overexpression of human amyloid precursor protein in drosophila

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Molecular Cell Biology Research Communications 4, 43–49 (2000)

doi:10.1006/mcbr.2000.0248, available online at http://www.idealibrary.com on

Overexpression of Human Amyloid PrecursorProtein in Drosophila

Yoshimasa Yagi,*,† Susumu Tomita,* Makoto Nakamura,‡ and Toshiharu Suzuki*,1

*Laboratory of Neurobiophysics, School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo113-0033, Japan; †Biooriented Technology Research Advancement Institution, 3-18-19 Toranomon, Minato-ku,Tokyo 105-0001, Japan; and ‡National Institute for Basic Biology, Myodaiji-cho, Okazaki 444-8585, Japan

Received July 6, 2000

Amyloid precursor protein (APP) is the precursor ofthe b-amyloid peptide which is associated with Alzhei-mer’s disease. The physiological function of APP is notwell understood. We have established model systemfor the analysis of APP function in Drosophila. In neu-al cells, overexpressed human APP was transportedo the synaptic terminal in a manner similar to itsocalization in human neurons, which suggested thathe Drosophila protein transport system localizes hu-an APP appropriately. Expression of APP in imagi-al discs resulted in a defect in adult cuticle secretionnd a blistered wing phenotype. The severity of theing blister phenotype was proportional to the APP

xpression level. These results suggested the presencen Drosophila wing tissue of a protein or protein(s)

which can interact with APP. © 2000 Academic Press

Deposition and accumulation of b-amyloid (Ab) inuman brain and cerebral blood vessels is a hallmarkf Alzheimer’s disease (AD). Ab is derived from amy-

loid precursor protein (APP) by proteolytic cleavageand is believed to be neurotoxic (reviewed in 1). APP isthought to have physiological function because of itsreceptor-like structure (reviewed in Refs. 2 and 3). Thestructural properties of APP is comparable with thoseof the epidermal growth factor (EGF) receptor and theinterleukin 2 (IL-2) receptor (4). Furthermore, cell-surface APP is expected to implicate in signaling path-ways in cortical development and in Alzheimer’s dis-ease (reviewed in Ref. 5). It has been, in fact, reportedthat cell-surface APP plays a role in neurite extensionof primary cultured neurons (6) and PC12 cells (7)through possible interactions with extracellular and/orcytoplasmic molecules, and the neurite outgrowth is

1 To whom correspondence should be addressed at Laboratory ofNeurobiophysics, School of Pharmaceutical Sciences, University ofTokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Fax: 181-3-5841-4805. E-mail: [email protected].

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inhibited by downregulation of APP expression (8).However, the biological role for APP as receptor hasnot been analyzed sufficiently.

Members of the APP protein family have been foundnot only in mammals but also in other vertebrates (9)and in invertebrates such as fruit fly (10) and nema-tode (11). The cytoplasmic domains of APP and APP-like proteins (APLPs) from a variety of organisms arehighly conserved (9, 12–14) and contains signal se-quences which are thought to be responsible for theintracellular metabolism (15–18) and function (7, 19) ofthe proteins. The sequence 653-YTSI-656 (humanAPP695 isoform numbering) contains a characteristicinternalization signal, YXXI (15, 18). The 667-VTPEER-672 contains a phosphorylation site, Thr668,in neuron-specific manner (20) and Arg672, which isimportant for regulation of APP metabolism (17). This667-VTPEER-672 motif consistent with an N-terminalhelix capping box structure (21). The sequence 681-GYENPTY-687 contains NPXpY element but Tyr687of APP is not phosphorylated. This motif binds to pro-teins regulating APP metabolism (22–26).

APP-like protein (Appl) in the fruit fly, Drosophilamelanogaster (10), is likely to be metabolized in a man-ner identical to the metabolism of mammalian APP/APLPs (27, 28). Moreover, human APP partially res-cued a behavioral deficit in Appl mutant flies (29).These observations suggest that a physiological systeminvolving APP and/or APP-like proteins is conservedacross a variety of cell types and a wide range of animalspecies. Therefore, analysis of the biological function(s)of the APP family using non-human animals may con-tribute to our understanding of the role of APP familyproteins.

Here we show that overexpressed human APP wastransported to synapses in Drosophila, in a mannersimilar to its transport in mammalian neurons. APPwas also distributed around the post- synapse of neu-romuscular junction (NMJ) when expressed in Dro-sophila muscle tissue. No significant morphological de-

1522-4724/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 4, No. 1, 2000 MOLECULAR CELL BIOLOGY RESEARCH COMMUNICATIONS

fects were observed in fly neurons expressing humanAPP. Overexpression of APP in imaginal discs caused acuticle secretion defect and blistered wing with apicaland basal surface localization of APP in wing disc cells.Our results suggest that Drosophila may express mol-cules which interact with APP, and thus provide aseful model system with which to identify the physi-logical function of APP.

ATERIALS AND METHODS

ly Stocks

Most of the fly strains used in this study have beenescribed elsewhere (30). The Actin-Gal4 line was gen-rated by flip out of the yellow cassette from the Ay-al4 line (31) using the b-tub flp line (32). The Gal4

lines used were 21B, 24B, 32B, 69B, 71B, elav-Gal4,pros-Gal4, sca-Gal4, G455.2, sev-Gal4, tsh-Gal4, T80-Gal4, ap-Gal4, ptc-Gal4, omb-Gal4, vg-Gal4, Appl-G1a(33) and GMR-Gal4. GMR-Gal4 was obtained fromDrs. M. Okabe and Y. Hiromi, 24B was obtained fromDr. A. Nose and Appl-G1a was obtained from Dr. K.White. Other stocks were obtained from Dr. S. Ha-yashi.

Transgenic Flies Expressing Human APP

The 695 isoform of wild-type human APP cDNA andmutant cDNAs [R672A, Swedish (K595N/M596L),

672A/Swedish double mutant, T668A and T668E, (7,7, 34)] were inserted into pUAST (35). A cDNA encod-ng a truncated form of APP 681stop, was constructedy introduction of a stop codon by PCR. A cDNA en-oding a deletion mutant which lacks 17 amino acidsncluding NPTY motif, D675–691, was also constructedy PCR. Both of these cDNAs were inserted intoUAST. Germline transformation was carried out asreviously described (36). Three independent P ele-ent insertion lines were used for the experiments.

henotype Scoring

We used homozygous viable UAS-APP insertionines. Gal4 lines used for calculating viability werealanced or X chromosome insertion lines. Flies carry-ng Gal4 and UAS constructs were identified by domi-ant markers on balancer chromosomes except for Xhromosome insertion lines. Viability was calculatedy dividing the number of flies without dominantarker by the number of flies with dominant marker.emales carrying the UAS construct were crossed withales with a P-Gal4 inserted X chromosome and the

emale/male ratio was recorded as viability. Three in-ependent P-inserted lines were used for APP expres-ion. Alteration of the phenotype induced by ap-Gal4-riven APP was scored by counting the number oflistered wings and dividing by the total number of

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ings observed. The UAS lines themselves do not ex-ibit the blistered wing phenotype.

istology

Larvae and white pre-pupae were dissected and fixedn 4% (w/v) para-formaldehyde/PBS for immunostain-

ing with UT-421 anti-APP antibody (37) and anti-FasIImonoclonal antibody. Signals were detected with theVectastain ABC Kit (Vector Lab) or FITC- or Cy3-labeled goat anti-rabbit IgG (Zymed) and Cy3-labeledgoat anti-mouse IgG (Amersham/Pharmacia). Z-sec-tions of imaginal discs were observed using a confocalmicroscope (LSM410, Carl Zeiss).

RESULTS

Expression of Human APP in DrosophilaNeural Tissues

Several groups created transgenic mouse expressinghuman APP or b-amyloid (Ab) to produce model ani-mal of amyloidogenesis and Ab-related disorders (re-viewed in Ref. 1). These transgenic mice expressingfamilial Alzheimer’s disease (FAD)-linked human APPshowed Ab deposits but did not show the complete ADpathology. However, studies to analyze physiologicalfunction of APP using human APP transgenic mousewere almost nothing. We attempted to express humanAPP in Drosophila using 19 Gal4 lines. Since Alzhei-mer’s disease is accompanied by a neurodegenerativedisorder and Appl, a Drosophila homologue of APP, isexpressed in neural tissues (27), it might be expectedthat overexpression of APP could induces phenotypicalterations in neural tissues. Even though we tested anumber of Gal4 drivers known to induce expression inneural cells, we could not find any visible effect onneural-cell and -tissue morphology by APP expression.Since APP is transported to axon in neural cells (38,39), we examined the localization of APP in neural cellsand the NMJ boutons of flies expressing APP (Fig. 1).Expressed human APP was transported to synapticterminals in central nervous system (Fig. 1A) and alsoto the axon terminal of motor neurons in NMJ (Fig.1B). We also investigated overexpression of APP inmuscle cells using 24B-Gal4. We found no morpholog-ical or lethal effect, but we observed that APP formedpatch-like granules and localized preferentially arounda subset of boutons (Fig. 1D). The localization of APPwas not altered by point mutations in the cytoplasmicdomain, truncation at amino acid residue 681 or inter-nal deletion of residues 675–691.

Overexpression of Human APP in NonneuralDrosophila Tissues

Expression of APP under the control of two tissue-specific drivers, T80-Gal4 and ap-Gal4, induced visible

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phenotype in adult flies (Figs. 2 and 3). T80-Gal4 in-duces ubiquitous expression of gene in wing and legdiscs of third instar larvae and ap-Gal4 induces geneexpression in dorsal part of wing disc of third instarlarvae. When APP expression was driven by T80-Gal4,many flies died soon after eclosion (Table I) as a resultof becoming trapped in food and showed defective cu-

FIG. 1. Localization of APP in Drosophila nervous system. Tntibodies. (A) APP expression was induced with ap-Gal4 and the Cetected in cell body (arrow) and axon (arrowheads). (B, C) APP expere observed by double staining with anti-APP (B) and anti-Fa

arrowheads). (D–F) APP expression in body wall muscle was indutaining was carried out. (D) Anti-APP, (E) anti-FasII, (F) merge oigration around a subset of NMJ boutons was observed (arrowhea

FIG. 2. APP overexpression using T80-Gal4. Wing (A, B) and leg(C, D) of wild-type (A, C) and yw; T80-Gal4/UAS-APP(wt)-1 (B, D)flies. Wing proliferation was inhibited and blistering was sometimesobserved as a result of APP expression (compare B with A). Legswere abnormally weak because of abnormal cuticle secretion (com-pare arrowheads in D with C). Scale bar, 400 mm.

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ticle secretion (data not shown) and malformed legsand wings (Fig. 2). The wings of these flies were notfully extended and their legs were abnormally weak.

When APP expression was driven by ap-Gal4, wingmalformation was observed (Fig. 3) but neither reduc-tion of viability nor any cuticle defect was observed inthese flies (data not shown). A “curl up” phenotype in

d-instar larvae expressing APP were dissected and stained withof a third-instar larvae were stained with anti-APP. Signals were

sion was induced with elav-Gal4 and the NMJ of third instar larva(C). APP signals were observed in FasII-positive axon terminalswith 24B-Gal4 and anti-APP (green) and anti-FasII (red) double

and E. APP signals appeared as patch-like granules (arrow). APP. Scale bar: 32 mm (A), 50 mm (B–F).

FIG. 3. APP overexpression using ap-Gal4. (A) Wild type, (B) w;ap-Gal4, UAS-APP(T668A)-3/1, (C) w; ap-Gal4, UAS-APP(T668A)-3/UAS-APP(wt)-1, (D) w; ap-Gal4, UAS-APP(T668A)-3/UAS-APP(T668A)-1. Low-level expression of APP caused the anterior andposterior edges of wings to curl toward the dorsal side (B, arrow-head). High-level APP expression caused blistered wing phenotype(C, D). Scale bar, 400mm.

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TABLE I


which the anterior and posterior edges of the wingscurled toward the dorsal side, was observed in most ofthese flies (Fig. 3B) and some flies showed a blisteredwing phenotype (Figs. 3C and 3D). The proportion ofblistered wing induced by ap-Gal4 differed betweenUAS-APP lines and the mutations we introduced intoAPP did not affect the blistered wing phenotype. Theblistered wings appeared more frequently in flies car-rying two copies of UAS-APP (Table II), which suggeststhat the blistered wing phenotype is affected by APPexpression levels. Deletion of the APP cytoplasmic do-main weakened the wing phenotypes (data not shown).

Cuticle is secreted from the apical side of epithelialcells, and the blistered wing phenotype suggests a de-fect in cell–cell interaction on the basal surface of wingcells. We observed localization of human APP on theapical and basal surfaces of wing disc cells (Fig. 4).These data suggest that human APP interferes withcuticle secretion on the apical surface and cell–celladhesion on the basal side of the wing cell.

DISCUSSION

In the present study, we showed that APP was trans-ported by a selective transport system in fly tissues. In

Viability of Flies Expressing APP

Ratio (Cy1/Cy2)a

Act-Gal4 T80-Gal4

APP(wt)-1 0.180 (484)b 0.675 (695)APP(wt)-2 0.888 (574) 0.788 (345)APP(wt)-3 0.731 (505) 0.786 (450)GFP 1.33 (452) 1.45 (362)

a Act-Gal4 and T80-Gal4 were balanced with CyO.b The total number of flies scored is indicated in parentheses.

TAB

Copy Number Dependency

Genotype Norm

Canton S 21ap-Gal4/UAS-GFP[S65T] 9ap-Gal4/UAS-APP(wt)-1ap-Gal4, UAS-APP(T668A)-1/1ap-Gal4/UAS-APP(T668E)-1ap-Gal4, UAS-APP(T668A)-3/1ap-Gal4, UAS-APP(T668A)-3/UAS-APP(wt)-1ap-Gal4, UAS-APP(T668A)-3/1; UAS-APP(T668E)-1/1

a Percentages of blistered wings.b Flies carry indicated number of UAS-APP.

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particular, APP was transported normally in fly neu-rons, in the same manner as APPL, a Drosophila ho-mologue of APP. This suggests that a common systemis used for the transport of APP and APPL. APP mi-gration to the post-synapse in rat has been reported(40), and APP migration around NMJ in Drosophilalarval muscle suggests that a conserved anterogradetransport system localizes APP to the post-synapse inboth vertebrates and invertebrates. Recently, anothergroup reported that APPL overexpression induces mor-phological changes in synaptic boutons in NMJ (28).We found no significant morphological defect in bou-tons of flies overexpressing APP though the localiza-tion patterns of APP and APPL in neurons were iden-tical. Functional conservation between APP and APPLwould be expected, but may be only partial.

FIG. 4. Localization of APP in imaginal disc. Wing discs of thirdinstar larvae carrying ap-Gal4 and UAS-APP (wild type or mutantform) were dissected and stained with antibody. (A) ap-Gal4-inducedAPP expression in dorsal part of the wing disc. A line indicates theplane of the section shown in B. Scale bar, 50 mm. (B) Transverseoptical section of A. Strong APP signals were observed on both apical(a) and basal (b) side of wing disc cells.

II

Blistered Wing Phenotype

Fly number

%a Copy No.bCurl up Blistered

0 0 0 00 1 1.0 0

306 40 12 1136 34 20 144 158 78 1

343 17 4.7 162 84 58 218 216 92 2

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The cuticle and wing phenotypes observed in thisstudy were similar to the “infantile” phenotype inducedby APPL overexpression (33). Fossgreen et al. recentlyreported wing blister formation in response to APPexpression driven by T80-Gal4 (42), although the blis-tered wing phenotype was not frequently observed inour study. The effect of T80-Gal4 on viability of APP-expressing flies differed between independent UAS-APP insertion lines and was affected by culture tem-perature (data not shown), which suggests that theexpression level of APP affects the degree of phenotypicalteration. T80-Gal4 can also induce expression in neu-ral cells from embryo but neural expression of APPappears not to reduce viability. It is more likely thatthe morphological defects in leg and wing cuticle re-sulted in the low viability of APP-expressing adultflies, because these flies could walk on dry vials and theviability of 3rd instar larva carrying T80-Gal4 andUAS-APP was not reduced (data not shown).

In MDCK cell, APP is transported to basolateralsurface (43, 44) by virtue of the basolateral sortingsignal sequence, which is present in human APP butnot in fly APPL (10, 11). APP overexpressed in Dro-sophila, was localized to both lateral and basal surfaceof wing discs of third instar larva. This localizationsuggests the existence of a conserved transport systemwhich localizes APP family proteins to the cell surface,further indicating that the APP basolateral sortingsignal may not function in Drosophila.

A number of proteins containing a phosphotyrosineinteraction domain (PID) bind to the 684-NPTY-687motif in the cytoplasmic domain of APP (22–24, 45). Weexamined whether the APP overexpression phenotypeinvolved protein-protein interaction mediated by the684-NPTY-687 motif. We found no remarkable differ-ences between the phenotype of flies expressing wild-type APP and those expressing the APP constructslacking the 684-NPTY-687 motif, 681stop and D675–691. It therefore appears that the wing blister pheno-type induced by APP overexpression is independent ofinteractions between the NPTY motif and any pro-tein(s) which may bind to it.

We utilized the blistered wing phenotype to identifygenes interacting with APP in Drosophila using defi-ciency lines covering about 78% of the genome and wefound five lines enhance the blistered wing phenotype(unpublished observation). Two types of proteins, inte-grins and presenilin, are believed to be involved in thephysiological function of APP. b-, a1- and a2-integrinmutants show the blistered wing phenotype in Dro-ophila, and co-localization of b1 integrin and APP has

been reported in mammals (46). Df(1)C128, b-integrindeleting deficiency, enhanced wing blister phenotype ofAPP overexpression fly but deficiency chromosomeswhich deletes a1-, a2-, and a3-integrin did not en-hanced the phenotype (data not shown). Further, wetested b-integrin mutant, mys[GX43] (obtained from

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Dr. Hama), but interaction with APP overexpressionphenotype was not observed (data not shown). Muta-tions in presenilin genes are associated with early-onset AD and affect processing of APP (1). In Drosoph-ila, PS (presenilin) mutant flies have a distinctive wingphenotype, and PS interacts with Notch (47, 48). Theresults of our genetic interaction screening do not in-dicate any genetic interaction between APP and PS, orbetween APP and gene(s) involved in Notch signaling.These observations indicate that APP does not interactwith integrins, PS or proteins of Notch signaling inDrosophila wing development.

Appl is not expressed in the wing disc of Drosophila(49). Flies carrying loss-of-function mutations in Applre viable and show no gross morphological defects29), whereas PS mutations are lethal with distinctorphological defects (47, 48). These observations re-

arding Appl also support our result that interaction ofPP with PS for APP processing may be one aspect of

he function of presenilins. Presenilins may have otherhysiological functions during early development (50,1) regardless of interaction with APP. We have foundve chromosomal deletions showing genetic interac-ion with the APP gene (unpublished observation), sug-esting at least five genes may interact with APP ining tissue. It may be possible to identify genes which

nteract with APP using flies showing the blistereding phenotype as a result of APP overexpression.

dentification and functional analysis of the geneshich interact with APP is expected to contribute tour understanding of the function of APP and patho-enesis of Alzheimer’s disease.

CKNOWLEDGMENTS

We thank Ms. Y. Morimoto and N. Kobayashi for technical assis-tance; Drs. S. Hayashi, M. Okabe, Y. Hiromi, A. Nose, T. Hama, K.White, and Bloomington Stock Center for fly stocks; Dr. A. Nose foranti-FasII antibody; Dr. S. Hayashi for critical reading of this manu-script; Dr. S. Goto for technical advice; and Dr. Y. Kirino for helpfuldiscussion and encouragement. This work was supported by a grantfrom the Program for Promotion of Basic Research Activity for In-novative Biosciences (T.S.).

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overexpression of human amyloid precursor protein in drosophila

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