©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Amyloid Protein (A) in Alzheimers Disease Brain
BIOCHEMICAL AND IMMUNOCYTOCHEMICAL ANALYSIS WITH ANTIBODIES SPECIFIC FOR FORMS ENDING AT Abeta40 OR Abeta42(43) (*)

(Received for publication, January 10, 1995)

Stephen A. Gravina (1) Libin Ho (1) Christopher B. Eckman (1) Kristin E. Long (1) Laszlo Otvos Jr. (3) Linda H. Younkin (1) Nobuhiro Suzuki(§) (2) Steven G. Younkin (1)(§)(¶)

From the  (1)Division of Neuropathology, Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, the (2)Discovery Research Division, Takeda Chemical Industries, Ltd., Wadai 10, Tsukuba, Ibaraki 300-42, Japan, and the (3)Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Biochemical and immunocytochemical analyses were performed to evaluate the composition of the amyloid beta protein (Abeta) deposited in the brains of patients with Alzheimer's disease (AD). To quantitate all Abetas present, cerebral cortex was homogenized in 70% formic acid, and the supernatant was analyzed by sandwich enzyme-linked immunoabsorbent assays specific for various forms of Abeta. In 9 of 27 AD brains examined, there was minimal congophilic angiopathy and virtually all Abeta (96%) ended at Abeta42(43). The other 18 AD brains contained increasing amounts of Abeta ending at Abeta40. From this set, 6 brains with substantial congophilic angiopathy were separately analyzed. In these brains, the amount of Abeta ending at Abeta42(43) was much the same as in brains with minimal congophilic angiopathy, but a large amount of Abeta ending at Abeta40 (76% of total Abeta) was also present. Immunocytochemical analysis with monoclonal antibodies selective for Abetas ending at Abeta42(43) or Abeta40 confirmed that, in brains with minimal congophilic angiopathy, virtually all Abeta is Abeta ending at Abeta42(43) and showed that this Abeta is deposited in senile plaques of all types. In the remaining AD brains, Abeta42(43) was deposited in a similar fashion in plaques, but, in addition, widely varying amounts of Abeta ending at Abeta40 were deposited, primarily in blood vessel walls, where some Abeta ending at Abeta42(43) was also present. These observations indicate that Abetas ending at Abeta42(43), which are a minor component of the Abeta in human cerebrospinal fluid and plasma, are critically important in AD where they deposit selectively in plaques of all kinds.


INTRODUCTION

In the brains of patients with Alzheimer's disease (AD), (^1)amyloid composed of 4-kDa amyloid beta protein (Abeta) is deposited in senile plaques and in blood vessel walls. Immunocytochemical studies using antisera to Abeta have established that Abeta is deposited not only in neuritic plaques, which are spherical clusters of altered neurites that in some cases surround well defined amyloid cores, but also in large numbers of diffuse plaques, which are poorly circumscribed, immunoreactive lesions showing minimal neuritic change(1, 2) . Recently, we and others have shown that normal processing of the large amyloid beta protein precursor (beta-APP) secretes 4-kDa Abeta that is readily detected in human cerebrospinal fluid and in medium conditioned by cultured cells(3, 4, 5, 6) . This soluble 4-kDa Abeta is primarily Abeta1-40, although minor amounts of Abeta1-42 and other species are also secreted(7, 8, 9) . Abeta1-42 has been shown to form insoluble amyloid fibrils more rapidly than Abeta1-40(10, 11, 12, 13) . Moreover, we have recently used transfected cultured cells to show that beta-APP mutations (DeltaI, DeltaF) linked to familial AD increase the percentage of long Abeta1-42(43) secreted(9) . Several groups have examined the insoluble Abeta that remains after the AD brain is extracted with high concentrations of SDS(14, 15, 16, 17, 18) . Recent reports using this approach, particularly those of Roher and his colleagues, indicate that the SDS-insoluble amyloid in senile plaque cores is primarily Abeta1-42(15, 17) , that diffuse plaques are primarily Abeta17-42(18) , and that vascular amyloid is a mixture of Abeta1-40 and Abeta1-42(16) . Thus, the minor, secreted Abeta1-42 may be critically important in the pathogenesis of AD.

Analysis of the SDS-insoluble Abeta in AD brain will substantially underestimate species ending at Abeta40 if these species are selectively solubilized in high concentrations of SDS. Thus, to obtain a better quantitation of the total Abeta in AD brain, we homogenized AD cerebral cortex directly in 70% formic acid or sequentially extracted first into Tris-saline buffer and then into formic acid and analyzed the resultant fractions with sandwich ELISAs that specifically detect Abetas ending at Abeta40 (Abeta) or at Abeta42(43) (Abeta). To determine where the Abetas that were quantitated are localized in AD brain, we immunostained sections with BA-27 and BC-05, the monoclonal antibodies in our sandwich ELISAs that specifically detect Abeta and Abeta, respectively.


EXPERIMENTAL PROCEDURES

Tissue Preparation

AD brains were diagnosed on the basis of classic pathology that included large numbers of senile plaques. Control brains were plaque-free. In our initial experiments, cerebral cortex (0.5 g; at -70 °C) from 3 AD and 3 control brains was Dounce-homogenized (10 strokes) in 4.0 ml of 70% glass distilled formic acid. After 15 min at 4 °C, homogenates were centrifuged at 100,000 times g for 1 h. The formic acid extract (which was between a thin overlying lipid layer and a very small pellet) was removed and separated into four aliquots, which were analyzed immediately or completely dried in a rotary vacuum and stored at 4 °C. The dried aliquots could effectively be resolubilized in 0.2 ml of 70% glass distilled formic acid by sonicating 2 times for 10 s at 50 watts and 70% amplitude (Microsonic Cell Disruptor, Kontes). No Abeta from the lipid layer or the pellet was detectable as estimated from 4G8-stained immunoblots. In our second series of experiments, the Abeta in 27 AD brains and 6 plaque-free control brains was examined by homogenizing cerebral cortex in Tris-saline buffer containing protease inhibitors (1 µg/ml pepstatin, 2 µg/ml N-p-tosyl-L-lysine chloromethyl ketone, 20 µg/ml aprotinin, 200 µg/ml phosphoramidon, 2 mM EGTA, and 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin) centrifuging at 100,000 times g for 1 h, removing the Tris-saline supernatant, homogenizing the pellet in 70% formic acid, and centrifuging again at 100,000 times g for 60 min. The Tris-saline supernatant was diluted 1:4 or 1:40 in buffer EC (0.02 M phosphate, 0.4 M NaCl, 2 mM EDTA, 0.2% bovine serum albumin, 0.05% CHAPS, and 0.05% NaN(3), pH 7). The formic acid supernatant was diluted 1:40 in 0.25 M Tris base (pH 8) containing 30% acetonitrile, neutralized with 5 M NaOH, and diluted in buffer EC (1:2.5-1:50). To assess Abeta recovery, synthetic Abeta (500 pmol of Abeta plus 500 pmol of Abeta, Bachem) was added to samples of cerebral cortex (0.5 g) from control brains prior to homogenization. This analysis showed that with formic acid extraction over 93% of the relevant Abetas were recovered when extracts were directly measured by any of the four sandwich ELISAs employed. In the more complicated experiments involving Superose 12 chromatography (see below), recoveries were 50-100%.

Sandwich ELISAs for Abeta

Sandwich ELISAs were performed as described previously(9) . Absorbances falling within the standard curve for each assay were converted to picomoles, averaged, corrected when necessary for the recovery observed with that assay, and expressed as pmol/g wet tissue. The monoclonal antibodies employed were BAN-50 (anti-Abeta1-16), 4G8 (anti-Abeta17-24)(19) , BA-27, which is specific for Abeta ending at Abeta40, and BC-05, which is specific for Abeta ending at Abeta42(43). We have previously shown that BAN-50/BA-27 and BAN-50/BC-05 ELISAs specifically detect Abeta1-40 and Abeta1-42 in medium conditioned by transfected cultured cells(9) . When BAN-50/BC-05 sandwich ELISA is used for analysis, Abeta1-42 is detected with a sensitivity 10-fold greater than that for Abeta1-43. When BAN-50/BC-05 assay is used for analysis, conventional Abeta1-42 (L-aspartate at Abeta1 and Abeta7) is detected with a sensitivity 20-fold greater than Abeta1-42 having L-isoaspartate at Abeta1 and Abeta7 (data not shown).

Size Exclusion Chromatography

In some experiments, the Abeta in supernatants obtained after homogenizing brains in 70% formic acid as described above was analyzed by Superose 12 (Pharmacia Biotech Inc.) size exclusion chromatography. An aliquot of each sample (0.15 ml) was passed through a Superose 12 column (30 times 300 mm) running at 0.2 ml/min, and 0.75-ml fractions were collected. Fractions were completely dried in a rotary vacuum and solubilized in 60% acetonitrile, 0.2% trifluoroacetic acid by sonicating as above. To determine where Abeta eluted, 5-µl aliquots from these fractions were diluted into 100 µl of buffer EC and analyzed by BC-05/4G8 and BA-27/4G8 sandwich ELISAs. Reactive fractions were pooled, dried in a rotary vacuum, resolubilized in 1.0 ml of 60% acetonitrile, 0.2% trifluoroacetic acid as above, diluted (0.2, 0.1, 0.01, or 0.001), and assayed by BC-05/4G8, BA-27/4G8, BAN-50/BC-05, and BAN-50/BA-27 sandwich ELISAs.

Immunocytochemistry

Formalin-fixed, paraffin-embedded blocks of human inferior temporal cortex were cut at 6 µm. Hydrated sections were treated with 3% hydrogen peroxide to inactivate endogenous peroxidase activity, and then with 70-99% formic acid for 5 min to enhance Abeta immunoreactivity. Sections were incubated with appropriate dilutions of primary antibody for 1 h at room temperature. Secondary affinity-purified biotinylated goat anti-mouse IgG (1:200) was applied for 20 min at room temperature, and tertiary streptavidin-horseradish peroxidase (1:200) was applied for 20 min at room temperature. Sections were washed with 1% normal goat serum/Tris-buffered saline after each incubation. Peroxidase was developed with 3,3` diaminobenzidine cosubstrate. Similar results were obtained using cortical blocks fixed in methacarn (60% methanol, 30% chloroform, and 10% acetic acid).


RESULTS AND DISCUSSION

Analysis of Abeta in Formic Acid Extracts

To quantitate all of the Abeta in AD brain quantitatively, we homogenized AD cerebral cortex directly in 70% formic acid, separated the Abeta in the formic acid supernatant from larger proteins by gel filtration, and analyzed the resultant fractions for Abeta using BA-27(anti-Abeta1-40)/4G8(anti-Abeta17-24) and BC-05(anti-Abeta35-43)/4G8(anti-Abeta17-24) sandwich ELISAs that discriminate synthetic Abeta1-40 from Abeta1-42 (Fig. 1, A and B). To evaluate the utility of this method, we analyzed 500 pmol of synthetic Abeta1-40 and Abeta1-42 added to 0.5 g of control cerebral cortex in 4 ml of 70% formic acid. After homogenization and Superose 12 chromatography, this synthetic Abeta was readily detected with recoveries of 50-100% by drying the formic acid fractions, resolubilizing in 60% acetonitrile plus 0.2% trifluoroacetic acid, and diluting at least 1:200 prior to sandwich ELISA (Fig. 1, C-F).


Figure 1: Analysis of formic acid-extractable Abeta in AD and control brain by Superose 12 chromatography. The results shown here for 1 AD and 1 control brain are typical of 3 AD and 3 control brains analyzed similarly. A, standard curve for BC-05/4G8 assay. B, standard curve of Abeta for BA-27/4G8 assay. circle, Abeta1-42; up triangle, Abeta1-40. C, BC-05/4G8 assay of Abeta in control brain. D, BA-27/4G8 assay of Abeta in control brain. E, BC-05/4G8 assay of control brain + synthetic Abeta. F, BA-27/4G8 assay of control brain + synthetic Abeta. G, BC-05/4G8 assay of AD brain. H, BA-27/4G8 assay of AD brain. Synthetic Abetas (500 pmol of Abeta1-40 and 500 pmol of Abeta1-42) were added to 70% formic acid at the same time as the control tissue, which was at -70 °C. Values for the standard curves represent the mean of three determinations ± S.E.



Having established the utility of this method, we used it to analyze the Abeta in 3 plaque-free control brains and in 3 AD brains with minimal cerebrovascular amyloid. All of the protein detected by our sandwich ELISAs eluted from the Superose 12 column at low molecular weight (Fig. 1, G and H) as previously reported for Abeta analyzed by other methods(8, 10) . Thus the sandwich ELISAs did not detect full-length beta-APP or large beta-APP fragments containing internal Abeta domains, as expected from the known specificity of BC-05 and BA-27 for species that end at Abeta40 or Abeta42(43). Very little Abeta was detected in plaque-free control brain (Fig. 1, C and D). In the 3 AD brains, the BA-27/4G8 ELISA showed a total of 93 ± 45 pmol of Abeta/g whereas the BC-05/4G8 ELISA showed 3200 ± 1350 pmol of Abeta/g (Table 1). Thus 97% of the Abeta in these AD brains ended at Abeta42(43). The Abeta-containing fractions were also analyzed using BAN-50(anti-Abeta1-16)/BC-05 and BAN-50/BA-27 sandwich ELISAs that we employed previously(9) . The BAN-50/BA-27 ELISA showed a total of 73 ± 28 pmol of Abeta/g in AD brain, whereas the BAN-50/BC-05 ELISA detected 725 ± 347 pmol/g (Table 1).



The sandwich ELISAs employing BAN-50 for capture do not provide information on the total Abeta in AD brain because BAN-50 (anti-Abeta1-16) cannot capture Abetas beginning at or beyond Abeta17. It was for this reason that we used horseradish peroxidase-linked 4G8, a monoclonal to Abeta17-24, for detection in BA-27/4G8 and BC-05/4G8 sandwich ELISAs. The increased amount of Abeta detected with 4G8 suggests that there may be considerable amino-terminally truncated Abeta in AD brain, consistent with the results of others who have examined the SDS-insoluble Abeta in AD brain(15, 17, 18) . This result should be interpreted cautiously, however, because other factors such as the presence of L-isoaspartates at Abeta1 and Abeta7 (15) may account for the diminished signal observed when BAN-50 was used for capture.

Analysis of Abeta in 27 AD and 6 Plaque-free Control Brains

Since the ELISAs employed specifically detect the Abeta in brain homogenates (Fig. 1), we sequentially extracted samples of cerebral cortex from 27 AD and 6 control brains first in Tris-saline containing protease inhibitors and then in 70% formic acid and directly analyzed the Abeta in the two supernatants after appropriate dilution and neutralization (see ``Experimental Procedures''). There was some Abeta in both the Tris-saline and formic acid extracts of the 6 plaque-free control brains analyzed. The amounts present were too small to quantitate accurately, but the total Abeta present in control brain was invariably less than 7 pmol/g. Based on this upper limit, the total Abeta in the 27 AD brains ranged from at least 500 to 4400 times that in plaque-free control brain. In the AD brains, 98.5% of the total Abeta present required formic acid for solubilization and only 1.5% was extracted into Tris-saline. The results of our analysis of the formic acid supernatants from the 27 AD brains are shown in Table 1and in Fig. 2, where the results of BC-05/4G8 and BA-27/4G8 analysis have been ordered in terms of increasing BA-27/4G8 signal. In many AD brains, virtually all Abeta was Abeta, the remaining brains had increasing amounts of Abeta, and large amounts of Abeta were deposited in some brains (Fig. 2).


Figure 2: Abeta detected by BA-27/4G8 and BC-05/4G8 ELISAs in 27 AD brains. Cases are ordered in terms of increasing BA-27/4G8 signal.



Direct analysis of the Abeta in 9 brains with minimal congophilic angiopathy (Table 1) gave results essentially identical to those obtained when Abeta was first fractionated by Superose 12 chromatography. Virtually all Abeta in the formic acid extracts from these 9 brains (96%) ended at Abeta42(43), and substantially more Abeta was detected with BC-05/4G8 (4663 ± 421 pmol/g) than with BAN-50/BC-05 (714 ± 68) assay. In the formic acid extracts of 6 brains with substantial congophilic angiopathy (Table 1), Abeta, analyzed by BC-05/4G8 (5089 ± 409 pmol/g) and BAN-50/BC-05 (826 ± 119 pmol/g) ELISA, was found to be present in amounts essentially identical to those measured in brains with minimal congophilic angiopathy. In these brains, Abeta, measured with BA-27/4G8 (13607 ± 3397 pmol/g) and BAN-50/BA-27 (5001 ± 2164 pmol/g) ELISAs, was dramatically increased. On average, 67% of the Abeta in brains with substantial congophilic angiopathy ended at Abeta40, and substantially more Abeta was detected with BA-27/4G8 than with BAN-50/BA-27 assay indicating that much of the Abeta ending at Abeta40, like the Abeta ending at Abeta42(43), is amino-terminally truncated or modified.

Using the BAN-50/BA-27 ELISA to analyze Abeta1-40 extracted into Tris-saline, Suzuki et al. previously showed a good correlation between the extent of congophilic angiopathy in AD brain and the amount of Abeta1-40 in the Tris-saline extract(20) . Consistent with this, the amount of Abeta extracted into Tris-saline was dramatically increased in brains with substantial as compared to minimal congophilic angiopathy when measured by either BAN-50/BA-27 (161 ± 60 versus 3 ± 1 pmol/g) or BA-27/4G8 (472 ± 103 versus 5 ± 1 pmol/g) assay.

Immunocytochemical Analysis

To show unequivocally that the large amount of Abeta detected by BC-05/4G8 sandwich ELISA is localized in plaques, we examined AD brains immunocytochemically with BC-05, 4G8, and BA-27 antibodies, pursuing observations made initially by Iwatsubo et al.(21) . Both 4G8 and BC-05 intensely labeled the AD brain, whereas BA-27 showed very little labeling (Fig. 3). 4G8 and BC-05 labeled uncored (diffuse and uncored neuritic) plaques, the cores of classic neuritic plaques, the region surrounding plaque cores, and vascular amyloid. BA-27 stained vascular amyloid quite well but, in contrast to BC-05 and 4G8, stained the cores of classic neuritic plaques less intensely and almost completely failed to stain uncored (diffuse and uncored neuritic) plaques or the region surrounding plaque cores. Essentially identical results were obtained using sections from tissue fixed in formalin or methacarn (data not shown). This finding and our observation that, in AD brains with minimal vascular amyloid, virtually all Abeta ends at Abeta42(43) provide strong evidence that immunocytochemical analysis with BA-27 and BC-05 is not distorted by selective destruction of the BA-27 epitope during tissue fixation or processing.


Figure 3: Immunostaining of AD brain. Similar regions of adjacent sections from the inferior temporal region of an AD brain fixed in formalin and photographed at magnification times 25 are shown after staining with: A, 4G8 (1:1000), which recognizes Abeta17-24; B, BA-27 (1:100), which is specific for Abetas ending at Abeta40; C, BC-05 (1:20,000), which is specific for Abetas ending at Abeta42(43)(9) . Closedarrows show the cores of classic neuritic plaques, and openarrows show vessels that are stained by the antibodies employed. Note the many uncored (diffuse and uncored neuritic) plaques that are intensely labeled by 4G8 and BC-05 but not BA-27.



The immunocytochemical results shown here and in a recent report by Iwatsubo et al.(21) demonstrate that BC-05 intensely stains all types of senile plaques, that vessels are stained by both BC-05 and BA-27, and that BA-27 stains plaques poorly, in many cases only faintly staining occasional cored plaques (Fig. 3). The apparent paucity of Abeta in senile plaques is remarkable because Abeta1-40 is the major Abeta peptide in human cerebrospinal fluid and it readily forms amyloid fibrils alone or in combination with Abeta1-42 in vitro. Thus it seemed there might be selective destruction of the BA-27 epitope during fixation that was causing misleading immunocytochemical data on the relative amounts of Abeta and Abeta in plaques and vessels. To examine this possibility, we focused our initial biochemical examination of Abeta on cases with minimal congophilic angiopathy where immunocytochemistry predicted a near absence of Abeta, and, to be sure that Abeta was not lost as tissue was processed for biochemistry, we used a procedure that evaluated the total Abeta in AD brain. Our results show unequivocally that in brains with minimal congophilic angiopathy virtually all Abeta ends at Abeta42(43). Thus in many AD brains virtually all Abeta that is deposited in senile plaques is Abeta.

Previous reports (14, 15, 16, 17, 18) have been confusing as to whether the Abeta deposited in AD brains is primarily Abeta or Abeta. Our analysis of the total Abeta in 27 AD brains showed that in 9 cases, all with minimal congophilic angiopathy, virtually all Abeta was Abeta, that the remaining brains had increasing amounts of Abeta, and that in 6 cases with substantial congophilic angiopathy Abeta was the major form deposited. Furthermore, the amount of Abeta in AD brains is at least 500-4400 times that in plaque-free control brains, and less than 2% of the total Abeta in AD brain is extracted into Tris-saline. This finding is consistent with previous reports (15, 17, 18) that most of the Abeta in AD brains is amino-terminally truncated or modified.

Remarkably, the amount of Abeta in AD brains with minimal congophilic angiopathy and little Abeta was similar to that in brains with substantial congophilic angiopathy and large amounts of Abeta. Thus, it appears that deposition of Abeta in senile plaques is fundamental to AD pathogenesis with additional deposition of Abeta, primarily in blood vessel walls, occurring in some cases. This view is supported by an immunocytochemical study of brains from trisomy 21 patients of various ages that was recently reported by Iwatsubo et al.(22) . In these brains, Abeta stained by BC-05 was deposited first in diffuse plaques, and there was essentially no staining by BA-27. Immature neuritic and mature cored plaques then developed along with congophilic angiopathy. As this occurred, BC-05 invariably showed intense staining of all plaques as well as some staining of blood vessel walls whereas BA-27 intensely stained blood vessels and showed increasing staining of Abeta in plaques, particularly mature cored plaques, although it never stained plaques to the extent observed with BC-05. Collectively, the findings reported here, the immunocytochemical data of Iwatsubo et al.(21, 22) , the recent analyses showing that most of the Abeta in isolated plaque cores ends at Abeta42(15, 17) , the demonstration that Abeta1-42(43) forms fibrils more rapidly than Abeta1-40(10, 11, 12, 13) , and the finding that the familial AD-linked beta-APP717 mutant (DeltaI, DeltaF) increases secretion of Abeta1-42 but not total 4-kDa Abeta (9) indicate that Abeta, which appears to be a minor component of the Abeta that is normally secreted, is critically important in AD where it accumulates selectively in senile plaques of all kinds.


FOOTNOTES

*
This work was supported by an ADRDA Zenith award and National Institutes of Health Grants AG06656 and AG12685. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Both authors contributed equally to this study.

To whom correspondence should be addressed: Institute of Pathology, Case Western Reserve University, 2085 Adelbert Rd., Cleveland, OH 44106. Tel.: 216-368-3381; Fax: 216-844-1810.

(^1)
The abbreviations used are: AD, Alzheimer's disease; Abeta, amyloid beta protein; beta-APP, amyloid beta protein precursor; ELISA, enzyme-linked immunoabsorbent assay; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.


ACKNOWLEDGEMENTS

We thank C. Cole, Y. E. Dietz, and P. L. Shaffer for technical assistance.


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