The Caenorhabditis elegans A�1– 42 Model of AlzheimerDisease Predominantly Expresses A�3– 42

*□S

Received for publication, June 1, 2009, and in revised form, June 29, 2009 Published, JBC Papers in Press, July 2, 2009, DOI 10.1074/jbc.C109.028514

Gawain McColl‡§1, Blaine R. Roberts‡§1, Adam P. Gunn‡§, Keyla A. Perez‡¶�, Deborah J. Tew‡¶�, Colin L. Masters‡§,Kevin J. Barnham‡¶�, Robert A. Cherny‡¶, and Ashley I. Bush‡¶2

From the ‡Mental Health Research Institute, Parkville, Victoria 3052 and the §Center for Neuroscience, ¶Department of Pathology,and �Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia

Transgenic expression of human amyloid � (A�) peptide inbody wall muscle cells of Caenorhabditis elegans has been usedto better understand aspects of Alzheimer disease (AD). Inhuman aging andAD, A� undergoes post-translational changesincluding covalentmodifications, truncations, and oligomeriza-tion. Amino truncated A� is increasingly recognized as poten-tially contributing to AD pathogenesis. Here we describe sur-face-enhanced laser desorption ionization-time of flight massspectrometry mass spectrometry of A� peptide in establishedtransgenicC. elegans lines. Surprisingly, theA�being expressedis not full-length 1–42 (amino acids) as expected but rather a3–42 truncation product. In vitro analysis demonstrates thatA�3–42 self-aggregates like A�1–42, butmore rapidly, and formsfibrillar structures. Similarly, A�3–42 is also the more potentinitiator of A�1–40 aggregation. Seeded aggregation via A�3–42is further enhanced via co-incubation with the transition metalCu(II). Although unexpected, the C. elegans model of A�expression can now be co-opted to study the proteotoxic effectsand processing of A�3–42.

Numerous studies support a role for aggregating A�3 inmediating the toxicity that underlies AD (1, 2). However,several key questions remain central to understanding howAD and A� pathology are related. What is the connectionbetween A� aggregation and toxicity? Is there a specific toxicA� conformation or species? How and why does aging impacton A� precipitation? Significant effort to address these ques-tions has been invested in the use of vertebrate and simpleinvertebrate model organisms to simulate neurodegenerativediseases through transgenic expression of human A� (3). Fromthese models, several novel insights into the proteotoxicity ofA� have been gained (4–7).

Human A� (e.g. in brain, cerebrospinal fluid, or plasma) isnot found as a single species but rather as diverse mixtures ofvarious modified, truncated, and cross-linked forms (8–10).Specific truncations, covalent modifications, and cross-linkedoligomers of A� have potentially important roles in determin-ing A�-associated neurotoxicity. For example, N-terminaltruncations of A� have increased abundance in AD, rapidlyaggregate, and are neurotoxic (9, 11). Furthermore, the N-ter-minal glutamic acid residue of A�3–42 can be cyclized to pyro-glutamate (A�3(pE)-42) (12), which may be particularly impor-tant in AD pathogenesis (13, 14). A�3(pE)-42 is a significantfraction of total A� in AD brain (15), accounting for more than50% of A� accumulated in plaques (16). A�3(pE)-42 seeds A�aggregation (17), confers proteolytic resistance, and is neuro-toxic (13). Recently, glutaminyl cyclase (QC) has been proposedto catalyze, in vivo, pyroglutamate formation of A�3(pE)-40/42(14, 18). A�1–42 itself cannot be cyclized by QC to A�3(pE)-42(19), unlike A� that commences with an N-terminal glutamicacid-residue (e.g. A�3–42 and A�11–42) (20). QC has broadexpression in mammalian brain (21, 22), and its inhibitionattenuates accumulation of A�3(pE)-42 into plaques andimproves cognition in a transgenic mouse model of AD thatoverexpresses human amyloid precursor protein (14). N-termi-nal truncations at position 3 have been reported in senileplaques (23, 24); however, the process that generates A�3–42 isunknown. Currently there are no reported animal models ofA�3–42 expression.

Advances in surface-enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF MS) analysisnow facilitate accurate identification of particular A� species.Using this technology, we examined well characterized C. el-egans transgenicmodels of AD that develop amyloid aggregates(25, 26) to see whether the human A� they express is post-translationally modified.

EXPERIMENTAL PROCEDURES

Strains—The strains N2, wild type; CL2006, dvIs2(pCL12-(unc-54:hu-A� 1–42) � pRF4) (25); CL2120, dvIs14(pCL12-(unc-54:hu-A� 1–42) � mtl-2:gfp); and CL2122, dvIs15(mtl-2:gfp) (26) were provided by theCaenorhabditisGenetics Center.All strains were cultured at 20 °C on 8P/22Namedium (27), andthen at the first day of adulthood (4 days old), were aged at 25 °Cas indicated.Reagents—Human A�1–40, A�1–42, and A�3–42 peptides were

synthesized by theW.M. Keck Laboratory (Yale University, NewHaven, CT). Peptides were dissolved in 60 mM NaOH at roomtemperature and thendiluted to1mg/ml indistilledH2Oand10�

* This work was supported by funds from the Australian Research Council,National Health and Medical Research Council, AGL Shaw Trust, Haroldand Cora Brennan Benevolent Trust, the Marian and E. H. Flack Trust,and the Operational Infrastructure Support program of the VictorianState Government.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: AIB, Mental Health

Research Institute, 155 Oak St., Parkville, VIC 3052, Australia. Fax: 61-3-93806182; E-mail: a.bush@mhri.edu.au.

3 The abbreviations used are: A�, amyloid �; AD, Alzheimer disease; SELDI-TOF MS, surface-enhanced laser desorption ionization-time of flight massspectrometry; QC, glutaminyl cyclase; MES, 2-(N-morpholino) ethanesulfo-nic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine; ThT, thioflavin-T; pE, pyro-glutamate; EM, electron micrograph.

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PBS (PBS is defined as 50mM sodiumphosphate, 2.7mMKCl, 137mM NaCl, pH 7.4) at a volume ratio of 2:7:1. Preparations weresonicated for 10 min in a water bath and then centrifuged at13,500 � g for 10 min at 4 °C; the supernatant was then filteredthrough 0.2-�mfilters (Supor, PALL) and kept on ice for immedi-ate use. Peptide concentration was determined by measuringthe absorbance value at 214 nm and applying the molarextinction coefficient values of 91,460 M�1 cm�1 for A�1–40and A�3–42 and 94,530 M�1 cm�1 for A�1–42. Molar extinc-tion coefficients were determined using amino acid analysis(Australian Proteome Analysis Facility Ltd.) and UV spec-trometry (Lambda 25 UV-visible, PerkinElmer LifeSciences).SELDI-TOFMass Spectrometry—Synchronized4-day-oldC. el-

egansadultswere filtered (toremoveeggsand larvae) through40-�mnylonmesh (BDBiosciences) prior to sonication in chilled TBS (100mMTris-Cl and150mMNaCl, pH8.0).After homogenizationwith aprobe sonicator, lysates were clarified by centrifugation (13,500 � gfor 5min), and then the supernatant was removed and then kept onice for immediateuseorstoredat�20 °Cforsubsequentanalysis.Forsynthetic A�1–42 and A�3–42 standards, 40 pmol was analyzed.SELDI-TOFMSwas performed using our established protocol (28).

Immunocapture used affinity-purifiedW0-2 (epitope: A� 5–8) (29) antibodycoupled to ProteinChip PS10 arrays(Bio-Rad), as described (28).A� Immunoblot Analysis—Insol-

uble A� species from C. eleganslysates (described above) were col-lected by centrifugation (100,000 � gfor 20 min). Pellets were then incu-bated in 70% (v/v) formic acid over-night at room temperature. A�species were resolved based on hy-drophobicity using bis/Bicine ureaPAGE as reported previously (30).Synthetic A� peptide standards wereprepared as described above, with75 ng of each loaded per lane. Fol-lowing electrophoresis, gels wereequilibrated (three washes for 5min at room temperature) in MESSDS buffer (50 mM Tris base, 50mM MES, 1 mM EDTA, 0.01% SDSat pH 7.3) prior to semidry transfer(iBlot, Invitrogen) to 0.2-�m nitro-cellulose membrane. Membraneswere then boiled in PBS for 3min bymicrowave. Affinity-purified pri-mary antibodies 4G8 (epitope: A�18–22, Signet) and 6E10 (epitope:A� 3–8, Signet) were used at 1�g/ml dilution in plus 0.05% Tween20 (Sigma-Aldrich). Chemilumines-cence (Pierce) was quantitatedusing a LAS-3000 Image Analyzer(Fuji).Prediction of Signal Peptide

Cleavage—SignalP 3.0 (31) was used to predict signal peptidecleavage site(s) from the 60-amino acid peptide encoded by thepCL12 open reading frame (25).Fluorometric Analysis of in Vitro A� Aggregation—For anal-

ysis of self-aggregation, we followed a derived protocol (32).Solutions of 5 �M A� peptide in PBS, pH 7.4, were incubatedat 30 °C in a 96-well microtitre plate (Wallac) with 20 �M

thioflavin-T (ThT, Sigma) at a volume of 150 �l for 20 h.Measurements of ThT binding to A� fibrils were obtainedusing a Flexstation 3 fluorescence spectrophotometer (MDSAnalytical Technologies) and measuring fluorescence at 482nm (excitation � 450 nm) with a 475 nm emission cut-offfilter. Data points were collected in 5-min intervals via topreading, with each cycle consisting of 3 s of orbital shakingimmediately followed by the fluorescence measurement.Plates were sealed with acetate adhesive seals (MP Biomedi-cals) to minimize evaporative loss. ThT binding was repre-sented as the mean relative fluorescent units from n � 6replicate wells following subtraction of the reaction vehiclebackground fluorescence. Mean lag time (tl) and aggregationrates (k) were determined as described previously (32). Fit-

FIGURE 1. C. elegans A� model accumulates truncated A�3– 42. A, SELDI-TOF MS analysis of lysate fromC. elegans expressing A�: panel i, in CL2006, the m/z of 4327.5 Da corresponds to A�3– 42 (calculated M�H�

4328.9), and similarly, in panel ii, in CL2120, the m/z of 4327.4 Da corresponds to A�3– 42. Panel iii, SELDI-MSanalysis of synthetic A�1– 42 (calculated M�H� 4515.1, observed m/z 4516.8) and synthetic A�3– 42 (calculatedM�H� 4328.9, observed m/z 4328.8). B, synthetic A� species resolved on the basis of hydrophobicity onbis/Bicine PAGE and detected using 4G8 as the primary antibody. C, the A� species from formic acid solubilizedC. elegans lysates resolved using 4G8 as the primary antibody. Synthetic standards of A�1– 40, A�1– 42,A�3– 42,and A�3(pE)-42 were loaded as a combined mixture (75 ng of each A� species) and A�3– 42 alone (75 ng) forcomparison. Shown are lysates from young (4-day-old) transgenic controls (CL2122), young A�-expressingCL2120, old (8-day-old) CL2120, and old (8-day-old) A�-expressing CL2006 C. elegans. D, schematic of SignalP3.0 output, indicating predicted cleavage of N-terminal synthetic signal peptide from the A�1– 42 construct. Asingle cleavage site is predicted (maximal Y-score) at position �3 of A�1– 42 (between residues 20 and 21 of thefull-length peptide), yielding A�3– 42.C-score, S-score, and Y-score indicate the likelihood of the residue positionbelonging to the (first amino acid of the) mature peptide, signal peptide, and the combined cleavage siteprediction scores, respectively. Scores are plotted against the amino acid sequence derived from the openreading frame of the A�1– 42 bearing plasmid, pCL12. Indicated are the synthetic signal peptide (boxed) andpredicted cleavage site (arrowhead) and mature A�3– 42 peptide (underlined).

C. elegans Model of A�3– 42

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ting curves were performed using Prism (version 5.01,GraphPad). tl error was calculated by the intersects of the95% confidence interval curves and reported as 2� S.E.

Seeded A�-aggregation was similarly analyzed. ThT bindingof 4.5�MA�1–40 (“substrate”) aggregationwith andwithout 0.5�M A�1–42 or A�3–42 (“seed”) was measured as above. In addi-tion, differing stoichiometric ratios of Cu(II), presented asCu(II)-glycine (1:6 metal/ligand molar ratio), were co-incu-bated with the reactions.Electron Microscopy of A� Fibrils—A� peptides alone (60

�M) or in seeded reactions (54 �M A�1–40 � 6 �M A�1–42 orA�3–42 with or without equimolar Cu(II)-glycine) were incu-bated in PBS, pH 7.4 (with 0.1% w/v NaN3) at 37 °C for 3 days.Samples were prepared for transmission electron microscopyby adsorbing 5 �l of reaction onto a carbon-coated Formvarfilm mounted on 300 mesh gold grids (ProSciTech). Prior toadsorption, the grids were rendered hydrophilic by glow dis-charge in a reduced atmosphere of air for 10 s. After 120 s ofadsorption, samples were blotted, washed twice with (18megaohms) milli-Q water (Millipore), and negatively stainedwith 1.5% aqueous uranyl acetate (ProSciTech). Transmissionelectron microscopy was performed using a Tecnai G2 TF30(FEI Co.) instrument operated at 200 kV. Images were acquireddigitally with an UltraScan 1000 (2k � 2k) pixels CCD camera(Gatan).Turbidometric Analysis of Seeded in Vitro Aggregation of

A�1–40—Comparison of seeded aggregation was based uponprevious protocols (33, 34). A�1–40 alone (22 �M) or 20 �M

A�1–40 with either 2 �M A�1–42 or 2 �M A�3–42 was co-incu-bated in sterile filtered PBS, pH 7.4, incubation at 37 °C, in thepresence or absence of (10�M) Cu(II)-glycine (1:6metal/ligandmolar ratio). Turbidometric measurements were then per-formed as described previously (33, 34) using clear flat-bot-tomed 96-well plates (Greiner). Absorbance at 400 nm, meas-ured using a Powerwave spectrophotometer (BioTek), wastaken immediately and then again every 24 h over 5 days fromn � 4 replicate wells. The differences in absorbance (�A400 nm)were then compared. An automatic 5-s plate agitation wasincorporated prior to the analysis to evenly suspend any aggre-gates. Incubation was performed in a humidified chamber toprevent evaporative loss.

RESULTS

The C. elegansModel of AD Produces A�3–42—To determinewhether A� is truncated or modified in the C. elegansmodel ofA� toxicity, we performed SELDI-TOF MS analysis usingW-02 (as the capture antibody) on C. elegans expressingA�1–42 (expected M�H� 4515.1 Da). Unexpectedly, neitherstrain CL2006 nor strain CL2120 expressed any detectableA�1–42 (Fig. 1A). However, in both strains, we detected amajorspecies that had an m/z consistent with A�3–42 (expectedM�H� 4328.9 Da, observed m/z of 4327.5 and 4327.4 inCL2006 and CL2120, respectively). Due to the inherent limita-tions in the accuracy of the SELDI-TOF technique and becausethe molecular mass of A�1–40 (4329.9 Da) is only 2 Da greaterthan that of A�3–42, we did not conclusively assign the speciesat m/z of 4327.5 and 4327.4 as A�3–42 (M�H� 4328.9) on the

basis of SELDI-TOF alone. No other species of A� weredetected.A� Immunoblot Analysis of C. elegans—To confirm whether

the species present in the C. elegans was A�3–42, we used bis/Bicine urea gels to resolve the A� species based on hydropho-bicity of the peptide (30). Analysis of synthetic A�1–40, A�1–42,A�3–42, and A�3(pE)-42 using this technique resulted in the res-olution of the A� peptides (Fig. 1B) in an order consistent withthe calculated grand average of hydropathy (GRAVY) (35)scores (A�1–40 0.057, A�1–42 0.205 A�3–42 0.258) calculatedusing ProtParam (36) and previous reports of A�3(pE)-42 (37).Analysis of A� in formic acid-soluble fractions using bis/Bicineurea gel combined with immunodetection with 4G8 confirmedthat the species observed in two C. elegans models of A�

FIGURE 2. Analyses of in vitro aggregation: A�1– 42 versus A�3– 42. A, self-aggregation of A�1– 40, A�1– 42, and A�3– 42. All peptides (at 5 �M in PBS, pH7.4) were incubated at 30 °C. Data presented are the mean relative fluores-cence units from ThT binding (�S.E., n � 6 replicate wells) measured in 5-minintervals over a 20-h period. AU, arbitrary units. B, EM at �31,000 magnifica-tion of self-aggregated A�1– 40 showing typical amorphous aggregates(arrows). C, self-aggregated A�1– 42 showing characteristic fibrils. D, self-ag-gregated A�3– 42 showing some long fibrils with many more smaller protofi-brillar aggregates. Bar denotes 100 nm. E, ThT binding (as above) of 4.5 �M

A�1– 40 aggregation with and without seeds of 0.5 �M A�1– 42 or A�3– 42, asindicated. F, A�1– 40 showing amorphous aggregates (arrow). G, A�1– 40seeded with A�1– 42 showing short protofibrillar structures. H, A�1– 40 seededwith A�3– 42 showing similar protofibrillar structures.

C. elegans Model of A�3– 42

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(strains CL2120 and CL2006) corresponds to A�3–42 and thatA�1–42 was not present (Fig. 1C). The background strainCL2122 possessed no A� immunoreactivity (Fig. 1C). Agingpopulations to 8 days, previously shown to exacerbate amyloidformation (38), did not detectably alter A� any further. Equiv-alent results were observed in the TBS-soluble fractions probedwith 4G8 (supplemental Fig. 1) or 6E10 (data not shown).Predicted Processing of Transgenic A� in C. elegans—When

originally constructed, the A� transgene (used in both CL2006and CL2120 strains) possessed an 18-amino acid synthetic sig-nal peptide N-terminal to A� (25), which, it was proposed,would be cleaved to yield A�1–42 (Fig. 1D).We therefore re-ex-amined how the translated protein in these transgenic C. el-egansmight be processed. Using SignalP 3.0 (31), a neural net-working predictor of eukaryotic secretory signal peptides, wedetermined the most likely position of cleavage to be betweenresidues 20 and 21 (Fig. 1D). Cleavage at this site results in thegeneration of A�3–42.In Vitro Amyloidogenesis of A�1–42 Versus A�3–42—The

amyloidogenic properties of A�3–42 are not well characterized.Therefore, we examined synthetic peptides in vitro using ThT,a diagnostic fluorescent dye that specifically binds �-sheets,such as those in amyloid oligomers. When bound, ThT under-

goes a characteristic blue shift of itsexcitation spectrum.We found thatfollowing incubation in PBS, pH 7.4,for 20 h at 30 °C, both A�1–42 andA�3–42 increased ThT fluorescence(Fig. 2A). Kinetic parameters foreach curve were then determined,showing that A�3–42 has a shorterlag phase and faster rate of aggrega-tion, where for A�1–42, tl � 8.41 �0.15 (�2 S.E.) h and k � 0.30 � 0.01(�S.E.) h�1, and for A�3–42, tl �6.25 � 0.15 h and k � 0.52 � 0.02h�1.

To examine the quaternarystructure(s) formed by aggrega-tion, we examined sample mor-phology via electron micrograph(EM) (Fig. 2, B–D). Consistentwith our ThT data, and as has beenreported previously, A�1–40 didnot form fibrillar structures at pH7.4; consequently, no aggregationkinetics were determined for A�1–40.A�1–42 formed long fibrils oflength in excess of 200 nm. In con-trast, A�3–42 formed fewer longfibrils but a greater number shorterfibrils and protofibrils (less than100–200 nm in length) with anincreased tendency to clumptogether. These data indicate thatunder these in vitro conditions,A�3–42, like A�1–42, self-aggregatesand forms amyloid fibrils.

In Vitro Aggregation of A�1–40 Seeded by A�1–42 and A�3–42—We then examinedwhetherA�3–42 could substitute forA�1–42

in the classical assay of nucleated precipitation (“seeding”) ofA�1–40 (39).We found that A�3–42, when comparedwithA�1–

42, is the more aggressive initiator of A�1–40 aggregation (Fig.2E). Kinetic parameters were determined, such that for A�1–

42-seeded aggregation, tl � 12.21 � 0.54 h and k � 0.46 � 0.03h�1, and for A�3–42-seeded aggregation, tl � 10.01 � 0.67 hand k � 0.25 � 0.01 h�1. EM analysis of aggregated A�1–40,seeded with either A�1–42 or A�3–42 (Fig. 2, F–H), revealedthat both seeded reactions produced similar morphology ofprotofibril and short fibrillar structures (less than 200 nm inlength), which appeared crooked or flexible.We have previously reported that Cu(II) accelerates, in vitro,

A�1–42-seeded aggregation of A�1–40 (34). To examine inter-actions between Cu(II) and A�3–42-seeded aggregation, wereplicated this experiment using both ThT and turbidometricanalyses (Fig. 3). We observed an A�:Cu(II) ratio-dependentsuppression of A�3–42-seededmature fibril formation (Fig. 3,Cand D). However, Cu(II) caused an increase in amorphous A�

aggregation (Fig. 3E) as observed by light scatter. These data areconsistent with the effects of Cu(II) on A�1–42 aggregation

FIGURE 3. Analyses of in vitro A�1– 40 aggregation in the presence of Cu(II) seeded with A�1– 42 or A�3– 42.A, ThT binding of 4.5 �M A�1– 40 aggregation with and without seeds of 0.5 �M A�1– 42, with differing stoichio-metric ratios of Cu(II). AU, arbitrary units. B, EM of aggregated A�1– 40 seeded with equimolar A�1– 42:Cu(II).Shown are two images with amorphous A� aggregates (arrows) and short protofibrils (arrowhead). C, ThTbinding of 4.5 �M A�1– 40 aggregation with and without seeds of 0.5 �M A�3– 42, with differing stoichiometricratios of Cu(II). D, EM of aggregated A�1– 40 seeded with equimolar A�3– 42:Cu(II). Shown are two images withexamples of amorphous A� aggregates (arrows) and short protofibrils (arrowhead). Bar denotes 100 nm at�31,000 magnification. E, turbidometric analysis of A�1– 40 aggregation demonstrating that seeding withA�3– 42 causes greater aggregation. Samples of 22 �M of A�1– 40 alone and 20 �M of A�1– 40 with 2 �M of A�3– 42,with and without 10 �M Cu(II)-glycine in PBS (pH 7.4) at 37 °C. Data presented are the mean differences inturbidity from the initial readings (�A400 nm, �S.D., n � 4 replicate wells).

C. elegans Model of A�3– 42

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reported previously (40) and with transition metals increasingthe �-helical content of A� (41).

DISCUSSION

The accumulation of A� truncations and modifications andtheir respective contribution to the pathogenesis of A�-medi-ated toxicity have not been studied comprehensively in simplemodel systems. Therefore, we initiated a study to delineate theA� variants that accumulate in established transgenic C. el-egans models expressing A�. Surprisingly, we discovered thatonly A�3–42 was expressed rather than the predicted A�1–42.We found concordant data using both SELDI-TOFMS, as wellas immunoblot analysis of samples resolved by hydrophobicity.Neither technique, nor the antibodies used, are expected to biasdetection of specific A� truncations. The detection of a singleA� species, rather than a range of variants, suggests simplepost-translational processing. Both the original A�-expressingstrain, CL2006 (25), and a derived strain, CL2120 (26),expressed only A�3–42. The likely cause is aberrant cleavage ofthe N-terminal signal peptide incorporated in the C. eleganstransgene (Fig. 1D). Although signal peptide cleavage (includ-ing that in C. elegans) is not fully understood, prediction accu-racy of cleavage sites has improved greatly (42), especially overthe 14 years since the A�-expressing C. elegansmodel was firstreported (25).We examined the accumulated A� in young (4-day-old) and

aged (8-day-old) post-reproductive C. elegans adults andobserved no additional truncations, covalent modifications(including pyroglutamate), or cross-linking. This model, there-fore, represents a relatively clean system in which further A�modifications could be studied via additional transgenics. TheC. elegans genome encodes a potential QC ortholog,H27A22.1(data not shown). However, it is not known whether an analo-gous enzyme activity is found in the same cell type thatexpresses the transgenic A� (body wall muscle cells). TheH27A22.1 promoter does not appear active in body wall mus-cles (43). Therefore, additional genetic manipulations maybe useful to examine interactions between QC and A�3–42 inC. elegans.Proteotoxicity of the (cytoplasmic) expressed A�3–42 in this

C. elegansmodel has been clearly demonstrated (6, 26, 38, 44).The expressed A�3–42 aggregates and forms amyloid in vivo.We observe that in vitro, A�3–42 forms fibrils. In addition, wefound that A�3–42-seeded aggregation of A�1–40 substrate ismore rapidly initiated when compared with A�1–42-seededaggregation. Furthermore, A�3–42-seeded aggregation is exac-erbated in the presence of Cu(II). These results are importantbecause of the increasing evidence that Cu(II) is constitutivelyenriched in the synapse and is dysregulated in AD, where itpools in the vicinity of the synapse and within plaques (45).Our observation that Cu(II) promotes in vitroA�3–42 aggre-

gation (Fig. 3, C and E) is consistent with the observation thatA� amyloid formation is induced in C. elegans (CL2120, thesame strain as used in our study) by exposing animals to Cu(II)and rescued by treatmentwith clioquinol (46). Clioquinol bindsCu(II), abolishes the redox activity of A�:Cu(II) complexes, res-cues amyloid neuropathology, and improves cognition in amy-loid precursor protein transgenic mice (28). Our data suggest

that A�3–42 exhibits similar interactions with metal ions thathave been characterized previously for A�1–42 (47, 48). This isconsistent with residues 3–9 of A� mediating a transition to�-sheet and insoluble aggregates upon Cu(II) binding (49).Although we examined the effects of Cu(II), it should be notedthat oligomerization of A� may be induced by other metals,such as synaptic zinc (50).In summary, we find that by serendipity aC. elegansmodel of

cytoplasmic A�3–42 expression and toxicity has been generatedthat has value as amodel for AD despite not expressing A�1–42.In AD, A�3–42 can bemodified toA�3(pE)-42 thatmay representa key species in A�-derived neurotoxicity. This C. elegansmodel can now be exploited to provide further insight into thebiology of this truncated A� in AD pathogenesis.

Acknowledgments—We thankChristopherD. Link (University of Col-orado), Simon James (MHRI), andmembers of theMasters, Barnham(University ofMelbourne), Cherny, and Bush laboratories (MHRI) forhelpful discussions and critical reading of this manuscript. In addi-tion, we acknowledge the technical expertise of the staff of the Univer-sity of Melbourne Bio21 EM Unit for their contribution to this work.All nematode strains were provided by the Caenorhabditis GeneticsCenter funded by the U. S. National Institutes of Health NationalCenter for Research Resources.

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Colin L. Masters, Kevin J. Barnham, Robert A. Cherny and Ashley I. BushGawain McColl, Blaine R. Roberts, Adam P. Gunn, Keyla A. Perez, Deborah J. Tew,

42−3βExpresses A Model of Alzheimer Disease Predominantly42−1βThe Caenorhabditis elegans A

doi: 10.1074/jbc.C109.028514 originally published online July 2, 20092009, 284:22697-22702.J. Biol. Chem. 

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