyrosine-phosphorylated CTFs

Both full-length APP (APPfl) and CTFs are presentin control and AD brain extracts as demonstrated byF25608 immunoprecipitation of APPfl and immuno-detection of CTFs with 4G8 (Fig. 5). Analysis ofbrain extracts with an antibody specific for phos-photyrosine residues revealed that at least someof the CTFs are phosphorylated. In fact, the immuno-staining with the phosphotyrosine antibody revealedtwo bands (B1 and B2) with estimated molecularweights of 22- and 12.5-kDa in both AD and controlsubjects. The antibody detected neither the full-lengthAPP holoproteins that migrate between 90 and 130kDa nor the polypeptides that migrate as bands B3–B5(Fig. 5).

FIG. 4. (a) Determination of CTFs and Ab beginning with L-aspartate or pyroglutamate in the brain membrane extracts. Immu-noprecipitation of CTFs with the antibody F2568 for the C-terminusof APP, followed by immunodetection with specific antibodies forthe L-aspartate-1 and pyroglutamate-3 residues of Ab identify the2.5-kDa band (B2) which contain both L-Asp1-100 and N3(pE)-100equences. (b) The antibodies aN3(pE) and aAbl-Asp detect Ab

isolated from AD brains as well. Triton-extracted brain fractions,immunoprecipitated with the antibody R3659 for Ab and immuno-detected with the antibodies 4G8 (lanes 1–4), aN3(pE) (lane 5), andaAbl-Asp (lane 6), identify Ab peptides in AD, DSa, and DSf brainss previously described (Kuo et al., 1997; Mann and Iwatsubo, 1996)bN3(pE)-40/42 and Abl-Asp1-40/42 peptides are detected

n DSa (data not shown) and in AD brains (lanes 5 and 6, respec-ively). No Ab is detected in control (CO, lane 3) cases with this

ethod.

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DISCUSSION

Proteolytic processing of APP is required for therelease of Ab peptides and their deposition intoamyloid lesions (Glenner and Wong, 1984; Selkoe,1998). Although the presence of amino-terminallytruncated Ab peptides is well documented in plaques,cerebrospinal fluid, and even in cells culture, onlyrecently some pyroglutamate-modified forms of Ab(AbN3(pE)-40/42 and AbN11(pE)-40/42) have beencharacterized in plaques, found in plasma, in the sol-uble fraction of brain extracts and quantified as prob-ably the major Ab specie in sporadic and familial AD(Saido et al., 1995; Russo et al., 1997; Kuo et al., 1997;Ancolio et al., 1999; Russo et al., 2000). In DS subjectsthe truncated Ab peptides increase with the progres-sion of the plaque formation and are much less repre-sented in young DS brains without plaques (Russo etal., 1997), suggesting that their presence may be cor-related with the severity and to the disease’s progres-sion (Russo et al., 1997, 2000; Ancolio et al., 1999). Inetal or early postnatal DS brains Ab 1-40/42 is the

major detectable peptide as shown in Fig. 4b, DSfsubject, while the formation of N-truncated Ab pep-tides increases with age (Russo et al., 1997), to anAD-like appearance in the adult life (Fig. 4b, DSacase). The Ab peptides with amino-terminal trunca-

FIG. 5. Tyrosine phosphorylation of the CTFs polypeptides in ADand control (CO) brain extracts. Immunoprecipitation of TritonX-100-extracted APP full-length (APPfl) and CTFs with the antibodyF2568, followed by immunodetection with the antibody 4G8, iden-tifies both APPfl and the CTFs in AD and control brains (CO). Thesame preparation probed with an antibody against phosphotyrosineresidues (pY), the C22 kDa fragment and the CTFs that migrate inB1 and in B2 can be seen. No tyrosine-phosphorylated APPfl couldbe visualized even after prolonged exposure.

tions are linked to the early-onset familial AD due toPS1 and APP mutations (Ancolio et al., 1999; Russo etal., 2000). However, the origin of these truncated Abpeptides is still unclear. Although they might be gen-erated also through a progressive proteolysis of thefull-length Ab1-40/42, heterogeneous b-secretasecleaved fragments have been previously identifiedboth in human brain and transfected cells. In addition,carboxy-terminal APP polypeptides starting at posi-tions 26 or 23 or truncated at residues 4, 11, 15, and7 have also been identified (Haass et al., 1992; Estus etl., 1992; Kammesheidt et al., 1992; Simons et al., 1996).

Recently identified b-secretases BACE 1 and 2 aretransmembrane proteins with an enzymatic activityhighly specific for APP (Vassar et al., 1999; Yan et al.,999; Sinha et al., 1999; Acquati et al., 2000). The sub-trate specificity of BACE corresponds, at least in vitro,o positions 1 and 11 of Ab. Our results indicate that in

human brain b-secretase might be cleaving APP atseveral different sites, and that amino-terminally trun-cated AbN3(pE)-40/42 are not generated in the hu-man brain by the progressive proteolysis of Ab1-40/42 as previously suggested (Saido, 1998), but theirformation is rather an event that precedes the g-secre-tase cleavage. The b-secretase cleavage of APP gener-ates CTFs polypeptides which contain the completeAb sequence and appear to accumulate intracellularlyin neurons expressing familial AD (FAD) mutants ofAPP. These polypeptides cause neurodegenerationwhen expressed in transfected neuronal cells (McPhieet al., 1997). Transgenic animals expressing these frag-ments in the brain also exhibit some neuropathologi-cal and behavioral AD-like deficits (Caputo et al., 1992;Oster-Granite et al., 1996; Neve et al., 1996; Berger-Sweeney et al., 1999). The a-secretase-derived CTFs donot contain the entire Ab sequence and therefore arenot considered amyloidogenic. Detergent-extractedCTFs were immunodetectable in all the subjects ana-lyzed and abundantly present in DS brains even infetal life. An abnormally increased formation of theseamyloidogenic fragments in young DS brains mayconstitute a pathogenic event directly correlated withthe early occurrence of amyloid deposits in these pa-tients. Indeed, higher levels of both b-secretase protein(BACE2 is encoded in the “Down critical region” in21q22.3) (Acquati et al., 2000) and substrate (APP),may be responsible for the increased formation ofCTFs and their toxic species in DS. The findings that inDSf subjects Ab1-42 is the major Ab peptide (Teller etal., 1996; Mann and Iwatsubo, 1996) and that N-trun-cated Ab peptides are increasingly present in an age-related manner (Mann and Iwatsubo, 1996; Russo et


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al., 1997) correlate well with our results in which aigh CTFs content is accompanied by a very lowxpression of N-truncated Ab peptides in DSf cases

(Fig. 4b). With age progression the formation of N-truncated Ab increases (Russo et al., 1997) culminatingin an AD-like pattern in the adult life (Saido et al.,1995; Teller et al., 1996; Mann and Iwatsubo, 1996;Russo et al., 1997) as shown in Fig. 4b, while CTFslevels decrease. This implies that in DS brains anage-related differential g-cleavage of CTFs might alsooccur.

The observation that in the DSa case CTFs levels aredecreased in comparison to the DSf group (albeit pre-liminary and nonconclusive since only one DSa sub-ject was available for this study), might suggest thatpart of the CTFs are cleaved by g-secretase to produceAb peptides abundantly present in DSa (Fig. 4b). Thesame mechanism might take place in AD leading tothe underestimation of the overall CTFs content incomparison to control cases, where the accumulationof g-secretase cleavage products is very low and Abpeptides are practically undetectable with our meth-ods (Fig. 4b). Moreover, the fact that the levels of the22-kDa APP fragment are not increased in DSf sug-gests that this polypeptide may have a different ori-gin, possibly through a non-b-secretase cleavage.

The finding that the 22 kDa and some of the otherCTFs are tyrosine-phosphorylated points to a differentaspect of the APP involvement in the AD pathogene-sis. Since our data show that the APP holoprotein isnot phosphorylated, the hypothesis is that the CTFsphosphorylation occurs after the APP’s cleavage. Al-ternatively, the phosphorylation of full-length APPmight constitute a signal that leads inevitably to itsfast proteolytic processing, eventually leaving no oronly transient tyrosine-phosphorylated APP. Whythese CTFs are tyrosine phosphorylated, and whatkinases are involved, are the important questions to beanswered in order to understand the APP physiolog-ical role and its degradation pathways. The phosphor-ylation might be required for the degradation of theCTFs fragment by g-secretase or caspases or for theactivation of a yet unknown intracellular pathway.Many receptors require this modification in order tobe activated and transfer a physiological signal intothe cell machinery. The receptor-like structure of APPhas been linked to the Go protein mediated signaltransduction, although the ultimate APP function isstill unknown (Okamoto et al., 1995). The intracellularportion of APP can be bound to different proteins likeFe65, X11, and APP-BPI (Fiore et al., 1995; Borg et al.,1996; Trommsdorff et al., 1998; Russo et al., 1998;


Duilio et al., 1998; Chen et al., 2000). However, the rolesof these interactions are not yet clear. The CTFs phos-phorylation might, therefore, play a role in the lateAPP processing or indicate abortive processing orsignaling events. Finally, a cellular role for phosphor-ylated CTFs that is independent of APP primary func-tion may also be considered.

ACKNOWLEDGMENTS

We thank Dr. Takaomi C. Saido, Riken Brain Science Institute,Saitama, Japan for generously providing antibodies aN3(pE) andaAbL-Asp, and Dr. Pierluigi Gambetti (Case Western Reserve Uni-versity) for advice and support. Brain samples used in this studywere kindly provided by the University of Miami Brain and TissueBank for Developmental Disorders. This represents a joint effort ofthe University of Miami and the University of Maryland Brain andTissue Bank through National Institute of Child Health and HumanDevelopment Contract N01-HD-33199. This work was supported byGrants from MISAN IRCCS Progetto Strategico Alzheimer, fromAngelini Ricerche, CDC grant CCU 515004, the Britton Fund, andfrom National Institute of Health Grants AG14359, AG08155,AG08012, and NS37392.

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Identification of Amino-Terminally and Phosphotyrosine-Modified Carboxy-Terminal Fragments of the Amyloid Precursor Protein in Alzheimer's Disease and Down's Syndrome Brain