Proc. Natl. Acad. Sci. USAVol. 90, pp. 6825-6828, July 1993Neurobiology

Alzheimer disease A68 proteins injected into rat brain inducecodeposits of j8-amyloid, ubiquitin, and al-antichymotrypsin

(tau/senile plaques/neuroflbrillary tangles)

RYONG-WOON SHIN, GREGORY T. BRAMBLETT, VIRGINIA M.-Y. LEE, AND JOHN Q. TROJANOWSKI*

Department of Pathology and Laboratory Medicine, Division of Anatomic Pathology, University of Pennsylvania School of Medicine, Hospital of theUniversity of Pennsylvania, Maloney Basement, Room A009, Philadelphia, PA 19104-4283

Communicated by Eliot Stellar, April 30, 1993

ABSTRACT Aberrandy phosphorylated tau proteins (i.e.,A68 or PHF-tau) and (-amyloid or A4 (13A4) peptides are majorcomponents of pathologic lesions in Alzheimer disease (AD).Although A68 and 3A4 colocalize in AD neurofibrillary tangles(NFTs) and amyloid-rich senile plaques (SPs), the mechanismsleading to the convergence of A68, lA4, and other proteins inthe same AD lesions are unknown. To probe the biologicalproperties of A68 in vivo, and to assess interactions of A68 withendogenous proteins in the rodent brain, we injected A68,dephosphorylated A68 (DEP-A68), and normal adult human tauprotein into the hippocampus and neocortex of rats. In markedcontrast to DEP-A68 and tau, A68 resisted rapid proteolysis andinduced codeposits of three rodent proteins-i.e., lA4, ubiqui-tin, and al-antichymotrypsin (ACT)-that accumulate in ADNFTs and SPs together with A68. These fidings suggest thatA68 may interact with PA4, ubiquitin, and ACT in neuronalperikarya as well as in the extraceflular space after release ofA68from degenerating neurons. The model system described herewil facilitate efforts to elucidate mechanis leading to theconvergence of A68, PA4, ubiquitin, and ACT in hallmrklesions of AD.

The most prominent abnormalities in the Alzheimer disease(AD) brain are neurofibrillary tangles (NFTs), neuropilthreads, amyloid-rich senile plaques (SPs), and the massiveloss of selected populations of central nervous system (CNS)neurons (1-3). Efforts to elucidate the composition of thepaired helical filaments (PHFs) that dominate in NFTs as wellas the subunits ofamyloid fibrils in SP cores have shown thatabnormally phosphorylated tau proteins (i.e., A68, PHF-tau)are the building blocks of PHFs and that f-amyloid (or A4)peptides (,8A4) form AD amyloid fibrils (1-3). Although A68and ,A4 assemble into morphologically distinct filamentsthat are the predominant structural elements in NFTs andSPs, respectively, a wide variety of other proteins accumu-late in these lesions. For example, ubiquitin and al-antichymotrypsin (ACT) colocalize with both A68 and ,BA4 inNFTs and SPs (4-13). How A68, f3A4, ubiquitin, and ACTaccumulate and converge in NFTs and SPs remains elusive.Furthermore, it is not entirely clear how A68 is generatedfrom normal adult tau protein or how ,3A4 is cleaved fromwithin the larger transmembrane ,BA4 precursor proteins(,BAPPs). Indeed, the selective loss ofCNS neurons providesa straightforward explanation for the clinical manifestationsof AD, but it is still uncertain how A68 and ,BA4 contributeto the degeneration ofneurons in AD. These issues have beendifficult to resolve due to the paucity of in vivo and in vitromodel systems in which to examine the pathobiology of A68and f3A4. For these reasons, we sought to develop a rodentmodel system in which to probe the biological properties ofA68 in vivo. Here we show that injections of A68 into the rat

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brain result in delayed proteolysis ofA68 and codeposition ofrat brain-derived B3A4, ubiquitin, and ACT, all of whichaccumulate in two hallmark brain lesions of AD-i.e., NFTsand SPs.

MATERIALS AND METHODSIsolation and Characterization of A68, Dephosphorylated

A68 (DEP-A68), and Normal Tau. A68 was purified from thebrains of four AD patients and one Down syndrome patientwith AD (14-16). To render A68 water soluble for injection,A68 was further purified as follows. After sucrose gradientcentrifugation (14, 15), the 1.25-2.0 M and 2.25-2.5 Msucrose fractions were extracted in 2 M guanidine isothio-cyanate at 37°C for 60 min, and the guanidine-insolublematerial was removed by further centrifugation for 30 min at100,000 x g. The supernatant was exhaustively dialyzedagainst distilled water, and the water-insoluble material wasremoved by centrifugation once again. The resulting super-natant was lyophilized and used for injection into rats as wellas for the generation of DEP-A68. This guanidine-extracted,water-soluble supernatant was shown by Western blots (seebelow) to contain purified A68. Although the A68 preparationcontained PHFs (14) prior to guanidine extraction, the guani-dine-extracted, water-soluble A68 did not reassemble intoPHFs or straight filaments in vitro (data not shown), asmonitored by negative staining and electron microscopy (14).DEP-A68 was generated from A68 by enzymatic dephos-phorylation after overnight incubation in type III-N Esche-richia coli alkaline phosphatase (20 units/ml) at 37°C asdescribed (14). Normal adult human tau was prepared exactlyas described (14, 15). Aliquots of the A68, DEP-A68, andnormal adult human tau preparations that were used forinjection were analyzed by gel electrophoresis and by West-ern blots with epitope-specific antibodies to A68 and tauaccording to described methods (2, 3, 14, 15). The anti-tauand anti-A68 antibodies used in this study included Alz5O toresidues 2-10; T60 to residues 119-150; T14 to residues141-178; T46 to residues 404-441; Taul, which recognizestau and DEP-A68, but not A68, and binds to a nonphospho-rylated epitope within residues 189-207; T3P, which recog-nizes A68, but not tau or DEP-A68, and binds to an epitopewithin residues 389-402 that contains a phosphate at Ser-396;and PHF1, which is similar to the T3P antiserum (for furtherinformation on these antibodies, see refs. 2, 3, and 14-21 andcitations therein; the numbering system for the amino acidsin tau referred to here is based on the largest tau isoform, asdescribed in ref. 22).

Abbreviations: ACT, al-antichymotrypsin; AD, Alzheimer disease;,3A4, ,B-amyloid/A4 peptide; BAPP, ,3A4 precursor protein; DEP-A68, dephosphorylated A68; mAb, monoclonal antibody; NFT,neurofibrillary tangle; PHF, paired helical filament; SP, senileplaque.*To whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 90 (1993)

Injections of A68, DEP-A68, and Tau into Rat Brain. FemaleSprague-Dawley rats (170-280 g) were anesthetized by in-traperitoneal injections of Ketamine (87 mg/kg) and xylazine(13 mg/kg), prepared for surgery, and placed in a stereotaxicinstrument (Kopf, Tujunga, CA). Multiple A68 samples fromeach of the five brains were injected into cerebral cortex andhippocampus at three separate sites on one side, while controlrats received similar injections of DEP-A68 and normal adulthuman tau. The three stereotaxic injection sites in each ratbrain were determined by using system B of Pellegrino et al.(23): -2.0 mm rostral-caudal (RC); 2.0 mm medial-lateral(ML); 3.0mm dorsal-ventral (DV); -3.0mm RC; 3.0mm ML;3.0 mm DV, and -4.0 mm RC; 4.0 mm ML; 4.0 mm DV. AUinjections were performed over 10 min with a 10-pl Hamiltonsyringe. After injection, the needle was left in place for another10 min and then was slowly removed. The concentration ofA68, DEP-A68, and tau was estimated by protein assay andimmunoblots and approximately similar amounts ofeach (i.e.,3 ,ug) were injected in 3 ,ul of phosphate-buffered saline (PBS)at each site.

Following different postinjection survival times, the ratswere deeply anesthetized and sacrificed by perfusion withPBS. The brains were removed and fixed by overnightimmersion in 70% ethanol and 150 mM NaCl. The postinjec-tion survival times for rats injected with A68, DEP-A68, ortau were as follows: A68, 1 hr (n = 5), 2 days (n = 5), 1 week(n = 6), 4 weeks (n = 5), 8 weeks (n = 3), 13 weeks (n = 2);DEP-A68, 1 hr (n = 4), 2 days (n = 4), 1 week (n = 4); normaladult tau, 1 hr (n = 4), 2 days (n = 3), 1 week (n = 3), 4 weeks(n = 3), 8 weeks (n = 1), 13 weeks (n = 1). Immunoelectronmicroscopy was performed on two additional rats that sur-vived 1 day after injections ofA68. These rats were sacrificedand perfused as described above and small pieces of thecortical injection sites were immersed for 3 hr in fixativecontaining glutaraldehyde (1%) and paraformaldehyde (4%).

Light and Electron Microscopic Immunohistochemical Pro-cedures. The methods for tissue processing and light micro-scopic immunohistochemical analysis have been described(7, 14, 15, 24). The antibodies used for immunohistochemis-try included those described above to tau and A68, as well asa monoclonal antibody (mAb) to ubiquitin (mAb5lO1, Sig-ma), six different mAbs and polyclonal antisera to (BA4(UP107, 2332, 1280, 2,84, AMY33, lOD5), and antibodies tonon-,BA4 domains in the f3APPs (LN21 and LN27, which bindto epitopes within the first 200 residues of the 3APPs;anti-C15, which binds to the last 15 residues in the ,BAPPs).The specificities ofthese antibodies have been described (seerefs. 7, 14, and 24-27 and citations therein). Finally, anti-

7 .

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B D

A B C )D E F

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FIG. 1. Biochemical and Western blot data on A68, DEP-A68,and normal adult human tau. (A) Coomassie brilliant blue-stainedSDS/l0o polyacrylamide gel. (B-F) Nitrocellulose replicas of gelscorresponding to lanes 1-3 inA (B), lanes 1 and 2 inA (C and D), andlane 1 in A (E and F). Lanes: 1, A68; 2, DEP-A68; 3, normal adulthuman tau. Molecular mass markers are 110, 84, 47, and 33 kDa (leftin A). Antibodies used to probe the gel replicas included T14 (B), T3P(C), Taul (D), Alz5O (E), and T60 (F).

bodies to ACT also were used (ICN and DAKO). Theabsorption of the antibodies for control experiments wasperformed as described (7, 28). Preembedding immunoelec-tron microscopy was performed with a Hitachi 600 electronmicroscope using the T3P and the anti-,3A4 antisera with5-nm gold particles (Amersham) and silver intensification asdescribed (14, 27, 29, 30). Formic acid pretreatment was usedto enhance 13A4 immunoreactivity (7, 31).

RESULTSThe properties ofA68, DEP-A68, and purified normal humantau were established by SDS/PAGE (Fig. 1A) and Westernblots (Fig. 1 B-F) using the panel of anti-tau and anti-A68antibodies described above. As shown in Fig. 1, the A68,DEP-A68, and adult tau proteins that were injected into ratbrains exhibited the distinct biochemical and immunochem-ical properties described in earlier reports (1-3, 14, 15). Forexample, A68 migrated more slowly than normal adult humantau and DEP-A68 (Fig. 1A), but each of these distinct tauphosphoisoforms was recognized by all of the antibodies thatbind to phosphate-independent tau epitopes (Fig. 1 B, E, andF). In contrast, the T3P antiserum only recognized A68 (Fig.1C), and the Taul mAb recognized normal tau (data notshown) and DEP-A68 but not native A68 (Fig. 1D).

Aliquots of each of these samples were injected into ratbrains and near serial sections ofthese brains (extending over

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FIG. 2. Photomicrographs of injected A68 (A-D), DEP-A68 (E and F), and normal adult human tau (G) at 1 hr (A, E, and G), 2 days (B andF), 1 week (C), and 4 weeks (D) postinjection survival times. Sections were probed with mAb T14. Arrows in F and G identify injection site.(A, B, F, and G, xll; C, x53; D, x85; E, x27.)

6826 Neurobiology: Shin et al.

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Proc. Natl. Acad. Sci. USA 90 (1993) 6827

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the entire extent of the three injection sites) were examinedby immunohistochemistry with the same anti-tau antibodiesdescribed above. At 1 hr and 2 days after the injections ofA68, aggregates of amorphous material were seen (Fig. 2 Aand B), and they gradually disappeared from 1 to 13 weekspostinjection as macrophages and reactive astrocytes accu-mulated in the injection sites (Fig. 2 C and D). In contrast,DEP-A68 (Fig. 2 E and F) and normal human tau (Fig. 2G)were eliminated from the injection sites within 2 days.

Specific domains in A68 were eliminated from the injectionsites in a distinct temporal sequence as defined by usingepitope-specific antibodies. The tau epitope containing phos-phorylated Ser-396 (i.e., the epitopes recognized by PHF1and T3P) persisted the longest in rat brain, whereas N-ter-minal tau epitopes [recognized by Alz5O (Fig. 3B) and T60]were eliminated completely at 1 hr and 2 days postinjection,respectively, even though the Alz5O and T60 epitopes werepresent in A68 before injection (Fig. 1 E and F). Tau epitopeslocated downstream of the Alz5O and T60 sites (recognizedby T14, Taul, T46, and Tau2) were extinguished moregradually, but none survived as long as the tau domaincontaining the phosphorylated Ser-396 site. As predictedfrom previous studies (14), Taul stained the A68 aggregatesonly after the sections were enzymatically dephosphory-lated, suggesting that rat brain phosphatases do not dephos-phorylate A68. The elimination of these tau epitopes in A68may involve the orchestrated activity ofa number ofdifferentcells, proteases, and other mediators. Notably, the temporalsequence of events described here has parallels in situ and invitro, where N-terminal tau epitopes are eliminated fromextracellular NFTs released by dying neurons (32) and fromisolated PHFs subjected to in vitro proteolysis (33, 34).To determine whether injected A68 interacted with other

proteins known to colocalize with A68 in NFTs and/or neu-ritic SPs in the AD brain (4-13), we probed adjacent sectionsfrom the same brains with antibodies to ubiquitin, fA4, andACT. At 1 hr postinjection, weak or no ubiquitin and ACTstaining in the A68 aggregates was noted, while intenselyimmunoreactive 3A4 was present throughout the aggregates.Immunoreactivity for these same proteins was seen consis-tently throughout the A68 aggregates in all five rats sacrificed2 days postinjection (Fig. 3 C, E, and G) and the immunore-activity for these proteins was removed by absorbing theantibodies with the corresponding antigen (Fig. 3 D, F, and H,respectively). These findings do not reflect contaminants inthe A68 preparations since no ubiquitin, PA4, or ACT immu-noreactivity was detected in aliquots of A68 analyzed byWestern blots or by ELISA with the same antibodies used inthe immunohistochemical studies. No deposition of fA4,

ubiquitin, or ACT immunoreactivity was observed in ratbrains injected with DEP-A68 or normal adult tau, except forl3A4 with DEP-A68 only at 1 hr postinjection. Furthermore,human high molecular weight neurofilament subunits and fetaltau also were injected into rat brains as additional controls, butthe results of these experiments were identical to those seenwith normal adult tau (R.-W.S., V.M.-Y.L., and J.Q.T.,unpublished data). Ubiquitin, ,BA4, and ACT immunoreactiv-ity colocalized with A68 as long as the A68 remained in thebrain (i.e., for 4 weeks). 13A4 and ACT immunoreactivity wasmore intense and abundant than the A68 and ubiquitin immu-noreactivity at 1-4 weeks, and immunoreactive ,3A4 and ACTremained at the injection site for up to 13 weeks postinjection.In contrast to the anti-j3A4 antibodies, none of the antibodiesspecific for N- and C-terminal epitopes in the I3APPs stainedthe A68 aggregates, and none ofthe aggregates was Congo redor Thioflavin S positive.

Finally, immunoelectron microscopy performed on corticalinjection sites containing A68 showed extracellular amorphousmaterial and tightly bundled 10- to 25-nm straight filaments. Theamorphous material was immunoreactive with both theT3Pandthe anti-PA4 antisera (Fig. 4), while the filaments were immu-

FIG. 4. Immunoelectron photomicrographs ofA68 injection sitesfollowing a 1-day survival labeled with antisera to A68 (T3P; A) or

,(A4 (1280; B) and silver-intensified immunogold particles. Thenucleus (N) ofthe intact cell in the lower right ofA andB is indicated.(A, x26,700; B, x44,500.)

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Neurobiology: Shin et al.

Proc. Natl. Acad. Sci. USA 90 (1993)

Sequential Events In Rat Brains Injected With A68

Injection 1 hour 2 days 13wks

AlzS0 T60 UBQ Tau2 T46 Taul T14 T3PPHFI.

A68 A68 A68,BAP I BIAP 1

FIG. 5. Schematic of the sequential events that occur afterinjections of A68 into rat brains over time (indicated by horizontalarrows) from 1 hr (left) to 13 weeks (right) postinjection. After A68forms clustered aggregates, it is gradually degraded and removedfrom the injection site. However, the tau and A68-specific epitopes(identified in the schematic by the code names of the antibodies thatrecognize them) in the injection sites are extinguished (indicated bydiagonally oriented arrows that point to an epitope-specific antibody)in a temporal sequence similar to that suggested by in vitro and in vivostudies of NFTs (32-34). During the early postinjection time period,A68 induces codeposits of ,BA4 (fBAP), ubiquitin (UBQ), and ACT(solid-line rectangles). Subsequently, immunoreactive A68 and ubiq-uitin are lost (dashed-line rectangles), but immunoreactive 13A4 andACT persist (solid-line rectangles) in the injection site.

noreactive with the T3P antiserum (Fig. 4A). Thus, at leastsome of the guanidine-extracted A68 proteins reassembled intofilaments similar to the staight filaments found along withPHFs in NFTs and dystrophic neurites (1-3).

DISCUSSIONThis study directly examines the biological properties ofhuman AD PHF proteins (i.e., A68) injected into rat brain. Inthe in vivo model system described here, we show thatabnormally phosphorylated A68 is more resistant to prote-olysis than" DEP-A68 and normal adult tau. More signifi-cantly, after injection into the rat brain, A68 assembled intostraight filaments and rapidly induced codeposits of ratbrain-derived 1A4 as well as ubiquitin and ACT. Fig. 5

illustrates the timing of the interactions of injected A68 withrodent 8A4, ubiquitin, and ACT as well as the temporalprofile for the sequential loss of specific A68 epitopes in therat brain injection sites. Since recent studies showed thatsoluble ,BA4 is secreted by cells in the normal and AD brain(35, 36), the rodent ,3A4 that accumulates at the A68 injectionsites in our experimental paradigm could come from theextracellular 13A4. The work described here is significantbecause it provides evidence that A68 specifically interactswith f3A4 in vivo. Thus, the accumulation of A68 as PHFs inneurons and the release ofA68 from degenerating neurons ortheir processes may serve as a nidus for the deposition of /A4(in addition to ubiquitin and ACT) in NFTs and in the coronaof neuritic SPs. However, it is uncertain whether A68 con-tributes to the formation of diffuse deposits of BA4 sincethese deposits are not thought to be associated with abnormalprocesses rich in A68 (7, 8).

Currently, mechanistic hypotheses that attempt to explainthe convergence of A68, PA4, ACT, and ubiquitin in SPs andNFTs (4-13), or the pathogenesis of SPs, NFTs, and otherAD lesions, are nearly impossible to validate solely bystudying postmortem human brain tissues. Hence, the ex-perimental approach described here may set the stage forfuture studies of the mechanisms whereby ,BA4 and A68accumulate together in NFTs and SPs. Indeed, this and otherin vivo models of AD brain pathology may provide opportu-nities to clarify the role ofA68 and A4 in the dysfunction anddegeneration of neurons in the AD brain.

We thank J. Martin, A. G. DiDario, and J. Mantione for assis-tance; Drs. J. Eberwine, S. Pleasure, M. L. Schmidt, and Ms. J. I.

Ko for comments; colleagues in the Departments of Pathology andLaboratory Medicine, Neurology, Psychiatry, and the Penn Alzhei-mer Center for assistance in obtaining tissue samples; and the

families of the patients. Drs. L. I. Binder, P. Davies, B. Greenberg,S. G. Greenberg, B. T. Hyman, D. J. Selkoe, and S. G. Younkinkindly provided antibodies, and Dr. H. Rubin and B. Coopermandonated recombinant human ACT. Dr. G. G. Pietra and Ms. J.Minda provided assistance with electron microscopy. This work wassupported by grants from the National Institutes of Health.

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