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Neurobiology of Disease 7, 9–22 (2000)d

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ehavioral Disturbances without Amyloideposits in Mice Overexpressing Human Amyloidrecursor Protein with Flemish (A692G) or Dutch

E693Q) Mutation

amir Kumar-Singh,*,1 Ilse Dewachter,†,1 Dieder Moechars,‡

rsula Lubke,§ Chris De Jonghe,* Chantal Ceuterick,¶

rederic Checler,( Asha Naidu,** Barbara Cordell,** Patrick Cras,§

hristine Van Broeckhoven,* and Fred Van Leuven†

Flanders Interuniversity Institute for Biotechnology, Born-Bunge Foundation, Department ofiochemistry, §Laboratory of Neurobiology, Born-Bunge Foundation, Department of Medicine,nd ¶Laboratory of Electronmicroscopy, Born-Bunge Foundation, Department of Medicine,niversity of Antwerp, Antwerp, Belgium; †Experimental Genetics Group, Center for Humanenetics, Flanders Interuniversity Institute for Biotechnology, K.U. Leuven, B3000 Louvain,elgium; ‡Department of Functional Genomics, Janssen Research Foundation, Beerse, Belgium;

Institut de Pharmacologie Moleculaire et Cellulaire, Valbonne, France; and **Scios, Inc.,unnyvale, California 94086

eceived April 20, 1999; revised July 10, 1999; accepted for publication September 9, 1999

The contribution of mutations in the amyloid precursor protein (APP) gene known as Flemish(APP/A692G) and Dutch (APP/E693Q) to the pathogenesis of Alzheimer’s disease and hereditarycerebral hemorrhage with amyloidosis of the Dutch type, respectively, was studied in transgenic micethat overexpress the mutant APP in brain. These transgenic mice showed the same early behavioraldisturbances and defects and increased premature death as the APP/London (APP V717I), APP/Swedish (K670N, M671L), and other APP transgenic mice described previously. Pathological changesincluded intense glial reaction, extensive microspongiosis in the white matter, and apoptotic neuronsin select areas of the brain, while amyloid deposits were absent, even in mice over 18 months of age.This contrasts with extensive amyloid deposition in APP/London transgenic mice and less pro-nounced amyloid deposition in APP/Swedish transgenic mice generated identically. It demonstrated,however, that the behavioral deficiencies and the pathological changes in brain resulting from animpaired neuronal function are caused directly by APP or its proteolytic derivative(s). These accelerateor impinge on the normal process of aging and amyloid deposits per se are not essential for this

oi:10.1006/nbdi.1999.0272, available online at http://www.idealibrary.com on

phenotype. r 2000 Academic Press

Key Words: Alzheimer’s disease; transgenic mice; amyloid precursor protein; Flemish APP

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mutation; Dutch APP mutation.

NTRODUCTION

Alzheimer’s disease (AD) is a progressive neurode-enerative disorder of the central nervous system andhe most frequent form of dementia afflicting the agingopulation. The main characteristics of AD brain

acg1These authors contributed equally to this work.

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969-9961/00 $35.00opyright r 2000 by Academic Pressll rights of reproduction in any form reserved.

athology are senile plaques and neurofibrillary tanglesomposed mainly of ,4-kDa amyloid-b peptide (Ab)nd hyperphosphorylated microtubule-associated pro-ein t, respectively, and associated with dystrophiceurites and neuronal cell loss (Price & Sisodia, 1994;elkoe, 1994). A primary pathogenic role in AD for
myloid-b precursor protein (APP) and its proteolyti-ally derived amyloid peptides (Ab40/42) is sug-ested by specific mutations in APP flanking the Ab

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equence that are tightly linked to autosomal domi-ant forms of the presenile AD (Van Broeckhoven,994). Concurrently in vivo mouse models overexpress-ng mutant forms of APP produce variable AD patholo-ies ranging from amyloid plaque deposition (Quon etl., 1991; Games et al., 1995; Moechars et al., 1998a) toascular amyloid (Walker, 1997), some form of tathology (Sturchler-Pierrat et al., 1997), cognitiveeficits (Hsiao et al., 1996; Moechars et al., 1998a),eurodegeneration, and neuronal loss (Calhoun et al.,998; Moechars et al., 1999b). Two mutants of APP haveeen most frequently used in reproducing the ADathology in transgenic mice, i.e., the Swedish muta-

ion (APP/Sw) located near the b-secretase site (Mul-an et al., 1992) and the London (APP/Lo) mutation(s)istal to the g-secretase cleavage site(s) (Goate et al.,991). APP/Sw causes more of total Ab productionhile APP/Lo is subject to altered cleavage by g-secre-

ase, thereby producing more of the amyloidogenicb42 peptide (Jarrett & Lansbury-PT, 1993; Citron et

l., 1996). While the b- and g-secretases are essential forhe production of Ab, a third secretase (a-secretase) isnown and its activity precludes the formation of Ab dueo cleavage within the Ab sequence (Anderson et al., 1991).

Patients presenting with mutations in APP residingn proximity of the a cleavage site present withifferent phenotypes (Hendriks et al., 1995). The DutchPP mutation (APP/Du) at codon 693 of APP (E693Q;umbering according to the largest APP isoform,PP770) causes hereditary cerebral hemorrhage with

myloidosis of the Dutch type (HCHWA-D) and segre-ates in an autosomal dominant pattern in a subset ofatients with cerebral amyloid angiopathy (CAA) (Vanroeckhoven et al., 1990; Levy et al., 1990). It isharacterized by extensive amyloid deposits in themall leptomeningeal and cortical arterioles, whicheads to an early death of those afflicted in their fifth orixth decade (Bornebroek et al., 1996). No such predilec-ion for blood vessels, however, is noted for thelemish APP mutation at the preceding codon (A692G)APP/Fl) with which patients present with a combinedathology of CAA and presenile AD, with the presencef both large-cored parenchymal senile plaques andxtensive amyloid deposits in blood vessels (Hendrikst al., 1992; Cras et al., 1998; van Harskamp et al., 1999).

oreover, on the basis of clinical features, HCHWA-Dan be considered to represent a mixed form of AD andulti-infarct dementia (Roos et al., 1991). These results

uggest that the clinically distinct entities, presenile

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D and CAA, can be caused by mutations in the APPene (Van Broeckhoven et al., 1990), causing, unlike the- and g-secretase-related presenile AD mutations, a

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opyright r 2000 by Academic Pressll rights of reproduction in any form reserved.

hanged specificity for a-secretase. To test this and totudy the processing of mutated human APP in vivo,e have generated transgenic mouse strains express-

ng human APP/Fl and APP/Du from the neuron-pecific mouse thy-1 gene promoter (Moechars et al.,996). Human APP was expressed in brains of allransgenic mouse strains analyzed. We also demon-trated an increased processing of human APP at the-secretase cleavage site causing increased formationf N-terminal fragments of Ab protein. However, theice did not develop amyloid plaques or t pathology,

ven by 22 months of age, nor was there any evidenceound in vasculature for circulatory Ab peptides.evertheless, behavioral, pharmacological, and patho-

ogical observations in these mice disclosed subtlespects of early and late onset AD similar to thosebserved in APP/Lo and other mutant and wild-typePP (APP/Wt) transgenic mice (Moechars et al., 1999a).hese defects comprised differential glutamatergic re-ponses, increased aggression, some form of neurode-eneration, occasional spontaneous seizures, and vari-ble premature death. These signs were largelyndependent of the actual mutant APP that was expressednd were not directly correlated with a singleAPP metabo-ite (Moechars et al., 1996, 1999a). The current modelsould shed more light on various other aspects of APP/Flnd APP/Du processing in rodent brain, but requiredditional investigation to understand the pathobiologicalathways leading to two distinct clinical entities.

ATERIALS AND METHODS

eneration of Transgenic Mice

The Flemish (A692G) and Dutch (E693Q) APP muta-ions were introduced in the human APP cDNA usingn vitro mutagenesis (Hendriks et al., 1995; De Jonghe etl., 1998). APP cDNA encoding these mutants werelaced under the transcriptional control of the mouse

hy-1 promoter (Moechars et al., 1996). From the 8.1-kbcoRI fragment comprising the mouse thy-1 gene, a.5-kb BanI–XhoI fragment was replaced by the respec-ive cDNAs employing blunt-end ligation. The re-laced fragment included the thymus-specific regula-

ory elements, present in intron 3 of the thy-1 gene. Therain-specific regulatory sequences in intron 2 wereetained (Moechars et al., 1996). Linearized constructs,evoid of all plasmid sequences, were purified

Kumar-Singh et al.

Sephaglass; Pharmacia) and microinjected into 1.5-day-ld prenuclear embryos isolated from superovulatedVB/N females according to standard procedures

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Hogan et al., 1994). The injected eggs were allowedvernight to reach the two-cell stage and subsequentlyransferred to pseudopregnant fosters (CD1). Off-pring were genotyped by Southern blotting of tail-iopt DNA restricted with KpnI and probed with aadiolabeled 490-bp PCR amplicon of human APPDNA with primers 58-CCGATGGGTAGTGAAG-AATGGTT and 58-TGTGCCAGCCAACACAGA-AAC, which were also used for subsequent routine

enotyping of offspring by PCR. Stable transgenic linesere established by breeding founders to FVB/N mice.

nalysis of APP Transgene RNA and Protein

Mouse brain RNA was analyzed by Northern blot-ing (Moechars et al., 1996) and relative mRNA levelsuantified densitometrically after autoradiography ory phosphorimaging. APP was analyzed by Westernlotting and immunoprecipitation with antibodies in-icated. Brain was homogenized in 15 vol of 50 mMris pH 7.4, 150 mM NaCl, and proteinase inhibitorsBoehringer Mannheim, Germany) and cleared by centrifu-ation (100,000g, 1 h, 4°C). The pellet was reextracted withhe same buffer containing 2% Triton X-100, 2% Nonidet-40 and centrifuged as before. Samples were denaturednd reduced (2% SDS, 1% 2-mercaptoethanol, 95°C, 5in), separated on polyacrylamide gels (4–20% Tris–

lycine), and transferred to nitrocellulose filters (Hy-ond-C; Amersham, UK) for Western blotting.

ntibodies

The monoclonal antibody (mAb) 1G5 (Athena Neu-osciences, San Francisco, CA) is directed againstmino acid residues 444–592 of human APP. Rabbitntisera B11/4 and B12/4 were generated against aynthetic peptide representing the 20 C-terminal resi-ues of APP, synthesized on Tentagel-MAP branchedesin (ABI, Foster City, CA). The antiserum reactsqually well with human and mouse C-terminal stubsDe Strooper et al., 1995). Rabbit antiserum R1736 isirected against a synthetic peptide representing partf the Ab peptide located between the a- and b-secre-ase cleavage sites (residues 595–611) (Haass et al.,992). C-terminal fragments were immunoprecipitatedith rabbit antiserum B11/4 and separated on 10–20%

ris–tricine gels and fragments detected via Western

myloid Precursor Protein Transgenic Mice

lotting by ECL chemiluminescence using B12/4 (1/000) as primary antibody. For immunohistochemistry,he following antibodies were used: mAb 4G8 for d

-amyloid (18–24 amino acid residues of Ab; Senetek),abbit antisera FCA3340 and FCA3542 for Ab residuesnding at 40 and 42 amino acids (Barelli et al., 1997),Ab 22C11 directed against N-term APP (Boehringerannheim), abnormally phosphorylated PHF-t (AT8;

nnogenetics), and glial fibrillary acidic protein (GFAP;ako). Blocking sera (rabbit and goat sera), link anti-odies (biotinylated rabbit anti-mouse and goat anti-abbit), and horseradish avidin–biotin complex weresed in recommended dilutions (Dako).

b Peptide Levels

Mouse brains were homogenized in 6.5 vol of 20 mMris–HCl, pH 8.5, 5 mM EDTA, 2 mM PMSF, 0.5µg/mleupeptin, 0.7µg/ml pepstatin, 0.1 mg/ml phenanthro-ine, and 0.1 mg/ml benzamidine. The homogenate wasleared (220,000g, 2 h, 4°C) and concentrated on Sepak C18artridges (Waters, Milford, MA). Sandwich ELISA wassed to measure Ab40, using mAb 1101.1 as capturing andA#1 as detecting antibody (Higgins et al., 1994).

esident Intruder Test

All mice were identically housed in a temperature-nd humidity-controlled vivarium, kept on a 12-hark–light cycle (light on 7:00 AM EST), with freeccess to food and water, except during the test. In thesolation-induced aggression test ‘‘resident’’ male non-ransgenic or transgenic mice were 10–12 weeks of aget the time of testing and were solitarily housed for 4eeks prior to the test. Bedding was changed once aeek without touching or handling the animal. ‘‘Intrud-

rs’’ were nontransgenic male FVB/N mice, 8–10eeks of age at the time of testing, housed in groups of

our since weaning. The test consisted of placing aandomly chosen intruder in the resident cage andggression of the resident was scored by measuring (i)ttack latency, i.e., the time in seconds between intro-uction into the cage and the first attack, and (ii) theumber of attacks during a 3-min session, with ‘‘at-

ack’’ defined as biting the intruder. One week followinghe first test, the same resident mice were tested a secondime with a different set of intruder mice. Statisticalnalysis of means was performed by Student’s t test.

lutamate Analogs

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N-Methyl-D-aspartate (NMDA) and kainic acid (KA),issolved in pyrogen-free 0.9% sodium chloride solu-

Copyright r 2000 by Academic PressAll rights of reproduction in any form reserved.

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ion, were injected intraperitoneally (ip) into mice of 3o 4 months. Reactivity of the mice was scored byetermining the LD50.

istopathology

Mice were anesthetized with ip injection of sodiumentobarbital (5 mg) and Ketamine (4 mg). Transcar-ial perfusion was performed in some animals with

ce-cold 0.01 M PBS (pH 7.4) followed by perfusionith 4% paraformaldehyde in PBS. The brains wereissected free from the skull by incising the cranialerves and spinal cord and placing them in saline. Forach specimen, half the brain, cut midsagittally orransversely, was fixed in methacarn overnight at 4°Cnd further processed for paraffin embedding, and theest was either processed for electron microscopy ornap frozen until further use.

Serial 4-µm parasagittal and/or transverse sectionsere made from embedded brain tissue specimens for

istological and immunohistochemical stainings withhe exception for Congo red staining (10µm). Classicalistological staining included hematoxylin–eosin, cre-yl violet, and Bodian and Congo red. For immunohis-ochemistry, sections were immersed in 0.03% hydro-en peroxide in methanol and incubated for 1⁄2 h tolock endogenous peroxidase followed by preincuba-ion with normal sera diluted 1:5. Sections were incu-ated overnight (16 h) at 4°C with the primary antibod-

es, followed by 1⁄2-h incubations with first biotinylatedecondary antibodies and then horseradish peroxidase-onjugated avidin–biotin diluted 1:1:100. Sections werenally treated with peroxidase substrate solution con-

aining 0.01% hydrogen peroxide and 0.05% diamino-enzidine tetrahydrochloride and counterstained witharris hematoxylin and coverslipped in DPX. Allilutions were made in 0.1 M PBS containing 0.1%ovine serum albumin. In situ detection of apoptoticells by terminal transferase incorporation of dUTPick end labeling (TUNEL) was performed on sectionsetrieved in citrate buffer (pH 6), microwaved, andtained according to the guidelines provided by theupplier (Boehringer Mannheim).

ransmission Electron Microscopy

To examine any fibrillar deposits in or around blood

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essels and to rule out minute amounts of diffuse Ab,issue was processed for electron microscopy andmmunoelectron microscopy. For the former, brain

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emiblocks were fixed with 4% glutaraldehyde solu-ion in phosphate buffer (PB; pH 7.2; 0.2 M) for 4 h at°C, washed in PB and water, and dehydrated in araded ethanol series (30, 50, 75, 90, and 23 100%), 15in each, at 220°C. Specimens for Unicryl embeddingere further processed according to guidelines pro-

ided by the manufacturer (British Biocel Internationaltd, Cardiff, UK). In short, tissue was brought immedi-tely from 100% ethanol to pure resin for an infiltrationeriod of 60 min and one infiltration overnight in freshure resin, both at 220°C. Tissue blocks were polymer-

zed using Philips LTD 15W/05 lamps for a period of 4ays, with a final curing period of 7 days at 4°C.ltrathin sections were cut and collected on nickelrids. For immunoelectron microscopy, tissues wererocessed as described above, except that they werexed with 2% paraformaldehyde solution in PB con-

aining 0.05% glutaraldehyde. Ultrathin sections wereollected on Formvar-coated nickel grids. The gridsere preincubated for 30 min on 20-µl drops of 20%

ormal serum in Tris-buffer saline (0.05 M, pH 7.6)ollowed by incubation with primary antibodies andetection with a 10-nm gold-labeled secondary anti-ody (AuroProbe EM; Amersham International plc,lough, UK). After staining, the sample grids wereostfixed in 2% glutaraldehyde for 5 min. Sectionsere thereafter stained with uranyl acetate and Rey-

old’s lead citrate (5 min each) and examined using aeol 100 C electron microscope (Japan).

ESULTS

ransgene Expression

Wild-type APP and Flemish and Dutch mutant APPere expressed in the brain of transgenic FVB/N mice

y the mouse thy-1 gene promoter (Moechars et al.,996). Founders and offspring in FVB/N backgroundere genotyped by Southern blotting over four to six

enerations, confirming stability of integration andendelian transmission of the gene. The 13 indepen-

ent transgenic lines expressed transgene mRNA atoderate to high levels (Table 1, Fig. 1).

iochemical Analysis of APP Intermediates

In brain of heterozygous transgenic mice, ages 6 to 8

Kumar-Singh et al.

eeks, full-length cell-associated APP and its catabo-ites were analyzed and quantified, comprising totalecreted and a-cleaved secreted APP, the residual

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-terminal fragments (C-stubs), and the Ab (1–40)eptide (Fig. 2, Table 2). Expression levels of mutantPP in brains of mice of lines APP/Fl/1 and APP/u/4 were 47 and 60%, respectively, of the expression

evel in APP/Wt/4 transgenic mice. Effects of thelemish and Dutch APP mutations on APP processingere analyzed by quantification of APP-processingroducts, normalized for expression level for humanPP/Wt transgenic mice. This revealed a significant

elative increase in b-carboxyl fragments in transgenicPP/Fl and APP/Du mouse brains (Fig. 2). Further-ore, standardization of a-secreted APP to total se-

reted APP revealed a consistent and significant de-rease in a secretion of APP in APP/Fl and APP/Duransgenic mice (Fig. 2). The same protocols and

ethods revealed a marked increase in b-carboxylPP fragments and decrease in a-cleaved secretedPP for transgenic mice bearing the Swedish APP

Moechars et al., 1998a).

ABLE 1

verview of Different Transgenic APP Mouse Lines

inigene Founders LineRNAlevel

Mortality(%)

APP/Fl 7 APP/Fl/1 14 31.4APP/Fl/3 2 7.7

APP/Du 6 APP/Du/4 5 31.6APP/Du/5 3 13.7

Note. Tabulation of transgenic founders generated from the twoinigenes and of some transgenic lines analyzed for RNA expres-

ion levels (relative to wt APP) and mortality in the different APPransgenic lines after 180 days.


IG. 1. Generation of APP transgenic mice. (A) Schematic representation orain RNA from the following transgenic mice: lane1, APP/Wt/4; lane 2, And lane 6, APP/Du/5. The 3.5-kb endogenous mouse APP transcript (mAP

henotypic Characterization

remature Death

FVB/N mice live in our conventional animal houseor more than 2 years, while APP transgenic mice inhis genetic background die variably but prematurelyMoechars et al., 1996), a phenomenon that was alsobserved by others (Hsiao et al., 1995; LaFerla et al.,995). Monitoring of 150 heterozygous mice from twoPP/Fl and two APP/Du transgenic strains, housedith 46 nontransgenic littermates for 6 months, re-

ealed that only 3% of nontransgenic mice died, aspposed to between 8 and 16% of the APP transgenicice (Table 1). Early mortality correlated with higher

evels of mutant APP expression as reported for mu-ine APP/RK transgenic mice (Moechars et al., 1996).remature death was absent in a large number of other

ransgenic mouse strains, housed under identical con-itions in the animal house, but overexpressing other

ransgenes unrelated to APP using the same mousehy-1 gene promoter and in the same genetic back-round (results not shown). Premature death is thuspecific for APP transgenic mice and occurs most of theime without overt signs of deteriorating health andery rarely with decreased bodyweight or muscleasting.

pontaneous Behavior and Seizures

Typical for the APP transgenic mice were alternatingpisodes of hyperactivity, anxiety, and aggression inhe home cage and in a minority of mice progressingnto seizures as reported before for other APP trans-

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f the mouse thy-1 gene promoter construct. (B) Northern blot of totalPP/Wt/2; lane 3, APP/Fl/1; lane 4, APP/Fl/3; lane 5, APP/Du/4;P) and the larger transgenic mRNA (hAPP) are indicated.


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enic mice (Moechars et al., 1996, 1999a). Offspring ofhe APP/Fl and APP/Du transgenic strains developednd behaved normally for 6–8 weeks after birth,

udged by weight gain, feeding, drinking, locomotion,nd climbing in the home cage. Progressively, moreice of both sexes in the different APP transgenic

trains displayed signs of increased agitation andouts of wild running with an age of onset of 8 weeksr more, depending on the expression level of theransgene. Subsequently seizures, a neurologic sign of

IG. 2. Analysis of APP-processing products. (A) Western blot of mice of lines APP/Wt/4, APP/Fl/1, and APP/Du/4 (n 5 8). Full-len

) was done by densitometric scanning in a linear range of the assaestern blots of soluble fractions of brains of nontransgenic and het

n 5 4). Total secreted APP was detected by 1G5, a-secretase-cleaved). (D) Detection of C-terminal fragments was done by immunoprecractions of brains of nontransgenic and heterozygous transgenic mlots were semiquantitatively analyzed (Results, see Table 2).

ABLE 2

iochemical Analysis of APP Metabolites

Expressionlevel b-COOH

b-COOHnormalizedexpression le

PP/Wt 100 6 0 100 6 0 100 6 0(n 5 8) (n 5 8) (n 5 8)

PP/Fl 47 6 6 102 6 23 215 6 49(n 5 8) (n 5 8) (n 5 8)

PP/Du 60 6 8 108 6 27 180 6 45

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Note. Tabulation of the levels of APP and APP processing intermediatesb40). Values represent mean values and standard errors of the mean of the


bnormal brain activity, became apparent in a subset ofhe transgenic mice. In about 15% of surviving micelder than 6 months, seizures that progressed fromild to severe tonic-clonic were observed and were

ndependent of the APP transgene. Mild seizuresonsisted of facial movements, forelimb clonus, andhole-body clonus lasting less than 10 s while severe

onic-clonic seizures lasted 30 to 60 s. Mice gasped androduced tonic seizures, stretching their limbs with

he tail upright, followed by whole-body clonic sei-

ne extracts of brains of nontransgenic and heterozygous transgenicnsgenic APP was detected by 1G5. Semiquantitative analysis (Table

rmined by measurement of a dilution curve, not shown). (B and C)ous transgenic mice of lines APP/Wt/4, APP/Fl/1, and APP/Du/4y R1736. Blots were semiquantitatively analyzed (Results, see Tablen with B11/4, followed by Western blotting with B12/4. Membranenes APP/Wt/4, APP/Fl/1, and APP/Du/4 (n 5 8) were analyzed.

b-Amyloid(Ab40)

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APPsa

normalizedfor total APPs

1.8, 2.6 1.8, 2.6 100 6 0(n 5 2) (n 5 2) (n 5 4)

1.2 6 0.2 2.6 6 0.4 77 6 10(n 5 3) (n 5 3) (n 5 4)0.8, 0.8 1.3, 1.3 83 6 11

Kumar-Singh et al.

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ures. Subsequently, mice became lethargic for varyingeriods.

ale Aggression

Transgenic mice expressing human APP displayedn intense aggressive behavior not observed in non-ransgenic littermates handled and housed under iden-ical conditions (Moechars et al., 1996, 1998a, 1999a).

ajor indications were wounds and bald patches inhe fur resulting from male–male but also male–femaleghting. This behavior was typical only for APP

ransgenic mice housed in rooms with many otherransgenic mice expressing unrelated minigenes fromhe same or other promoters, but was independent ofhe actual isoform or mutant of APP expressed. Aug-

ented aggressive behavior in male mice of linesPP/Fl/1 and APP/Du/4 was tested, under strictly

ontrolled conditions, in comparison to nontransgenicice, employing isolation-induced aggression. ‘‘Resi-

ent’’ males were housed in isolation for 4 weeksefore being confronted with male nontransgenic ‘‘in-ruders’’ who were reared ‘‘socially’’ in groups. In eachest, aggression was scored over a 3-min observationeriod by (i) measuring the latency of the first attacknd by (ii) recording the number of attacks.Transgenic residents expressing mutant APP at-

acked the intruder significantly sooner than nontrans-enic FVB/N resident males. Furthermore, the num-er of attacks by all APP transgenic mice wasignificantly higher than in nontransgenic mice andas also experienced by the observer to be ‘‘moreerce.’’ A second test of the same residents withifferent intruders, 1 week after the first test, demon-trated a considerable increased intensity of aggres-ion, both for nontransgenic and for transgenic resi-ents (Fig. 3). This is consistent with the fact thatggressiveness increases with fighting experience.uantitatively, in the first test 78 and 86% of thePP/Fl and APP/Du transgenic residents, respec-

ively, attacked the intruder in the 3-min test period aspposed to only 11% of nontransgenic male mice.etween 33 and 44% of the transgenic residents of theifferent transgenic strains attacked within the firstinute, which was never observed for nontransgenic

esidents in the first test. In the second test, theercentage of transgenic mice with short-latency at-


ack was increased to between 71 and 84% in APP/Flnd APP/Du residents compared to only 16% inontransgenic residents.

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istopathology and Ultrastructural Examination

Brains of APP/Fl (n 5 18) and APP/Du (n 5 18)ransgenic mice, including those of two mice that diedrematurely from both groups, along with those of wtVB/N mice (n 5 5) were subjected to histological and

mmunohistochemical examinations (Fig. 4). On grossxamination, brains of transgenic mice were not differ-nt from age-matched wt mice nor was any grossathology of any other organ evident. In the majorityf the APP/Fl and APP/Du animals, a characteristic

oosening or spongiosis of the neuropil was noticed in

IG. 3. Male aggressive behavior. Resident–intruder test of aggres-ion with nontransgenic FVB/N mice (n 5 15) and heterozygousransgenic mice from lines APP/Fl/1 (n 5 9) and APP/Du/4 (n 5 7).ttack latency (top) and number of attacks (bottom) were scored in

wo consecutive tests (black bar, test 1; open bar, test 2) over 3 minnd results are means with standard errors. Two-tailed Student’s test indicated most differences with nontransgenic mice to beignificant (at least P , 0.05).

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he frontal cortical regions and parahippocampal anderebellar areas (Fig. 4E). Associated with it was anntense glial response, demonstrated by a dense stain-


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ng pattern with a GFAP antibody, present in cerebralortical areas and cerebellar white matter, as well as inippocampus and hilus of dentate gyrus, and extend-

ng to the granular cell layer. In animals experiencingeizures and premature death a more intense reactivestrogliosis was demonstrated by GFAP immunostain-ng (Figs. 4F and 4G). Reactive gliosis was present inhe brain of all APP transgenic mice independent of the

utation, while absent in transgenic mice overexpress-ng unrelated genes under control of the thy-1 pro-

IG. 4. Histology and immunohistochemistry of the mouse braighlighting occasional condensed nuclei in the granular layer of thurther in (B). Note here a neuron with karyorrhexis (arrow). Similaroticed like here in a section of APP/Du mouse brain (C, arrow). TUrain in the region of the granular layer and polymorphic layer of th

IG. 5. Electron microscopy. Nuclear changes seen on light micros-opy were also confirmed ultrastructurally in which nuclear balloon-ng was noticed in APP/Fl (A, original magnification 340,000) as

ell as in APP/Du mice section (B, original magnification 340,000)ith occasional deposition of lipofuschin consequent to aging (20onths). Granular cell layer of the dentate gyrus was normal in this

ection of APP/Du mouse brain (C, original magnification 340,000)nd no amyloid-like fibrils were noticed in or around blood vesselsn a nonperfused specimen of APP/Du mouse brain specimen (D,riginal magnification 380,000).


rain (E). Reactive gliosis in cerebellar cortex illustrated by GFAP immunmmunostaining in APP/Du brain specimen in the region of cerebral cortexesult of background (I).

oter, in the same genetic background. Reactive glialells were also observed ultrastructurally. No viral-likearticles were noted in these areas, and axons wereppropriately myelinated (Fig. 5).Both APP/Fl and APP/Du transgenic mice were

oticed to harbor occasional abnormal neurons withense chromatin and condensed nucleus in the cere-ral cortex especially in layers II, IV, and V and in theentate gyrus. Abnormally condensed neurons werelso evident in similar areas in mice that died prema-urely. Although rare, parenchymal lacunas associated

ith disintegrating neurons were sometimes noticed,specially in the mice with premature death (Figs. 4Ao 4C). Ultrastructurally, ballooning of nucleus waslso noticed occasionally (Fig. 5A). TUNEL stainingould demonstrate a considerable number of positiveells in these areas (Fig. 4D). However, only a minorityf these neurons were shown to have a typical morphol-gy of an apoptotic cell ultrastructurally (Figs. 5A toC).No parenchymal or vascular amyloid deposits were

oticed on Congo red staining or on immunohistochem-stry with mAb 4G8 and rabbit sera FCA3340 andCA3542. Although some amyloid staining existed,specially with 4G8 and for hyperphosphorylated tAT8) (Fig. 4I), neither amyloid fibrils nor pairedelical filaments were noted within neurons ultrastruc-

urally (Fig. 5) nor was any reactivity on immunoelec-ron microscopy (data not shown). Some blood vessels

ere dilated, perhaps due to the perfusion performedrior to fixation. Nonetheless both APP/Fl andPP/Du transgenic mice were shown to express ele-ated amounts of APP compared to the age-matchedontrols. Using mAb 22C11, recognizing both mousend human APP, expression of APP was noticed notnly in neurons but also in glial cells, ependymal cells,pithelium of the choroid plexus, and occasionally ineptomeninx. Neuronally, expression was strongest inerebral cortical layers II, IV, and V; pyramidal layer ofippocampus; olfactory bulbs; nucleus basis pontis;ranial nerve nuclei; and weakly in Purkinje andranular cell layers of the cerebellum (Fig. 4F).

staining of APP/Fl mice (A) overviewing hippocampal region,ate gyrus and pyramidal layer of hippocampus, which are detailedof neurons, consequent to possible apoptosis, was also occasionallyaining recognizing apoptotic neurons in a section of APP/Fl mouseate gyrus (D). Cerebellar white matter spongiosis in APP/Fl mouse

17

in. HEe dently lossNEL ste dent

ostaining in APP/Fl (F) compared with FVB/Wt (G). APP (22C11)(H) and staining of blood vessels for Ab (4G8) in APP/Fl mice as a


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eactivity to Glutamate Analogs

To address neuropathological changes at the molecu-ar level, we studied reactivity of the glutamate neuro-ransmitter system, known to be involved in mecha-isms of learning and memory as well as ineurodegeneration and excitotoxicity. The present hu-an APP mutant transgenic mice behaved very simi-

arly to the mutant APP/RK and other previouslyeported human APP transgenic mice in response tohe glutamate analogs NMDA and KA (Moechars et al.,996, 1999a). Dose–response curves for KA-inducedortality clearly indicated that all APP transgenicice were more sensitive to KA than nontransgenic

VB/N mice: the LD50 for the APP/Fl/1 and APP/u/4 mice was situated between 28 and 32 mg/kgody weight, while all nontransgenic FVB/N miceurvived the dosage of 32 mg/kg (Table 3). In contrasto the hypersensitivity to KA the present APP trans-enic mice were hyposensitive toward NMDA as werehe previously reported APP transgenic mice (Moecharst al., 1996, 1999a). The LD50 in nontransgenic FVB/Nice of 3 to 4 months was about 70 mg NMDA/kg. All

ge-matched APP transgenic mice were less sensitiveith LD50 values that ranged between 160 and 200 mgMDA/kg (Table 3B).

ISCUSSION

So far, transgenic mouse models have not yet suc-eeded in fully recapitulating all aspects of AD pathol-gy, a problem that cannot be judged yet to be

ABLE 3

ortality Resulting from Administration of KA and NMDA

KA 24 mg/kg

VB/N 0% (n 5 5)PP/Du/4 0% (n 5 2)PP/Fl/1 25% (n 5 4)

NMDA 40 mg/kg 80 mg/kg

VB/N 0% (n 5 15) 67% (n 5 21)PP/Du/4 0% (n 5 2) 0% (n 5 2)PP/Fl/1 0% (n 5 2) 0% (n 5 2)

Mortality resulting from intraperitoneal administration of KA andequals the number of mice tested.

8

mpossible to reach. The ultimate development of thehenotype in any mouse model will depend on many

actors, i.e., the transgene copy number, the specific

otl


romoter used to drive the transgene, the geneticackground of the mouse strain, the relative preponder-nce of the Ab subspecies, etc. (reviewed in Price &isodia, 1994). Even additional genetic or environmen-al factors in humans affect the development of ADvan Duijn et al., 1994; Finch & Tanzi, 1997). Therefore,

direct extrapolation of a human phenotype into aouse model should be carefully considered. Develop-ent of amyloid plaques has now been demonstrated

n several different models of transgenic mice and theroblem is now evidently shifted to determining what

heir relevance and precise contribution is, if any, to thehenotype and the pathology.Despite the early behavioral disturbances and de-

ects, increased premature death and subtle pathologi-al changes like astrogliosis and microspongiosis withndications for limited apoptosis, neither APP/Fl norPP/Du transgenic mice produce appreciable amy-

oid plaques. We hypothesize that APP is not efficientlyirected into the amyloidogenic pathway by theseutants as it is by other clinical mutants, APP/Lo andPP/Sw, similarly expressed by the same promoter in

he same mouse strain that do produce Ab depositsMoechars et al., 1999a). Mutations in APP/Du andPP/Fl transgenic mice do, however, significantlyecrease a-secreted APP while increasing b-secretase-leaved C-terminal fragments in mouse brain, relativeo wild type and normalized for total secreted APP.his effect was similar, although less pronounced than

n APP/Sw transgenic mice and conforms to thenown metabolic effects of mutations in APP on itsetabolism in vitro in which the mutations located

ear the a-secretase cleavage site also favor b cleavage

28 mg/kg 32 mg/kg

0% (n 5 8) 0% (n 5 10)33% (n 5 6) 87% (n 5 8)37% (n 5 8) 75% (n 5 8)

120 mg/kg 160 mg/kg 200 mg/kg

100% (n 5 16) 100% (n 5 2) 100% (n 5 2)0% (n 5 8) 29% (n 5 7) 100% (n 5 5)0% (n 5 7) 43% (n 5 7) 100% (n 5 5)

, to non-transgenic, heterozygous APP/Du/4 and APP/Fl/1 mice.

Kumar-Singh et al.

f APP (Haass et al., 1994, Mc Phie et al., 1997). Despitehis shift from a to b cleavage, there was no increase inevels of Ab peptides in APP/Fl transgenic mouse

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rain, whereas in the APP/Du transgenic mice, rela-ive b-amyloid levels were even lower. Interestingly,his parallels our earlier experiments with transfectedHO-K1 cells, HEK-293 cells, and human neuroglioma4 cells in which APP/Fl increased the production ofb while Ab levels in the supernatants of APP/Du-

ransfected cells were slightly lower than in APP/Wt-ransfected cells (De Jonghe et al., 1998).

Absence of any vascular Ab deposits in the brains ofPP/Du and APP/Fl transgenic mice could not beue to the use of a specific neuronal (thy-1) promoter,s APP mice using the same promoter have beenhown to develop vascular amyloid deposits (Calhount al., 1998). Moreover, the only other study withPP/Du transgenic mice (Howland et al., 1995) also

ailed to demonstrate any amyloid deposition in brain.t was suggested that E693Q and A692G substitutionsn the APP/Du and APP/Fl mutants, respectively,ould alter the secondary structure and aggregatingroperties of the Ab peptide, thereby contributingifferently to the final phenomenon. In the APP/Du

soform, an accelerated amyloid fibril formation rela-ive to the native sequence was noticed (Wisniewski etl., 1991; Clements et al., 1993). For APP/Fl mutation,owever, no significant change in the rate of in vitromyloid aggregation was detected. In fact, the aggrega-ion of mutant Ab amyloid fibrils bearing the A692G

utation, was slightly slower than that of wild-typeb (Clements et al., 1993). The clinical cases of APP/Du

nd APP/Fl mutations could thus result either fromonelevated but highly fibrillogenic Ab in the formerr from mild to moderately elevated Ab levels in the

atter, but extended over a much more prolongederiod, which is obviously not possible in rodents.lternatively or in addition, the mutations might

ause a reduced clearance of the Ab in humans whileice might remain equally efficient in clearing theseutant Ab peptides.Occurrence of behavioral disturbances in these trans-

enic mice is not surprising as neuropsychiatric abnor-alities in the form of increased irritability and aggres-

ive behavior are present in a large percentage of ADatients (Hope et al., 1999) and also remain the majornderlying cause for caregivers to place these patients

n an institution (Stoppe et al., 1999). Moreover, sei-ures in both sporadic and familial AD patients as wells in patients with HCHWA-D has been frequentlyecorded, especially in the later stages of the diseaseRomanelli et al., 1990; Martin et al., 1991; Kennedy et


l., 1993; Mullan et al., 1993; Lampe et al., 1994; Silbert etl., 1995). Due to the poor visual performance of FVB,easurement of memory and learning abilities in the

ewo

orris Water Maze is not possible on this mouse strainSmith et al., 1997). However, in the resident–intruderest, APP transgenic mice displayed increased aggres-iveness compared to the nontransgenic mice. Also,eizures were one of the major symptoms in bothPP/Fl and APP/Du mice. Earlier, excitotoxicity me-iated by the glutamate neurotransmitter system was

mplicated in the physiology of hyperactivity, seizures,eurodegeneration, and neuronal cell death in ADGreenamyre & Young, 1989; Koh et al., 1990; Albin &reenamyre, 1992; Rothman & Olney, 1995), as well as

n more general phenomena like learning and memoryMalenka, 1994; Collingridge & Bliss, 1995). Moreover,PP was also recently shown to participate in the

egulation of extracellular levels of excitotoxic neuro-ransmitters like glutamate, by controlling their uptakento astrocytes (Masliah et al., 1998). Thus we testedensitivity toward KA and NMDA, as these are theajor glutamate agonists in the CNS. LD50 was taken

s a pharmacological end point in these experiments,s both KA and NMDA are known to induce a wideange of reactions, which are difficult to monitor. Usinghis criterion, increased sensitivity against excitotoxicnsults was apparent by hypersensitivity of APP trans-enic mice toward KA and of hypofunctional NMDA-ignaling pathways. Interestingly, NMDA binding isecreased in specific brain regions in AD cases (Greena-yre et al., 1987; Jansen et al., 1990; Rothman & Olney,

995) while KA binding is increased, at least in theuter molecular layer of the dentate gyrus (Geddes etl., 1992). However, absence of changes in NMDAeceptor density or distribution in APP/RK transgenic

ice eliciting similar glutamatergic drug responseMoechars et al., 1996) points toward subtle functionalisturbance in the glutamate neurotransmitter system

Moechars et al., 1998b).Nevertheless, the fact that transgenic mouse strains

verexpressing APP display neuronal degeneration asell as behavioral disturbances in the absence of

myloid deposits or neurofibrillary pathology, as dem-nstrated here, in previous studies (Moechars et al.,996, 1999a), and by others (Hsiao et al., 1995; LaFerlat al., 1995; Moran et al., 1995), suggests a neurotoxicffect of either APP or its C-stubs, which is indepen-ent of the amyloid plaque or its major constituent Ab.art of this phenomenon is also explained by an

ncreased sensitivity of certain mouse strains to overex-ression of APP in brain (Hsiao et al., 1995; Moechars etl., 1999a). However, since the consistent and common

19

arly defects shown here in APP/Du and APP/Fl miceere never observed in other transgenic mouse strains

verexpressing proteins unrelated to APP and driven


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y the same recombinant thy-1 promoter construct,nd such defects were also essentially similar to thoseocumented previously in at least two different mouseenetic backgrounds (Moechars et al., 1996), the speci-city for and by APP is guaranteed. A direct role ofPP or its intermediates in the development of theresent phenotype is supported by recent data onPP/Lo transgenic mice in which a decrease in theensity of presynaptic terminals and neurons, and arominent deficit in hippocampal synaptic transmis-ion on electrophysiology, occurs well before theseice develop amyloid plaques (Moechars et al., 1999a;sia et al., 1999).In summary, the results presented here on humanPP/Fl and APP/Du transgenic mice demonstrate a

hift from a to b cleavage of APP. Ab levels did,owever, remain low, not exceeding threshold levelsor amyloid precipitation and formation of amyloideposits. In concordance with earlier reports, overex-ression of APP and/or its proteolytic products causesn AD-related neuronal pathology. Further studies areeeded to address the distinct and common mecha-isms that underlie AD as well as HCHWA-D patho-hysiology.

CKNOWLEDGMENTS

We are grateful to Hubert Backhovens and Jef Boons for technicalssistance. The work was funded in part by the Fund for Scientificesearch–Flanders (FWO), the Interuniversity Attraction Poles

IUAP-P4117), the International Alzheimer Research FoundationIARF), the EU-BIOTECH program (BIO2-CT960743), NFWO-Lotto,he Action Program for Biotechnology (IWT/VLAB, COT-008), theooms-fund, and Leuven Research and Development.

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Behavioral Disturbances without Amyloid Deposits in Mice Overexpressing Human Amyloid Precursor...

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