Characterization of stable complexes involving apolipoprotein E and the amyloid β peptide in...

Neuron, Vol. 15, 219-228, July, 1995, Copyright 0 1995 by Cell Press

Characterization of Stable Complexes Involving Apolipoprotein E and the Amyloid B Peptide in Alzheimer’s Disease Brain

Jan NPslund, l Johan Thyberg,t Lars 0. Tjernberg,’ Christer Wernstedt,+ Anders Ft. Karlstrom, Nenad Bogdanovic,Il Samuel E. Gandy,# Lars Lannfelt,ll Lars Terenius,’ and Christer Nordstedt’ ‘Laboratoryof BiochemistryandMolecular Pharmacology Section of Experimental Alcohol and

Drug Addiction Research Department of Clinical Neuroscience tDepartment of Cell and Molecular Biology Medical Nobel Institute Karolinska Institute S-171 77 Stockholm Sweden *Ludwig Institute for Cancer Research Biomedical Center S-751 24 Uppsala Sweden BDepartment of Biochemistry Biopharmaceuticals, Pharmacia S-l 12 87 Stockholm Sweden IIDepartment of Geriatric Medicine Huddinge Hospital S-l 41 86 Stockholm Sweden #Department of Neurology and Neuroscience Cornell University Medical College New York. New York 10021

Summary

Genetic evidence suggests a role for apolipoprotein E (apoE) in Alzheimer’s disease (AD) amyloidogenesis. Here, amyloid-associated apoE from 32 AD patients was purified and characterized. We found that brain amyloid-associated apoE apparently exists not as free molecules but as complexes with polymers of the amyloid g peptide (Af3). Brain Af%apoE complexes were detected irrespective of the apoE genotype, and similar complexes could be mimicked in vitro. The fine structure of purified A3-apoE complexes was fibrillar, and immunogold labeling revealed apoE immunoreactivity along the fibrils. Thus, we conclude that AP-apoE complexes are principal components of AD-associated brain amyloid and that the data presented here support a role for apoE in the pathogenesis of AD.

Introduction

Alzheimer’s disease (AD) is an aging-related dementia without known treatments. One invariant neuropathologi- cal feature of AD is deposition of intracerebral and menin- geal amyloid (reviewed in Selkoe, 1991). The predominant component of AD-associated amyloid is the amyloid 6 peptide (A& which is mainly 40-42 amino acid residues in

length (Glenner and Wong, 1984; Masters et al., 1985; Roher et al., 1993; Naslund et al., 1994). A8 is derived by proteolytic processing of an integral membrane protein, the j3-amyloid precursor protein (Kang et al., 1987). The A8 peptide is detectable in soluble form in plasma and cerebrospinal fluid (Seubert et al., 1992) and in medium from cell cultures (Haass et al., 1992) whereas in amyloid deposits, the A6 peptide forms an insoluble and protease- resistant core (Masters et al., 1985). The physicochemical properties of A6 enable it to form polymers and fibrils spon- taneously (Kirschner et al., 1987; Nordstedt et al., 1994). It has, however, been suggested that other proteins present in, or in the vicinity of, the amyloid may act as”pathological chaperones,” facilitating A8 fibrillogenesis and deposition (Wisniewski and Frangione, 1992). A number of proteins unrelated to A8 have been detected immunohis- tochemically in AD-associated amyloid. These include heparan sulfate proteoglycans (Snow et al., 1990), al-antichymotrypsin (ACT; Abraham et al., 1988), amyloid P component (Coria et al., 1988) complement proteins (Roze- muller et al., 1989), and, most notably, apolipoprotein E (apoE; Namba et al., 1991; Wisniewski and Frangione, 1992).

apoE is a 299 residue protein with a molecular mass of approximately 34 kDa. In plasma, apoE associates with lipoproteins and mediates transport of lipids and choles- terol into the cells by binding to the low density lipoprotein receptor or the low density lipoprotein receptor-related protein/az-macroglobulin receptor (for review, see Weis- graber, 1994). apoE exists in three major isoforms, apoE2, apoE and apoE4, which are products of the ~2, ~3, and ~4 alleles, respectively. The isoforms differ in arginine or cysteine content at positions 112 and 158. Recent genetic studies suggest a role for apoE in the pathogenesis of AD. The frequency of the ~4 allele is significantly higher in sporadic (Mayeux et al., 1993; Rebeck et al., 1993; Saun- ders et al., 1993) and familial late onset AD (Corder et al., 1993; Strittmatter et al., 1993a) than in the general population. In addition, brain tissue from AD patients car- rying the ~4 allele contains more amyloid than brain tissue from AD patients with other apoE genotypes (Rebeck et al., 1993; Schmechel et al., 1993; Berr et al., 1994) implying a direct role for apoE in cerebral amyloidogenesis. How apoE might promote accumulation of amyloid and development of AD is still not understood. Recent in vitro experiments suggest that both apoE and apoE bind A8 peptide variants in a f3-mercaptoethanol-sensitive manner (Strittmatter et al., 1993b; LaDu et al., 1994). The complexes formed are stable to boiling in SDS, implying strong intermolecular binding. Electron microscopic analysis of these in vitro complexes demonstrated that both apoE and apoE interact with A8 to form monofibrillar structures, distinct from the twisted ribbons formed by synthetic A8 alone (Sanan et al., 1994). It has also recently been shown that apoE (Maet al., 1994; Wisniewski et al., 1994) and ACT (Ma et al., 1994) enhance polymerization of A8 in vitro, thereby providing a direct link between these pos- tulated pathological chaperones and amyloid formation.

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AD AD Ctrl Ctrl

6ElO + + + + 2El + + + +

200-

97.4- 69- 46- 30-

21.5- 14.3- 6.5- 3.4- -

Figure 1. lmmunoblot Analysis of Amyloid-Enriched HFIP Extracts from AD Patients and Nondemented Controls Amyloid-enriched HFIP extracts from 2 AD patients and 2 nondemented controls, respectively, were separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and probed with A6-specific antibody 6ElO or apoE-specific antibody 2El. The total amount of protein in each lane was 60 pg. Positions of the molecular mass markers in kilodaltons are shown at left.

Amyloid-associated apoE purified from human brain tissue has not previously been studied biochemically. We have extracted AD-associated amyloid deposits from pa- renchymal brain tissue and report here that a C-terminal fragment of apoE apparently exists in a complex with A6 polymers of varying sizes. Considerably more AD-apoE- immunoreactive complexes were recovered from AD brains than from brains of elderly, nondemented controls. The A6-apoE complexes were detectable in brain extracts from all 32 AD patients examined, without apparent rela- tionship to the apoE genotype (~31~3, .53/~4, or ~41~4). The purified AD-apoE complexes were resistant to various agents previously shown to dissociate A8 polymers, implying strong intermolecular association. When viewed by negative stain electron microscopy, the AD-apoE complexes appeared as bundles of tightly packed amyloid fibrils morphologically similar to those of noncomplexed A8 purified from AD brain. lmmunogold labeling of the purified complexes demonstrated that apoE immunoreactivity was distributed along the fibrils and fibril bundles. Complexes formed in vitro between synthetic A6 and recombinant apoE showed chromatographic, immunoreactive, and morphological features indistinguishable from the Aj3- apoE complexes purified from AD brain.

Results

Detection of Highly Insoluble AP-apoE Complexes in AD Brain Tissue Amyloid-enriched brain tissue fractions from 32 AD patients and 4 nondemented controls were extracted with hexafluoro-Ppropanol (HFIP). Extracted proteins from 2 representative patients in each group were analyzed by immunoblotting using the A8-specific antibody 6E10 or the apoE-specific antibody2El (Figure 1). In the samples from the AD patients, but not from the nondemented controls,

a prominent band with an apparent molecular mass of - 4.5 kDa was labeled by antibody 6ElO. This species was identified as a mixture of intact and N-terminally truncated A8(1-40) and A8(1-42) variants by microsequencing and electrospray mass spectrometry (Naslund et al., 1994). In addition to small quantities of A8 oligomers, the AD samples contained high molecular mass forms of A8 ranging in size from 22 to 24 kDa to the separation limit of the gel, corresponding to A3 polymers of varying sizes. Immu- noblotting of the same extracts using the apoE-specific antibody 2El yielded a staining pattern very similar to that obtained with 6E10, except that 2El did not label monomeric and oligomeric A6 (Figure 1). Hence, 2El-immune reactive material was apparently comigrating with A8 polymers in SDS-PAGE. The immunoreactivity of both antibodies could be abolished by preabsorption with their respective antigens. The similar appearances of the AD- and apoE-immunoreactive material indicate that the two antibodies may have bound to the same structures, i.e., SDS-resistant complexes formed by apoE and polymers of A6. Hereafter, these structures will be referred to as A6-apoE complexes. As seen in Figure 1, the apparent mass of the smallest A8-apoE complex is 22-24 kDa, which is below the nominal 34 kDa mass of apoE, suggesting that the apoE component of the complexes is truncated (see below).

Purification of the Ap-apoE Complexes from AD Brain Tissue To enable biochemical and morphological studies of the AD-apoE complexes, a protocol for partial purification was developed. Proteins from an amyloid-enriched AD brain tissue fraction were first separated by gel filtration on a Superose 12 column (Figure 2A). Using this method, proteins smaller than 22 kDa (including monomeric and oligo- merit A6 peptide not associated with apoE) were effi- ciently separated from the remainder of the material. The amount of A8 immunoreactivity apparently residing in apoE-complexed form varied, but was densitometrically determined to constitute at least 50% of the total A6 immunoreactivity in each case (Figure 28). Fractions containing the Aj3-apoE complexes (fractions 2-3) were pooled and then subjected to reverse-phase liquid chromatography (RPLC; Figure 2C). Previous experiments had shown that noncomplexed A6 elutes at - 18% acetonitrile (ACN) in this RPLC system (data not shown). The Af3-apoE complexes eluted as a broad peak between 24% and 29% ACN (Figure 2D). It is noteworthy that all fractions containing A8 polymers also displayed apoE immunoreactivity. Fractions 7-l 0 from the RPLC sessions were pooled and used in the subsequent characterization and dissociation experiments.

Immunological and Biochemical Characterization of the Afl-apoE Complexes A set of different antibodies was used to investigate whether the A6-apoE complexes harbored molecules other than A6 and apoE and to rule out cross-reaction between antibodies. As seen in Table 1, the only antibodies labeling the complexes were those raised against A8 or

AD-apoE Complexes Purified from Human Brain 221

Figure 2. Fractionation of an Amyloid-Enriched HFIP Extract by Gel Filtration on a Superose 12 Column

(A) Absorbance profile at 280 nm of the HFIP extract eluted in 70% formic acid. Fractionation started after an elution volume of 6 ml. Mono- meric, noncomplexed A6 eluted at - 13.5 ml (fraction 9) in this system. (6) lmmunoblot analysisof the Superose 12fractions. Aliquots of each fraction were separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and probed with Af3-specific antibody 6ElO (closed circles) or apoE-specific antibody 2El (open circles). The resulting autoradiograms were quantified by densitometry. Scanning was performed over the full length of each lane, and therefore optical density values refer to the total immunoreactivity in each sample. (C) AB-apoE complexes recovered from the Superose 12 column were further purified by RPLC on a Vydac C4 column and developed with a linear gradient of buffer B (see Experimental Procedures for details). (D) Each RPLC fraction was probed with AD-specific antibody 6E10 (closed circles) or apoE-specific antibody 2El (open circles) and subjected to the same analysis as in (E).

apoE. Interestingly, antibody 1 D7, recognizing an epitope overlapping the LDL receptor-binding domain located between residues 142-158 of apoE, did not label the complexes. In control immunoblot experiments, 1 D7 intensely labeled recombinant apoE. This, together with the positive labeling obtained with antibody 3Hl (recognizing an epi-

Table 1. lmmunoreactivity of the AD-apoE Complexes As Determined by lmmunoblotting

Antibody Specificity Reactivity .-

MAB 6ElO AP + MAb 4G6 AP fa G504 AD +a Anti-A6 AP +a MAb 2El apoE + MAb 1048 apoE +b MAb 3Hl apoE i” MAb lD7 apoE MAb 22Cll APP MAb Al290 APP 369A APP -

MAb 56-1 APP (KPI) Anti-ACT ACT Anti-ubiquitin ubiquitin Anti-TTR l-m MAb tau-1 tau MAb AT-8 P-tau

a Similar staining as with 6ElO. ’ Similar staining as with 2El. ACT, rkantichymotrypsin; APP, 6-amyloid precursor protein; KPI, Kunitz protease inhibitor; P-tau, phosphorylated tau; TTFI, transthyretin.

tope between residues 220-272) suggests that the Af3- apoE complexes are composed of the C-terminal part of apoE. Although material reactive with antibodies to the f3-amyloid precursor protein, ACT, ubiquitin, and phosphorylated tau could be recovered from AD brain using the same extraction protocol, none was associated (i.e., copurified) with the AD-apoE complexes.

Microsequencing confirmed that A6 was a component of the complexes. A Lys-C digest contained two peptides with A6 sequences, one in the 0.1% trifluoroacetic acid (TFA)-soluble fraction and one in the formic acid-soluble fraction (see Experimental Procedures). The sequence obtained in the 0.1% TFA-soluble fraction was XXIIGLMV (eluting at 32% ACN), corresponding to residues 29-36 of Af3. In the formic acid-soluble fraction, the sequence XAEFRHDSGYEVH (eluting at 35% ACN), corresponding to residues l-l 3 of A6, was obtained. We were unable to obtain any sequence corresponding to apoE, either by trypsin or by Lys-C digestion. This was interpreted as evidence either that apoE was present in levels too low to allow microsequencing or that the apoE moiety of the complex adopts a conformation too dense to allow penetration of the proteases used.

Association of Ap-apoE Complexes by Strong Intermolecular Bonds It was hypothesized that the A6 and apoE components in the complexes might be separated if the material were exposed to strong dissociating agents. The A@apoE complexes were therefore treated with acids, bases, deter- gents, chaotropic or denaturing agents, or organic sol- vents (see Experimental Procedures), some of which have previously been shown to dissociate A6 polymers purified from AD brain (Masters et al., 1985). The incubates were separated by SDS-PAGE, electroblotted onto nitrocellu-

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Figure 3. Congo Red-Stained AP-apoE Complexes Demonstrate Green Eirefringence When Viewed under Polarized Light

Purified AD-apoE complexes were suspended in 50% ethanol containing 1% Congo red, smeared onto glass slides, and viewed under polarized light at a magnification of 200 x

lose membranes, and analyzed with antibody 6ElO. The criteria used when determining whether the complexes had dissociated were reduced amounts of AD-apoE complex immunoreactivity with a molecular mass higher than 22-24 kDa and appearance of detectable amounts of A6 immunoreactivity within the 3-5 kDa region. Either crite- rion would indicate that the A6 peptide had been released from the complexes. None of the agents employed induced any measurable dissociation.

AJI-apoE Complex Formation and apoE lsoforms Af3-apoE complexes were detected in brain extracts from all 32 AD patients studied. In 21 of the patients, the apoE genotype was known, showing the following distribution: Es/E3 (n = 8) Es/Ed (n = 12), and E4/E4 (fl = 1). No AD patient with an ~2 allele was present in this study, probably owing to the reported low frequency of this allele among AD patients (4.4%; Peacock and Fink, 1994).

Morphological Examination of Afi-apoE Complexes and Noncomplexed AP The tinctorial properties of the purified Af3-apoE com-

Figure 4. Negative Stain Electron Microscopy of Aggregated AB- apoE Complexes and Noncomplexed Af3 (A) The purified AD-apoE complexes were incubated in TBS for 1 hr, negativelystained with2% uranyl acetate, andexaminedinanelectron microscope. (6) Less dense fibril matrix found in another region of the same specimen as in (A). (C) Noncomplexed A!3 purified from AD brain tissue incubated and stained as in (A). Bars, 100 nm.

plexes were indistinguishable from those typical of tissue amyloid (Glenner, 1980); i.e., when stained with Congo red, they manifested green birefringence under polarized light (Figure 3). The fine structure of the Af3-apoE complexes was investigated further by electron microscopy. After negative staining with uranyl acetate, tight bundles of fibrils were found (Figure 4A). The strictly parallel ar- rangement of the fibrils resembles that previously observed in AD brain tissue (Davies and Mann, 1993). In some regions, the bundles had apparently separated, and finer substructures became evident (Figure 48). Thus, a web-like matrix could be seen, which in its finest exten-

A&apoE Complexes Purified from Human Bram 223

B

Figure 5. lmmunogold Labeling of the Ab-apoE Complexes (A) Labeling of Ab-apoE complexes with antibody against apoE (2El), followed by negative staining. (B) Labeling of the same specimen as in (A) with normal mouse IgG as a control for nonspecrfic binding of the primary antibody. Bars, 100 nm.

sions consisted of one or a few fibrils twisted around each other. The fibrils had a diameter of 6-8 nm, in good accordance with published data on the dimensions of A8 fibrils (Sanan et al., 1994). The fine structure of noncomplexed A8 purified from AD brain (see Figure 2A, fraction 9) is shown in Figure 4C. As seen: the general morphology of noncomplexed A8 was very similar to that of the Af3-apoE complexes. Hence, the morphology of A8 fibrils is similar in the presence or absence of apoE.

lmmunogold labeling of the A6-apoE complexes with antibody 2El revealed that both the large fibril bundles and the dispersed fibrils were strongly immunoreactive (Figure 5A). This suggests that apoE was present not only on the surface of the bundles but also between the individ-

Figure 6. lmmunoblot Analysis of AD-apoE Complexes Formed In Vitro

lmmunoblot analysis of coincubates between Ab and apoE and corresponding controls. Incubations were performed for 4 days at 37% either with (B) or without (A) 10 mM DTT. The products were collected by centrifugation and treated with HFIP and 9 M urea before electrophoresis. Positions of the molecular mass markers in kilodaltons are shown at left.

ual fibrils. Some scattered gold particles could also be seen in close proximity to the fibril bundles. These particles probably represent labeling of material disrupted from the original structures during sonication, since the overall background labeling was very low (Figure 5B).

Reconstitution of Afl-apoE Complexes In Vitro To establish further that the Af3-apoE complexes are distinct chemical entities and not artifacts attributable to the extraction or purification procedures, the formation of Af3- apoE complexes was also characterized in vitro. Recombi- nant apoE and synthetic Af3( l-40) or A/3( 1-42) were coin- cubated under the conditions described in Experimental Procedures. Without addition of reducing agents to the incubation medium, apoE and apoE interacted with both A6 variants and formed heterologous complexes with a molecular mass of -40 kDa (Figure 6A). In agreement with previous reports, the complexes were SDS-stable (Strittmatter et al., 1993b; LaDu et al., 1994) and, in addition, were resistant to the HFIP and 9 M urea used in the present protocol. It was further evident that only a fraction of the total apoE reacted with A6 to form these avid complexes (varying between 5% and 20010, as determined by

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Figure 7. Electron Micrographs of At%-apoE Complexes and High Mo- lecular Mass Ab Aggregates Generated In Vitro

(A) Ab-apoE complexes generated in vitro by coincubation of Ab(i- 40) and apoE in the presence of 10 mM DTT. The complexes were purified by gel filtration, polymerized in TBS for 1 hr, and negatively stained. (B) lmmunogold labeling with 2El of the same specimen as in (A), followed by negative staining. (C) High molecular mass aggregates of Ab(l-40) that were treated as the sample in (A). Bars, 100 nm.

densitometry). If, however, the incubation was performed in the presence of a reducing agent (10 mM dithiothreitol [OTT]), the complex formation proceeded beyond the stoi- chiometric 1 :l complex, and higher molecular mass complexes labeled with both 6ElO and 2El were formed (Fig- ure 6B). In the case of A6(1-40), it is interesting to note the stepwise 4 kDa mass increments of the complexes, suggesting the successive incorporation of A6 monomers. The A@apoE complex bands became less distinct and more similar to the in vivo material when using synthetic A6(1-42) indicating that the higher hydrophobicity of the bound A8 induced aberrant, smear-like migration of the complexes in SDS-PAGE. No AB-apoE complexes were

detected at zero-time incubations (data not shown), con- firming that the complexes formed are not artifacts caused by postincubation sample treatment.

The AD-apoE complexes formed under reducing conditions in vitro were separated from residual noncomplexed apoE and A6 by gel filtration in 70% formic acid and examined by electron microscopy after negative staining and immunogold labeling. As for the A6-apoE complexes found in vivo, the high molecular mass products from the in vitro coincubations principally formed dense aggregates of parallel fibrils. However, in certain regions, a lattice of fibrils with extensive crossover characteristics could be found (Figure 7A). In addition, immunogold labeling of the in vitro coincubates using anti-apoE antibody demonstrated the same pattern of gold particle decoration, albeit at a lower density, as that observed for the Af3-apoE complexes formed in vivo (Figure 78). The lower density of immunogold labeling of the in vitro Af3-apoE complexes might be an effect of the limited time of incubation, or it may bethatefficient, invivo-likecomplexformation requiresan additional, unknown factor. Differences in A6 fibril morphology attributable to either apoE isoform were not observed. When A8(1-40) or Af3(1-42) was incubated alone, both peptides formed high molecular mass products that, when subjected to negative staining, displayed the same kind of fibril bundles as the in vivo and in vitro AP-apoE complexes (Figure 7C). These fibrils were not immunoreactive to the apoE-specific antibody 2El. Thus, the influ- ence of apoE on the final pattern of A6 fibril morphology under these experimental conditions seems to be negli- gible.

Discussion

Here we present evidence that complexes between A8 and apoE are components of AD amyloid. The complex formation occurs with either the apoE or the apoE isoform, as determined by apoE genotype analyses of the AD patients and by in vitro reconstitution studies. The A8- apoE complexes are joined by uncommonly strong intermolecular bonds, since they were resistant to treatment with a number of potent dissociating agents, some of which have previously been shown to dissociate A6 polymers from AD brain (Masters et al., 1985). lmmunoblot analysis suggests that the complexes are composed of the C-terminal part of apoE, a finding supported by recent in vivo studies (Wisniewski et al., 1995) and in vitro binding studies (Strittmatter et al., 1993b). The relatively broad retention profile of the Af3-apoE complexes in the RPLC system used is probably attributable to heterogeneity of the complexes, with the smallest complexes being composed of one apoE and one A6 molecule and the large complexes of severalfold more A6 than apoE molecules. The molar excess of A6 may also explain why microsequencing of the complexes yielded A8 fragments but no apoE fragments. The retention times of the Af3-apoE complexes were consistently longer compared with that of noncomplexed A(3 purified from AD brain tissue. These longer retention times indicate that the Af3-apoE complexes are more aggregable than A8 alone. Furthermore,

Ap-apoE Complexes Purified from Human Brain 225

all A3 eluting as polymers in the present chromatography system is associated with apoE, suggesting the coexis- tence of two populations of A6 in AD amyloid: one that is dissociable in HFIP and formic acid and another, the apoE-bound, that is resistant to dissociation. This conclu- sion is in part supported by immunohistochemical data, where antibodies against apoE stain some, but not all, Af3-immunoreactive deposits (Strittmatter et al., 1993a; Kida et al., 1994).

It has been suggested that pathological chaperones may mediate amyloid formation of polypeptide fragments (Wisniewski and Frangione, 1992). The Af3-apoE interaction described here isof such apparent avidity that dissociation of the components under physiological conditions is unlikely. The presence of apoE within or at the surface of the A/3 fibrils is therefore not surprising. However, another candidate pathological chaperone protein, ACT(Ma et al., 1994) which by immunohistochemistry has been shown to colocalize with Af3 in AD amyloid (Abraham et al., 1988), was apparently not bound to A[3 using the present extraction and purification protocol. This may reflect a more tran- sient mode of ACT interaction with AD.

The fine structure of the aggregated Af3-apoE complexes was similar to that of noncomplexed AD, as determined by negative stain electron microscopy. The similar morphology suggests that the A(3 components of the in vivo complexes might be composed primarily of Af3 variants ending at residue 42, since At3 variants ending at residue 42 are principal components of AD-associated amyloid (Roher et al., 1993; Naslund et al., 1994) and since coincubations between Af3(1-42) and apoE in vitro yielded a product with an immunoblot appearance nearly identical to that of the complexes recovered from AD brain. The in vitro-generated, high molecular mass polymers of Ab showed the same pattern of tightly aligned fibrils, irrespective of the presence or absence of apoE. These results, and other recent findings of Lansbury and coworkers (Ev- ans et al., 1995), are indicative of a minor role for apoE in determining the morphology of A6 fibrils and fibril bundles. Rather than influencing the fibril morphology per se, apoE may affect the rate of Af3 fibril formation, as in vitro data suggest (Ma et al., 1994; Sanan et al., 1994; Wisniewski et al., 1994; S. E. G., unpublished data).

It is particularly interesting that we have detected highly stable Af3-apoE complexes in light of the genetic evidence that apoE isoforms are an important determinant of an individual’s risk for AD. In our study, we observe that these complexes can include either apoE or apoE isoforms. Moreover, the apoE part of the AB-apoE complexes ap- pears to be composed of a C-terminal fragment truncated at a site C-terminal to residue 158. This complexed apoE fragment is therefore identical in primary structure in all three apoE isoforms. A direct interaction between Ab and the polymorphic sites in apoE may not be critical, since amino acid substitutions at residue 112, the single site at which apoE and apoE differ, have been shown to affect the structural properties of the C-terminal domain of apoE (Dong et al., 1994). The Af3-binding capacity of apoE may therefore be governed by domain interactions, similar to what has previously been shown to determine the isoform-

specific lipoprotein preferences of apoE and apoE (Dong et al., 1994). It is not clear, though, whether cleav- age of apoE occurs before or after A3 binding.

Since the extraction yield of the AD-apoE complexes is not known, and possibly fluctuates between preparations, no quantitative interindividual comparison in the amount of the complexes was made. However, examination of the immunoblot data from the 32 AD patients does not indicate that there was a correlation between the apoE genotype and the amount of Af3-apoE complexes extracted. It will now be important to determine whether quantitative differences can be observed when different isoforms are compared, orwhether apoE isoforms have adifferential affinity for associating secondarily with deposited Af3. Of possible relevance to this issue, differential association with soluble Ab has been observed for denatured, delipidated apoE isoforms (Strittmatter et al., 1993b; Ma et al., 1994; Sanan et al., 1994; Wisniewski et al., 1994) and physiological apoE isoforms (LaDu et al., 1994). Such differences between apoE isoforms in their association with A9 may lead to differential proficiency in catalyzing fibril formation (Ma et al., 1994; Sanan et al., 1994; Wisniewski et al., 1994) thus possibly accounting for the overrepresentation of the apoE isoform seen in both sporadic (Mayeux et al., 1993; Saunders et al., 1993) and familial AD (Corder et al., 1993; Strittmatter et al., 1993a). Alternatively, the isoforms could differ in their ability to stabilize the 6 structure of A6 in amyloid, since the deposition of Af3 in amyloid has been shown to be a dynamic, reversible process (Maggio et al., 1992).

The effect of inclusion of DlT in order to obtain in vitro A&apoE complexes with similar chromatographic and immunoreactive characteristics as the complexes formed in vivo superficially contradicts the results reported by Stritt- matter and colleagues, who observed that inclusion of reducing agents abolished AD-apoE complex formation (Strittmatter et al., 1993b; Sanan et al., 1994). The discrep- ancy in results is most likely explained by differences in the protocols used for complex solubilization prior to SDS- PAGE. Since A6 and apoE lack cysteine residues, the DTT effect seen in the in vitro coincubates cannot be ac- counted for by keeping sulfhydryl groups in a reduced state. DlT may instead inhibit the formation of methionine sulfoxides and so enhance the ability of apoE to bind oligomers and polymers of A(3.

Complex formation between Ab and apoE in vivo might have functional consequences related to the ability of apoE to bind glycosaminoglycans. ApoE contains two distinct heparin-binding sites, one of which is located in the C-terminal part of apoE between residues 202 and 243 (Cardin et al., 1966). This site does not overlap the postu- lated AD-binding site in apoE, located between residues 244 and 272 (Strittmatter et al., 1993b). The apoE moiety of the AD-apoE complexes may therefore provide an “an- chor”forA(3 polymers, binding them to the glycosaminogly- can-rich extracellular matrix of the brain tissue. These an- chored Ab polymers can in turn serve as nuclei for further A(1 polymerization and growth of amyloid deposits (Jarret and Lansbury, 1993).

Since the AD-apoE complexes were found in significant

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levels in all 32 AD brains examined and virtually identical results were obtained when analyzing amyloid derived from leptomeningeal AD tissue (J. N., unpublished data), it is proposed that the formation of AD-apoE complexes is an invariable feature of AD amyloidogenesis. These in vivo observations may be relevant to an elucidation of the molecular events that underlie the pathophysiology of cerebral amyloidogenesis and to improving our understand- ing of the role of apoE in this process.

Experimental Procedures

Human Brain Tissue and Antibodies Autopsy material from AD individuals (n = 32) was obtained from the Huddinge Brain Bank, Huddinge Hospital, Sweden. AD diagnosis was confirmed according to standard protocols (McKhann et al., 1984). The AD cases had an average age of 78 years (range = 57-94 years). The control cases (n = 4) were obtained from the Department of Foren- sic Medicine, Karolinska Institute, Stockholm, Sweden. Neither gross pathological examination nor clinical records revealed any sign of dementia or other neurological disorders, and the average age of the control cases was 75 years (range = 60-86 years). Occipital cortex (IO-40 g) from both AD and control cases was removed at autopsy and stored at -70°C until further use. Occipital cortex was used for reasons of availability. ApoE genotypes were determined from brain tissue as described (Wenham et al., 1991). The antibodies used in this study were either from commercial sources or have, with one exception, been described previously. The monoclonal antibodies used were 6ElO (Kim et al., 1990), 4G8 (Kim et al., 1988), 2El (Boeh- rmger), 1048(Chemicon), lD7and 3Hl (Milneetal.. 1981; Weisgraber et al., 1983), 22Cll (Boehringer), Alz 90 (Boehringer), 56-l (Wunder- lich et al., 1992), tau-l (Boehringer), and AT-8 (Innogenetics). The following antisera were also used: G504 (see below), anti-AD (Boeh- ringer), 369A (Buxbaum et al., 1990), anti-ACT (Calbiochem), anti- ubiquitin (Boehringer), and anti-TTR (Sigma). The general specificities of these antibodies are given in Table 1. Polyclonal antiserum G504. a gift from Dr. Toshiharu Suzuki, Rockefeller University, was raised in rabbits against AP(l-40) and purified by affinity chromatography using Ae(l-40) Immobilized on a Hi-Trap NHS column (Pharmacia).

Amyloid Extraction Brain tissue was extracted as described (Ntislund et al., 1994). Bnefly, repeated extraction of the tissue in a buffer containing 1% SDS, 0.1 mM phenylmethylsulfonyl fluoride, 150 mM NaCI, and 50 mM Tns- HCI (pH 7.4) yielded a SDS-insoluble pellet that In turn was extracted with 1 ,I ,1,3,3.3-hexafluoro-2.propanol (HFIP). After removal of debris and delipidation, the HFIP extract wasdried under a stream of nitrogen Protein determination was performed using the bicinchonlnic acid assay (Pierce). Samples were initially dissolved in 9 M urea, 50 mM Tris-HCI (pH 7.4), boiled for 5 min, and diluted to 3 M urea with water prior to protein determination.

SDS-PAGE and lmmunoblotting Samples were dissolved in 9 M urea and sonicated (Branson Sonifier 250) before addition of 3 x SDS-PAGE sample buffer. The samples were boiled and resolved on a 54/o-18% Tris-Tricine gradient gel system. After electrophoresis, the proteins were electroblotted onto nitrocellulose membranes (0.2 urn; Schleicher & Schuell) or polyvinylidene difluoride membranes (0.45 pm; Millipore). Membranes were probed with monoclonal antibodies or antisera for 3-12 hr at room temperature, and antibody binding was detected by adding horseradish perox,- dase-linked sheep anti-mouse, donkey anti-rabbit (both supplied by Amersham), or rabbit anti-goat (Sigma) immunoglobulin, depending on the host animal providing the primary antibody. The blots were visualized usmg the enhanced chemiluminescence system (Amer- sham) according to the instructions of the manufacturer. Quantitatlon of the blots was performed using a Bio-Rad Model GS-670 densitome- ter and the accompanying Molecular Analyst software.

Purification of the Afl-apoE Complexes The dry HFIP extracts were dissolved in 70% (v/v) distilled formic acid, centrifuged at 10,000 x g for 2 min (to remove insoluble material), and purified by gel filtration on a 1 x 30 cm Superose 12 column (Pharmacia) developed with 70% formic acid at a flow rate of 200 pII min (Roher et al., 1993). Absorbance was monitored at 280 nm, and aliquots of the collected fractions were dried by vacuum centrifugation after addition of SDS (0.1% final concentration). The dried aiiquots were subjected to immunoblotting, the fractions containing the AD- apoE complexes and noncomplexed AD were pooled separately, and the solvent was evaporated. The Afl-apoE complexes were redis- solved in 70% formic acid and further purified by RPLC using a 0.46 x 15 cm Vydac 214 TP C4 column developed over 30 min with a linear gradient of O%-80% buffer B, where buffer A was 60% formic acid in 0.1% TFA and buffer B was buffer A containing 40% ACN (Heukeshoven and Dernick, 1985). Flow rate was set at 500 jdlmin, and absorbance was monitored at 280 nm. Aliquots of the collected fractions were evaporated after addition of SDS (0.1% final concentration) and analyzed by immunoblotting. Fractions containing Ap-apoE complexes were pooled, evaporated, and used for immunological characterization, digestion, dissociationstudies, and lightandelectron microscopy (see below).

Enzymatic Digestion and Microsequencing of the Ap-apoE Complexes The partially purified AD-apoE complexes were alkylated with Cvinyl- pyridine according to standard procedures. Desalting of the alkylated proteins was performed on a 0.32 x 10 cm FastDesalting column (Pharmacia) developed with 20% ACN, 0.1% TFA at a flow rate of 100 pllmin, while monitoring absorbance at 254 and 280 nm. The void fraction was evaporated to dryness, and the alkylated AD-apoE complexes were resuspended in 10 pl of 9 M urea. After dilution to 1 M urea with 0.1 M ammonium bicarbonate buffer (pH 7.8), digestions were performed with endoproteinase Lys-C (Boehringer) or modified trypsin(Promega) for 18 hrat 37OCat anenzyme:substrate ratio(mass/ mass) of approximately 1:20. The incubations were terminated by the addition of TFA (0.1% final concentration). After evaporation, the di- gests were resuspended in 0.1% TFA and centrifuged at 10,000 x g for 2 min. The resulting supernatant and pellet were treated separately. The peptides in the supernatant were separated by RPLC using a Pharmacia SMART system equipped with a 0.21 x 10 cm C2-Cl8 mRPC column (Pharmacia) developed over 40 min with a linear gradient of O%-50% ACN in 0.1% TFA at a flow rate of 100 pllmin. The pellet was dissolved in 70% formic acid and diluted with 0.1% TFA to a final formic acid concentration of 10% before separation on a 0.21 x 15 cm Vydac 214 TP C4 column developed with the same solvent system as for the supernatant peptides. N-terminal sequencing of the peptide fragments was performed on an Applied Biosystems Model 477 A gas-phase microsequencer using procedures according to the manufacturer.

Dissociation Studies of the Ap-apoE Complexes One milliliter of each of the agents listed below was added to the purified AD-apoE complexes. The complexes were incubated in poly- propylene tubes with each agent for 12-48 hr. The samples were then diluted with water and dialyzed, using benzoylated dialysis tubing (Sigma), against 0.1 M ammonium bicarbonate (pH 7.8), before evaporation and analysis by immunoblotting. The agents used were SDS (lo%), urea (10 M), guanidine-HCI (6 M), phenol (90%), HFIP (1 OO%), ACN (lOO%), NH,OH (25%). NaOH (1 M), TFA (30%), and formic acid (70%100%).

Light and Electron Microscopy Purified A@apoE complexes were suspended in 50% ethanol containing 1% Congo red (Bush et al., 1994). The suspension was smeared onto glass slides and viewed under polarized light. For electron microscopy, the AD-apoE complexes and noncomplexed AD purified from AD brain (see Figure 2A, fraction 9) were dissolved in HFIP before dilution in Tris-buffered saline (TBS) to a final concentration of 5% HFIP. The samples were incubated in TBS (pH 7.4) at 37OC for 1 hr and pelleted by centrifugation at 20,000 x g for 20 min using a

Af3-apoE Complexes Purifted from Human Brain 227

Beckman TLA 45 rotor. The pellets were washed twice with water and resuspended in a final volume of 50 aI of water. Following sonication (Branson Sonifier 250) 5 ul aliquots were placed on copper grids covered by a carbon-stabilized Formvar film, and 5 ul of freshly pre- pared 2% uranyl acetate in water was added. After l-2 min, excess fluid was removed with a filter paper and the grids were air-dried. The specimens were examined and photographed in a JEOL EM 1OOCX at 60 kV. For immunogold staining, A6-apoE complexes were ad- sorbed to nickel grids covered by a carbon-stabilized Formvar film and air-dried. After washing in TBS for 2 min, nonspecific binding was blocked by incubation in TBS with 3% bovine serum albumin (BSA) for 30 min. The grids were then placed on a droplet of either the apoE-specific antibody 2E1 (1:50) or normal mouse IgG for 60 min (both diluted in TBS, 0.1% BSA), passed over six droplets of washing solution for 2 min each (TBS., 0.1% BSA), placed on a droplet of anti- mouse IgG conjugated to 10 nm colloidal gold particles for 30 min (Sigma; diluted 1:20 in TBS, 0.1% BSA). and passed over another six droplets of washing solution. The grids were fixed in 2% glutaralde- hyde in 0.1 M sodium cacodylate-HCI buffer (pH 7.3) for 5 min, washed under a jet of water from a Pasteur pipette for 15-20 s, and air-dried. Before the final examination in the electron microscope, the specimens were negatively stained with 2% uranyl acetate in water.

Generation of Ap-apoE Complexes In Vitro A6(1-40) and A6(1-42) were dissolved at 200 uM in 20 mM HEPES, 150 mM NaCI, 0.05% CHAPS (pH 6.90) by sonication. To create reducing conditions, 10 mM DTT was included in the buffer. Recombinant apoE (PanVeraCorp., Madison, Wisconsin) at 1 mglml in 0.7 M ammonium bicarbonate was added to a final concentration of 50 ug/ml (1.47 KM), which adjusted the pH of the incubation solution to 7.4. The incubation volume was 50 ul, and the amount of each reactant was corroborated by amino acid analysis (apoE) and RPLC (A6). After 4 days of incubation at 37% in a shaking water bath, the A6-apoE complexes were pelleted by centrifugation at 20,000 x g for 20 min in a Beckman TLA 45 rotor (4%). The pellets were resuspended in HFIP for 30 min at room temperature and dried, before analysis by immunoblotting. To remove noncomplexed apoE and A5 before electron microscopy, the HFIP-treated and dried pellets were dissolved in 70% formic acid. The AD-apoE complexes were purified by gel filtration in 70% formic acid on a 0.32 x 30 cm Superose 12 column (Pharmacia) using the SMART system. The fractions containing the A6-apoE complexes, as determined by immunoblotting, were pooled, dried, and preparedforelectron microscopyasdescribedforthe invivo samples. Samples in which A6 was incubated alone were subjected to thesamepurificationprocedure, andthesamefractionswerecollected as above.

Acknowledgments

We thank Drs. T. Suzuki and T. Ramabhadran for providing antiserum G504 and monoclonal antibody 56-1, respectively; Drs. K. S. Kim and H. M. Wisniewski for providing ascites 6ElO and 4G6; and Dr. Yves L. Marcel, Lipoproteinand AtherosclerosisGroup, Universityof Ottawa Hear-l Institute, Ottawa, Ontario, Canada, for providing ascites 1 D7 and 3Hl. We also thank lnga Volkmann, Lena Lilius, and Karin Blom- gren for expert technical assistance. AD brain tissue was obtained from the Huddinge Brain Bank, Huddinge Hospital, Sweden. This study was supported by National Institutes of Health grants HL49571-01 (L. T.) and AG-11506 (S. E. G.), The Swedish Medical Research Council (J. T. and L.T.), The C. V. Starr Foundation and The Starr Program for Neurogeriatric Research (S. E. G.), The Swedish Heart Lung Foun- dation (J. T.), and the King Gustaf V 60th Birthday Fund (J. T.). J. N. is supported by a fellowship from The Swedish Board of Industrial Development. C. N. is a recipient of fellowships from The Berth von Kantzow Foundation, The Swedish Society for Medical Research, and The Nicholson Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 USC Section 1734 solely to indicate this fact.

Received February 2, 1995; revised April 6, 1995

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