Induction of cerebral �-amyloidosis: Intracerebralversus systemic A� inoculationYvonne S. Eiselea,b, Tristan Bolmonta, Mathias Heikenwalderc, Franziska Langera,b, Laura H. Jacobsond, Zheng-Xin Yane,Klaus Rothe, Adriano Aguzzic, Matthias Staufenbield, Lary C. Walkerf, and Mathias Juckera,g,1

aDepartment of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tubingen, D-72076 Tubingen, Germany; bGraduate School forCellular and Molecular Neuroscience, University of Tubingen, D-72074 Tubingen, Germany; cDepartment of Pathology, Institute for Neuropathology,University Hospital, Zurich, CH-8091 Switzerland; dNervous System Research, Novartis Institutes for Biomedical Research, CH-4002 Basel, Switzerland; eSMPGmbH, D-72072 Tubingen, Germany; fYerkes National Primate Research Center and Department of Neurology, Emory University, Atlanta, GA 30322; andgGerman Center for Neurodegenerative Diseases, 72076 Tubingen, Germany

Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved June 4, 2009 (received for review March 25, 2009)

Despite the importance of the aberrant polymerization of A� in theearly pathogenic cascade of Alzheimer’s disease, little is known aboutthe induction of A� aggregation in vivo. Here we show that inductionof cerebral �-amyloidosis can be achieved in many different brainareas of APP23 transgenic mice through the injection of dilute A�-containing brain extracts. Once the amyloidogenic process has beenexogenously induced, the nature of the induced A�-deposition isdetermined by the brain region of the host. Because these observa-tions are reminiscent of a prion-like mechanism, we then investigatedwhether cerebral �-amyloidosis also can be induced by peripheral andsystemic inoculations or by the intracerebral implantation of stainlesssteel wires previously coated with minute amounts of A�-containingbrain extract. Results reveal that oral, intravenous, intraocular, andintranasal inoculations yielded no detectable induction of cerebral�-amyloidosis in APP23 transgenic mice. In contrast, transmission ofcerebral �-amyloidosis through the A�-contaminated steel wires wasdemonstrated. Notably, plasma sterilization, but not boiling of thewires before implantation, prevented the induction of �-amyloidosis.Our results suggest that minute amounts of A�-containing brainmaterial in direct contact with the CNS can induce cerebral�-amyloidosis, but that systemic cellular mechanisms of prion uptakeand transport to the CNS may not apply to A�.

Alzheimer’s disease � amyloid � prion � sterilization � transmission

A number of degenerative neurological and systemic diseasesare characterized by the aberrant polymerization and ac-

cumulation of specific proteins. In the CNS, these proteopathiesinclude Alzheimer’s disease (AD), Parkinson’s disease, fronto-temporal dementia, and the prion diseases, among others (1). Itis now established beyond a reasonable doubt that prion diseasescan be induced by exogenous, conformationally variant prionproteins (2–7). Other proteopathies have been considered non-transmissible, but there is convincing evidence indicating thatsystemic amyloid A and apolipoprotein AII amyloidoses can beinduced in susceptible hosts (8, 9).

We have previously shown that intracerebral injection of A�-containing extract from AD brain or transgenic mouse brain caninduce cerebral �-amyloidosis and associated pathology in thehippocampus of young APP transgenic mice (10, 11). �-amyloidosiscould be effectively blocked when brain extracts were A�-immunodepleted or proteins were denatured. Because syntheticA� failed to act as an amyloid-inducing factor, an A� confor-mation different from that of synthetic A� and/or additionalchaperones have been suggested to influence the amyloid-inducing activity (11). The exogenous induction of cerebral�-amyloidosis is not limited to transgenic mice, but also has beendemonstrated in nonhuman primates (12, 13).

Thus, similar to prion diseases, cerebral �-amyloidosis mayqualify as an infectious disorder mediated by protein structure andstipulated in the modified postulates of Koch (14, 15). However, amore widely used definition of infectivity is the occurrence of lateraltransmission, as observed for prion disease (4, 16). It has been

shown that an infectious prion particle can infect the host by variousperipheral application routes. This observation suggests a transportprocess of the infectious particles (or at least of prion infectivity)from the periphery to the brain (4, 6, 17).

In the present work, we demonstrate that cerebral �-amyloidosiscan be induced by a single intracerebral injection of a �-amyloid-containing brain extract not only in the hippocampus, but also inother brain regions of susceptible host mice. In contrast, peripheraladministration of the brain extract does not induce cerebral �-amyloidosis, despite the close procedural adherence to the inocu-lation routes used for the experimental transmission of priondisease. However, similar to previous findings regarding prion-contaminated stainless steel surfaces and the transmission of priondisease through contaminated surgical instruments (18–21), wedemonstrate the ability of �-amyloid-contaminated stainless steelwires to induce cerebral �-amyloidosis when in direct contact withthe brain of susceptible host mice.

ResultsInduction of A� Deposition in Different Brain Regions. A�-containingbrain extract from aged APP23 transgenic mice (Tg extract) wasinjected into different brain regions of young, predepositing 2- to5-month-old APP23 mice. Analyses performed 3 or 6 months laterrevealed that Tg extract, but not extract from nontransgenic (wild-type) control mice (WT extract) or PBS injections, induced �-amyloidosis in the young APP23 mice (Fig. 1).

Although all injected brain regions (i.e., olfactory bulb, parietalcortex, entorhinal cortex, striatum, and hippocampus) carry thepotential for the exogenous induction of A� deposition, differenceswere noted in the amount and type of amyloid induction among thevarious brain areas (Fig. 1). For example, in the entorhinal cortexand hippocampus, amyloid induction was strong and significantlycongophilic, while in the striatum, far less amyloid was induced andthe deposits were mainly diffuse (Fig. 1). These regional differencesclosely resemble the normal age-related deposition of A� in thebrain of APP23 mice (22, 23).

Induction of A� deposition was observed already 3 months afterinjection in the proximity of the injection site, and it increased andspread into adjacent brain areas 6 months after injection. Forexample, injections in the entorhinal cortex induced A� depositionin the molecular layer of the dentate gyrus as well, while injectionsinto the striatum also induced A� deposition in the overlyingneocortex (data not shown), suggesting that spreading can occuralong fiber tracts.

Author contributions: Y.S.E., M.H., L.C.W., and M.J. designed research; Y.S.E., T.B., M.H.,F.L., and L.H.J. performed research; Z.-X.Y., K.R., A.A., and M.S. contributed new reagents/analytic tools; Y.S.E. and M.J. analyzed data; and Y.S.E., M.H., M.S., L.C.W., and M.J. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: mathias.jucker@uni-tuebingen.de.

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The injected Tg extract also induced congophilic A� depositionin the vasculature, predominantly in the walls of pial and thalamicvessels. This induction in the vasculature was observed independentof the injection site. As in the case of parenchymal deposits,longer postinjection times further increased cerebrovascular A�deposition.

No A� deposits were found when APP23 host brains wereanalyzed 1 week after Tg extract injection (data not shown),suggesting that the immunoreactive A� deposits are not theinjected material itself (11).

In agreement with previous studies (11), analysis of the Tg brainextract revealed monomeric, dimeric, trimeric, and some largeroligomeric species (Fig. 1M). Quantitative A� analysis of 2 repre-sentative Tg extracts revealed total A� concentrations of 4.4 and 8.0ng/�L using immunoassay measurements and 8.8 and 9.2 ng/�Lusing densitometry of Western blots. These values again are verysimilar to those reported previously (11).

Peripheral Administration of Tg Extract Does Not Induce Cerebral A�Deposits. Because the induction of A� deposits by intracerebralinjection is reminiscent of intracerebral prion transmission, thepossibility that cerebral �-amyloidosis could be induced by periph-eral exposure, similar to prion diseases, was investigated. Prede-positing 2- to 5-month-old APP23 transgenic mice were given Tg orWT extracts through the oral, i.v., intraocular, or intranasal route(Table 1). These peripheral applications closely resembled theinoculation routes and procedures used for the experimental trans-mission of prionoses. Again, similar to the findings in prion trans-mission studies, the total amount of extract was several-fold higher(up to 2E4-fold for oral intake) compared with that used inintracerebral administration. Analysis at 4–8 months postinocula-tion revealed that none of the peripheral application routes yielded

detectable induction of cerebral �-amyloidosis in any brain region,including retina and optic nerve (Table 1). Special attention wasgiven to the brain areas closest to and/or in synaptic contact with theinjection site, that is, the olfactory bulb for intranasal applicationand the retino-recipient nuclei for intraocular application. For oraland i.v. administration, serum A� antibody titers were monitored;no appreciable A� antibody titers relative to nonexposed controlmice were detected.

Stainless Steel Wire Coated With Tg Extract Induces Cerebral A�Deposition. Prion diseases can be transmitted via steel surgicalinstruments contaminated with minute amounts of prionotic tissue(18, 19). To determine whether A� deposition might be similarlyinducible, stainless steel wires were immersed in A�-rich brainextract, dried, and permanently implanted unilaterally into thehippocampus and overlying neocortex of predepositing 4- to5-month-old APP23 transgenic mice. Analysis 4 months laterrevealed strong induction of A� deposits in brains implanted withTg extract-coated wire, but no amyloid induction in brains im-planted with WT extract-coated wire (Fig. 2; Table 2). Although A�deposits were seen primarily in the vicinity of the implanted wire,smaller A� deposits were found throughout the hippocampus, aswell as in thalamic and pial vessels of the hemisphere containing thewire. Most of the induced A� deposits near the wire were Congored-positive (Fig. 2C), while more distant A� deposits were Congored-negative.

When Tg extract-coated wires were subjected to plasma steril-ization before implantation, induction of A� deposition was com-pletely blocked (Table 2). In contrast, heating the wires in PBS for10 min at 95 °C did not diminish the amyloid-inducing activity of theimplanted wires (Table 2). Consistent with these observations,measurement of the concentrations of A� bound to the wires before

A B

C D

E F

G

I

K

H

J

L

M

Fig. 1. Induction of A� deposition in different brain regions of young APP23 mice. �-amyloid-containing brain extract from aged APP23 transgenic mice (Tg extract)or extract from age-matched nontransgenic (wild-type) control mice (WT extract) was injected into the hippocampus, entorhinal cortex, parietal cortex, striatum, orolfactorybulbof2- to5-month-oldAPP23mice.Themicewerethenanalyzed3to6monthspostinjection.A� immunoreactivitywasdetectedinallbrainregions injectedwith the Tg brain extract at 3 months (data not shown) and 6 months (A, C, E, G, I, and K), but not in regions injected with the WT extract (B, D, F, H, J, and L). Thepostsurgery interval for the olfactory bulb was 4 months. The induction of A� deposits was most robust in proximity to the injection site, but was observed throughoutthe injected brain region. Some of the induced amyloid deposits in the hippocampus, neocortex, and entorhinal cortex were congophilic, while only diffuse A� depositswere found in the striatum (inserts in A, C, G, I, and K). A� deposition was not induced in the APP23 mice after PBS injections or when Tg extract was injected intonontransgenic littermate control mice (data not shown). The number of mice was n � 3 for each region and time point and type of extract. (M) Brain extracts wereanalyzed by Western blot. A� bands (monomer and oligomer bands denoted by asterisks) were detected in the Tg extract, but not in the WT extract, using the humanA�-specific antibody 6E10. A mixture of synthetic A�1–40 and A�1–42 (syn A�) was used as a positive control. (Scale bar: 200 �m; inserts, 20 �m.)

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implantation (by boiling the extract-coated wires in sample bufferwith 1.32% SDS and 3.33% 2-mercaptoethanol, with subsequentWestern blot analysis) revealed �0.2–0.5 ng A� (corresponding to0.2–0.5 � 10�5 g total brain or 0.02–0.05 �L of 10% Tg brainextract). Similar values were found for the wires that had beenboiled in PBS. Very little A� was detected on the surface of wiresafter plasma sterilization (Fig. 2D).

DiscussionDespite the importance of the aberrant polymerization of A� inthe early pathogenic cascade of AD (24), surprisingly little isknown about the induction of A� aggregation in vivo. In thepresent study, we have extended our previous findings of �-amyloid induction in the hippocampus (10, 11) and demon-strated that the injection of minute amounts of an A�-rich brain

A B

C D

Fig. 2. Stainless steel wire coated with A� rich brain extract induced cerebral �-amyloidosis. Stainless steel wires were immersed for 16 h at room temperature withbrain extract from aged APP23 transgenic mice (Tg extract) (A and C) or aged wild-type control mice (WT extract) (B). After air-drying, wires were implanted unilaterallyinto the hippocampus and overlying cortex of APP23 hosts (stereotaxic coordinates: AP, �2.5 mm; L, �2.0 mm; DV, �1.8 mm). Immunohistochemical analysis withanti-A�antibodyrevealedstronglocal inductionofA�deposits inthevicinityof theTgextract-coatedwire (arrowheadinA).Thenumberofmiceusedwasn�8.Highermagnification of the dentate gyrus region of adjacent sections double-stained with anti-A� antibody and Congo red (C) revealed spreading of A� deposition alongthe hippocampal fissure and dentate gyrus molecular layers (distance between sections shown, 600 �m). No A� deposits were observed when the wire was coated withWT extract (B). The number of mice used was n � 4. Bicine-tris urea SDS/PAGE of coated wires and subsequent Western blot detection with 6E10 antibody (D) revealeddetectable amounts of A� on wire (WTg; lane 2), corresponding to approximately 0.02–0.05 �L of 10% seeding extract (reflecting 0.2–0.5 � 10�5 g of total brain) (lane1). Similar amounts of A� were detected after the wires were heated in PBS for 10 min at 95 °C (WTg 95 °C; lane 3). After plasma sterilization (WTg pl; lane 4), no A�40or A�42 was left on the wire, but a weak higher-molecular-weight band appeared that likely represents A�-specific signal, because plasma-sterilized wires coated withWT extract exhibited no bands (WWT pl; lane 5). (Scale bar: 1,000 �m in A and B; 200 �m in C.)

Table 1. Induction of cerebral �-amyloidosis in APP23 mice

InoculationTotal amount (�L)

of Tg extractRelative amount

of Tg extractNumber of mice with A�

deposits/total mice*

Intracerebral 0.125 1 11/11†

Oral 2,500 2E4 0/10Intravenous 10 80 0/14Intraocular 1 8 0/6Intranasal 40 3.2E2 0/6

APP23 host mice were inoculated with either A� -containing extract from aged transgenic APP23 mice (Tgextract) or age-matched nontransgenic wild-type control mice (WT extract). The total amount of 10% extractgiven to each animal is indicated. Intracerebral injections were done by bilateral injection of 2.5 �L of 0.5%extract, yielding 0.125 �L of 10% extract into the hippocampus. Oral intake was 500 �L for 5 consecutive days. Forintravenous injections, extract was diluted 1:100 in sterile PBS before administration. Five 200-�L doses were givenwithin 10 days. Intraocular injections were done unilaterally with a single dose of 1 �L. For intranasal adminis-tration, each mouse received 20 �L of extract per nostril in a single session. The number of animals with inducedA� deposits out of the total number of animals inoculated per group is indicated for the Tg extract. A similarnumber of mice were inoculated with the WT extract that induced no cerebral � -amyloid deposits in any of theconditions.*Half of the mice were sacrificed after 4 months; the other half, after 6–8 months.†n includes the 5 mice published in Meyer-Lühmann et al. (11).

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extract can induce A� amyloidosis in other brain areas as well,including the entorhinal and parietal cortices, striatum, andolfactory bulb. In each of the injected brain areas, the amyloidinduction was time-dependent; that is, the amount of inducedamyloid was greater at 6 months postinjection than at 3 monthspostinjection. Although the amyloid induction in all examinedbrain areas appeared to spread from the site of injection to moredistal regions, it is not clear whether ‘‘true’’ spreading of theamyloid induction occurred (i.e., the induced amyloid formednovel seeds), or whether the apparent spreading simply reflectsthe concentration gradient of the injected material away fromthe injection site.

By injecting brain extract into different APP transgenic mouselines, we previously found that the exogenous induction of cerebral�-amyloidosis is governed in part by intrinsic properties of the host(11). This conclusion can now be refined by the observation that thenature and efficiency of amyloid induction is also brain region-dependent and closely resembles the normal endogenous age-related deposition of A� in the APP23 host (22, 23). For example,exogenously induced A� deposits in the striatum are largely diffuseand sparse compared with those in the other injected brain regions,despite the use of the same brain extract for seeding. The endog-enously generated amyloid deposits in the striatum of aged APP23mice also are diffuse and less frequent compared with those inthe neocortex and hippocampus (22, 23). The regional hetero-geneity of lesions might reflect the lower transgene expressionlevels in the striatum (22) compared with the neocortex andhippocampus (and thus less available endogenous A� to build onthe injected ‘‘seeds’’), but also might result from region-specificintrinsic factors, because the striatum also is not a primary siteof �-amyloid deposition in AD (25).

Interestingly, the induction of A� deposition in the pial andthalamic vasculature appears to be relatively independent of thesite of extract injection. This finding suggests that vascularamyloid can be induced in regions far away from the injectionsite, possibly by transport or passive diffusion of the amyloid-inducing activity/amyloid seeds over significant distances alongperivascular drainage pathways (26). Moreover, the observationthat the induced A� deposition in the vasculature is significantlycongophilic reinforces our conclusion that the brain compart-ment of the host determines the nature of the A� depositiononce the amyloidogenic process is exogenously activated.

In contrast to the intracerebral inoculations, none of theperipheral applications of the �-amyloid-containing brain ex-tract yielded detectable induction of cerebral A� deposition.This was so despite the administration of 8 to 2E4 more brainextract by these routes compared with the amount used in

intracerebral injections. Moreover, the postinoculation periodfor these peripheral application routes was up to 8 months,whereas amyloid induction in APP23 mice after intracerebralextract injection was noted starting at 8 weeks postinoculation(11). Because the amyloid-inducing brain extracts were preparedfrom the same inbred mouse line that also served as the host,putative transmission barriers due to amyloid strains wereavoided (4, 11). Nevertheless, we cannot rule out the possibilitythat longer incubation times and/or more extract may be requiredto induce cerebral A� deposition after peripheral inoculations.

Systemic or peripheral inoculations of prion disease is rare inhumans, but has been documented in cases of ritualistic canni-balism, contaminated human growth hormone, blood transfu-sion, and prion-contaminated food consumption (27–30). Suchlateral transmission appears to occur more often in deer andsheep with chronic wasting disease and scrapie, respectively,although the mechanism of transmission often is not clear (31).Under experimental conditions, prion transmission has beendemonstrated by systemic and peripheral inoculation routes,including oral, i.p., i.v., intranasal, intraocular, intranerval, andvia scarified skin and tongue infection (6, 32–34). By far the mostpotent inoculation route is intracerebral, followed, in order, byintranerval, i.v., intranasal, and i.p. In rodents, oral transmissionof prion infectivity is estimated to be 10E5–10E9 less efficientthan intracerebral inoculation, although these values are highlyvariable and depend on the susceptibility of the host, as well ason the tropism and type of the inoculated prion strain (6, 34).

The transport of prion infectivity from peripheral sites to thebrain and its spread within the CNS are dependent on theendogenous generation of PrPc by the host (17, 35, 36). Byanalogy, the lack of human APP transgene expression in theperiphery in APP23 mice may be responsible for the failure of�-amyloid induction after peripheral inoculations. Although thispossibility cannot be ruled out, the absence of amyloid inductionafter intraocular and intranasal administration argues against it,because both the retina and the olfactory bulb, as componentsof the CNS, express the human APP protein in APP transgenicmice using neuronal promoters (37, 38).

Thus, our results provide no evidence that cerebral A�deposition can be induced by any route other than the directinoculation of the extract into the brain. However, in theory, anyparticle that can mimic the �-amyloid-inducing activity of A�from brain extracts could trigger the process of amyloid induc-tion. It has been shown that the induction of amyloid A amy-loidosis can be triggered in Tg mice by heteronucleants such assilk (39, 40), and heteronucleation of apolipoprotein AII amy-loidosis also has been demonstrated in mice (41). In light of thefact that the vast majority of AD cases are idiopathic in nature,a brain-penetrant environmental factor with A�-nucleatingproperties remains a potential candidate for the initiation of theA� cascade (8).

Of immediate clinical interest is our finding that stainless steelbinds the amyloid-inducing agent and can induce cerebral �-amyloidosis when in direct contact with the CNS. From thepresent work, it is not possible to conclude whether the amyloid-inducing activity desorbs from the wire or initiates induction andspreading amyloid from the bound state. The finding of lessextract on the wire than what appears to be necessary for similaramyloid induction using intracerebral inoculation may suggestthat the immobilization of the extract on the wire prolongs thepresence of the ‘‘seed.’’ Prions also bind to stainless steel, andprion-contaminated stainless steel wires or surgical instrumentscan transmit prionosis to mice (20, 21, 42). In addition, prioninfectivity through prion-contaminated surgical instruments hasbeen demonstrated in both nonhuman primates and humans (18,19). Thus, transmission of �-amyloid-inducing activity to pa-tients through the use of neurosurgical instruments contami-nated with �-amyloid-containing brain extract is a theoretical

Table 2. Induction of cerebral �-amyloidosis by implantationof a stainless steel wire

InoculationNumber of mice withA� deposits/total mice

WT-extract-coated wire 0/4Tg-extract-coated wire 8/8Tg-extract-coated wire after 10 min at 95 °C 4/4Tg-extract-coated wire after plasma

sterilization0/4

Stainless steel wires were incubated in A� -containing extract from agedtransgenic APP23 mice (Tg extract) or age-matched nontransgenic wild-typecontrol mice (WT extract) for 16 h at room temperature and then air-dried. Forheat-denaturation, wires were boiled in PBS for 10 min at 95 °C. Plasmasterilization was done in a STERRAD sterilization system. Wires were im-planted unilaterally into the hippocampus and overlaying cortex. Four monthsafter implantation, mouse brains were analyzed immunohistologically usingan antibody against human A�. The number of animals with induced A�

deposits out of the total number of animals inoculated per group is indicated.

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possibility, particularly if the instruments are improperly cleanedand sterilized. Such transmission in humans has not been doc-umented, but it may be masked by a very long incubation time.The exogenous induction of cerebral �-amyloidosis has beenfound to take several years in nonhuman primates (i.e., marmo-sets) (12, 13), and thus it may take decades in humans, obscuringcausal links between exposure and illness. Moreover, althoughour data indicate that cerebral �-amyloidosis can be actuated intransgenic mice by contaminated steel, there remains no evi-dence indicating that the complete clinicopathologic manifes-tations of AD, which occur only in humans, can be transmittedin this way.

We have previously reported that heating of the amyloid-containing brain extract to 95°C does not entirely eliminate theinductive potential of the extract; this is also true for theamyloid-inducing agent bound to stainless steel. Plasma steril-ization of stainless steel wires has been shown to be an effectivemethod of prion inactivation (42), and we used this sterilizationtechnique in the present study to prove conceptually the possi-bility of inactivation of �-amyloid-inducing activity on the wire.Further identification of the conditions required to eliminate theamyloid-inducing activity of contaminated surgical instrumentsappears to be necessary.

Materials and MethodsMice. APP23 transgenic mice (22) were backcrossed with B6 mice for �10generations [C57BL/6J-Tg(Thy1-APPK670N;M671L)] (23). In all experiments, 2- to5-month-old male or female mice were used. At this age, APP23 mice do notyet deposit A� in the brain (11, 22). All mice were kept under specific patho-gen-free conditions. All experimental procedures were conducted in accor-dance with the veterinary office regulations of Baden-Wurttemberg (Ger-many) and Basel (Switzerland) and approved by the local Animal Care and UseCommittees.

Brain Tissue Extracts. Unless stated otherwise, brain extracts were derivedfrom aged (22- to 28-month-old) amyloid-depositing male or female APP23transgenic mice and age-matched nontransgenic, control mice. The cerebel-lum and lower brainstem were removed, and the forebrains were frozen in dryice and stored at �80 °C. Tissue was then homogenized at 10% (wt/vol) in PBS,vortexed, sonicated for 3 � 5 s, and centrifuged at 3,000 � g for 5 min (11). Thesupernatant was then aliquoted and frozen. For all experiments, the 10%extract was used unless stated otherwise.

A� Quantification in the Extract by Electrochemiluminescence-Linked Immuno-assay. A� levels were determined using the MSD 96-well MULTI-SPOT Human(6E10) A� Triplex Assay (Meso Scale Discovery). Extracts were treated withformic acid (minimum 95%), sonicated for 30 s on ice, and centrifuged at25,000 � g for 1 h at 4 °C. Supernatants were neutralized (1 M Tris base, 0.5 MNa2HPO4, 0.05% NaN3). A� detection was performed according to the man-ufacturer’s instructions. The plates were read on a Sector Imager 6000.

A� Quantification by Western Blot Analysis. Total A� levels were determinedon 10–20% Novex Tricine Gels using Novex Tricine sample buffer includingreducing agent (Invitrogen). To determine A�40 and A�42 levels, 10% bicine-Tris, 8 M urea SDS/PAGE was used as described previously (43). To measure A�

concentrations on wires, the wires were heated in 100 �L of sample buffer ina 1.5-mL microfuge tube at 95 °C for 5 min. Synthetic A�1–40 and A�1–42(American Peptide) were used as controls. Proteins were transferred onto anitrocellulose membrane, probed with 6E10 antibody (Signet Pathology Sys-tems), and visualized with chemiluminescence using SuperSignal West Pico(Thermo Scientific). Densitometric values of band intensities were analyzed usingthe public domain software ImageJ, version 1.34 (http://www.rbs.info.nih.gov/ij).

Stereotaxic Injection of Extract Into Different Brain Regions. APP23 host micewere anesthetized with a mixture of ketamine (100 mg/kg body weight) andxylazine (10 mg/kg body weight) in saline solution. Stereotaxic injections of2.5 �L of brain homogenate were placed with a Hamilton syringe into thehippocampus (AP, �2.5 mm; L, �2. 0 mm; DV, �1.8 mm), parietal cortex (AP,�1.0 mm; L, �2.0 mm; DV, �1.2 mm), striatum (AP, �1.0 mm; L, �2.0 mm; DV�3.3 mm), entorhinal cortex (AP, �3.6 mm; L, �3.8 mm; DV, �4.5 mm), orolfactory bulb (AP, �4.6 mm; L, �1.3 mm; DV, �1.0 mm). For all regions exceptthe olfactory bulb, the injections were bilateral. The rate of injection was 1.0

�L/min, and the needle was kept in place for 2 min after injection before beingslowly withdrawn. The surgical area was cleaned with sterile saline solution,the incision was sutured, and the mice were monitored until recovery fromanesthesia.

Oral Feeding of Extract. APP23 transgenic mice were given 500 �L of brainextract orally daily for 5 days. The extract was delivered via a flexible polypro-pylene catheter placed over the tongue about 1–2 cm into the esophagus. Halfof the mice were given human brain tissue extracts derived from an autopsysample of the superior frontal gyrus from an 85-year-old female AD patient,and the other half were given the aforementioned APP23 mouse brainextract.

Intravenous Inoculation. For i.v. injections, APP23 mouse brain extract wasfurther diluted 1:100 in sterile PBS, yielding a 0.1% extract. APP23 transgenicmice were given five 200-�L injections of the diluted extract within a maxi-mum of 10 days, corresponding to a total of 10 �L of 10% mouse brain extract.

Intranasal Inoculation. Host mice were anesthetized with a mixture of ket-amine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) in salinesolution. With the mouse held upright, mouse brain homogenate was pipet-ted dropwise directly onto one nostril, and inhalation was monitored. A totalof 10 �L of homogenate was given within 5 min, followed by a break of 10 minand then inoculation of the other nostril. This procedure was then repeated,so that each mouse received a total of 40 �L (20 �L per nostril). Betweenapplications, the mice were kept under an infrared warming lamp.

Intraocular Inoculation. Host mice were anesthetized with a mixture of ket-amine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) in salinesolution. Each mouse received a unilateral injection of 1 �L of mouse brainhomogenate into the vitreous cavity using a 32-gauge needle attached to a10-�L Hamilton syringe. After injection, the needle was left in the vitrealchamber for 1 min to minimize leakage of the inoculum.

Intracerebral Stainless Steel Wire Inoculation. Stainless steel wire segments(0.25 mm diameter, 2 cm long) were autoclaved before coating. The sterilestainless steel wires were placed in 0.5-mL microfuge tubes containing 50�L of brain homogenate (10%) for 16 h at room temperature and thenair-dried. The wires were either heated in PBS at 95 °C for 10 min orplasma-sterilized before implantation. To do this, the wires were sealed ina STERRAD Tyvek pouch and sterilized in a STERRAD 100 S hydrogenperoxide gas plasma sterilizer (Advanced Sterilization Products) using theroutine long cycle as programmed by the manufacturer (43). The wireswere then implanted unilaterally into the hippocampus using a stereotaxicapparatus (AP, �2.5 mm; L, �2.0 mm; DV, �1.8 mm). The wire was insertedthrough a hole in the skull, and the dorsally projecting end was adhesivelybound to the skull and sealed using GLUMA Comfort Bond light-curing,one-component adhesive (Heraeus).

Serum Titers of A� Antibodies. Antibody titers were monitored in bloodsamples collected by cardiac puncture at the time of sacrifice using standardELISA procedures (11).

Histology and Immunohistochemistry. Brains were immersion-fixed for 48 h in4% paraformaldehyde (PFA) in PBS, then cryoprotected in 30% sucrose. Thei.v.-injected mice and mice with stainless steel wire implants were killed byperfusion with PBS, followed by 4% PFA under deep ketamine/xylazine an-esthesia (ketamine 400 mg/kg, xylazine 40 mg/kg). The perfused brains wereimmersion-fixed for an additional 24 h in 4% PFA, then cryoprotected in30% sucrose. Serial 25-�m-thick coronal sections were cut using a freezing-sliding Microtome and immunohistochemically stained as described previ-ously (44). Polyclonal antibodies NT12 [similar to NT11 (22)] or DW6 (cour-tesy of D. Walsh) to A� were used. Congo red staining was done accordingto standard protocols.

ACKNOWLEDGMENTS. We thank L. Behrends, J. Coomaraswamy, I. Guhl,S. Kaser, J. Odenthal, U. Obermuller, T. Rasse, C. Schafer (all of Hertie Institute,Tubingen), P. Mauz (Tubingen), M.-J. Runser (Basel), M. Meyer-Luhmann(Boston, MA), D. Walsh (Dublin), and F. Heppner (Berlin) for their experimen-tal help. This work was supported by grants from the Alzheimer Association(ZEN-06–27341, to M.J.), the German National Genome Network (BMBF-NGFNPlus, to M.J.), the German Competence Network on Degenerative De-mentias (BMBF-01GI0705, to M.J.), the Prof. Max Cloetta and Bonizzi-ThelerFoundation (to M.H.), and the National Institutes of Health (RR-00165,to L.C.W.).

12930 � www.pnas.org�cgi�doi�10.1073�pnas.0903200106 Eisele et al.

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