American journal ofPathology, Vol. 137, No. 1,July 1990 Copyright (6) American Association ofPathologists

Ultrastructural Localization of the Putative Precursors of the A4 Amyloid Protein Associated with Alzheimer's Disease

Nicoletta Catteruccia,* Julia Willingale-Theune,t Dirk Bunke,* Reinhard Prior,* Colin L. Masters, Andrea Crisanti,* and Konrad Beyreuther* From the Ceniterfor Molecuilar Biology, University oJ Heidelberg (ZMBII)*; Max-Planck Institut (MPI)ffir Zellbiologie, Rosenhoft Ladenburg/Heidelberg, West Germany; atnd the Departmenit of Pathology, Univeirsity ofMelbourne, Parkville, Victoria 3052, A istraliat

Any explanation of the causes of Alzheimer's disease and of its unique cerebral pathologic features must take into account the distribution and ultrastructurallocalization of the pre-A4 amyloid proteins in tissues and organs. The authors have analyzed the expression of thepre-A4 amyloid proteins in several tissues by immunogold electron microscopy and by immunofluorescence. For this purpose, they have used a mouse monoclonal antibody and a guinea pig antiserum raised against two syn-

thetic peptides corresponding to two different sequences common to all the full-length forms of the A4 amyloid precursors. They observed a tissue-specific distribution of the secreted and the transmembraneform of the precursors. The authors could determine that the secreted form is generated in vivo within the cytoplasm. In the salivary glands and in the adenobypophysis, all the immunoreactivity is associated with the process of secretion, whereas in the muscle, a staining pattern compatible with the presence of the pre-A4 amyloid proteins in the sarcoplasmic reticulum has been observed. This difference in the localization may reflect tissue-specific processing pathways and suggests that posttranslational modifications such as proteolytic removal of the transmembrane and cytoplasmic domains contribute to the structural and thus functional diversity of the A4 amyloid precursors. (AmJPathol 1990, 13 7:19-26)

Alzheimer's disease, the most common form of demen-

tia,1 is characterized by brain deposits of amyloid within

neurons (neurofibrillary tangles), extracellularly (senile plaques), and within the walls of the blood vessels.2 The fibrillar constituent of the amyloid, at least in the senile plaques and around the blood vessels, is the preA4 amyloid protein, a peptide of 42 to 43 amino acids3-5 that is a part of the sequence of several proteins (pre-A4 amyloid 695, pre-A4 amyloid 751, pre-A4 amyloid 770, described as putative A4 amyloid precursors or pre-A4 amyloid proteins) representing different splicing products of the same gene.6-9 Although the presence of amyloid deposits is not sufficient for the diagnosis of Alzheimer's disease, as they are found also in the brains of normal aged adults, a high density of the lesions and a high degree of A4 amyloid protein accumulation is characteristics for the brain of Alzheimer patients and used for the postmorten diagnosis. 10-12 The transcripts for the putative A4 amyloid precursors are thought to encode integral, glycosylated membrane proteins.61314 The pre-A4 amyloid mRNAs are ubiquitously expressed in tissues and organs of all individuals.15-17 The function of these proteins is not known, and their distribution in tissues and ultrastructural localization has not been firmly established. Recently, polypeptides thought to originate from the A4 amyloid precursors by processing pathways that remove the transmembrane and cytoplasmic domains have been biochemically identified in the supernatant of different cultured cell lines and in human cerebral spinal fluid (CSF).14,18,19 Both secretion and release from degenerating cells may account for such results. Alternatively, a secretory product could be directly derived from an additional splicing form.20 The various possibilities could be differentiated by immunolocalizing the precursors in the cells of secretory organs. For this purpose, we focused our attention on secretory cells from both endocrine and exocrine glands. We also have analyzed skeletal muscle cells to test whether the generation of the secreted form is an ubiquitous process or is confined to cells specialized Supported by grants from the DFG through SFB 74/A2 and 317, the Bundesministerium fOr Forschung und Technologie, and the Cusanus Werke. Accepted for publication February 13, 1990. Address reprint requests to Nicoletta Catteruccia, Center for Molecular Biology, University of Heidelberg (ZMBH), Im Neuenheimer Feld 282, D-6900 Heidelberg 1, FRG.

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for secretion. We have studied tissues and organs by immunofluorescence (IF) and immunogold electron microscopy (IEM), employing the monoclonal antibody (MAb) 2-2 G8 raised against a synthetic peptide of the pre-A4 amyloid proteins corresponding to a sequence (aa 506523 of the 695 pre-A4 amyloid) within the putative extracellular domain.6 A polyclonal guinea pig antiserum that recognizes part of the cytoplasmic domain14 also was used in IF. Both antibodies recognize sequences that are common to all the transmembrane forms of the A4 amyloid precursors.

Materials and Methods Immunofluorescence Microscopy Psoas muscle was removed and immediately frozen in isopentane. For IF, 7- to 1 0-w-thick cryostat sections were fixed in 1 % formaldehyde (FA) and 0.02% glutaraldehyde (GA) in phosphate-buffered saline (PBS) for 10 minutes. Nonspecific binding was blocked by preincubating the sections with PBS containing 10% fetal calf serum (FCS) for 30 minutes. The primary antibodies (culture supernatant for the MAb 2-2 G8 or a 1/2000 dilution of the guinea pig antiserum in PBS 10% FCS) were allowed to react for 2 hours at room temperature (RT), and the secondary fluorescinated antibodies for 40 minutes. Peptide inhibition (50 ,g/ml) of antibody binding was performed for each in situ experiment.

Immunogold Electron Microscopy For IEM, tissue blocks (1 mm3) were incubated with 3% FA and 0.05% GA in PBS for 3 hours. The tissues were dehydrated through an increasing ethanol series, embedded in LR White, and cured at 550C for 24 hours. Nonspecific binding was blocked by preincubating the ultrathin sections (70 to 90 nm) with PBS/FCS 10% for 1 hour. The sections then were incubated with MAb 2-2 G8 culture supernatant for 2 hours at RT. Bound antibody was revealed by incubating the sections with a biotinylated antimouse antibody for 1 hour, followed by incubation with streptavidin gold (15-nm gold particles) for 30 minutes. The sections were counterstained with uranyl acetate and lead citrate and examined using a Philips 400 T electron microscope at an acceleration voltage of 80 kV. Controls for each experiment were conducted using peptide inhibition (50 ,g/ml) of antibody binding or an irrelevant mouse monoclonal primary antibody (Culture supernatant from the cell line N-S.4. 1, American Type Culture Collection [ATCC] TIB 110, producing a MAb (IgMk) directed against sheep red blood cells).

Immunoblotting Cell lysates from skeletal muscle, salivary gland, and brain were obtained by treating with sodium dodecyl sulfate (SDS) (2%) organ homogenates. Escherichia coli cells were transfected with a plasmid that allows expression of the pre-A4 amyloid 695 as a fusion protein of the Fd domain of the mouse immunoglobulin heavy chain.14 Sodium dodecyl sulfate lysate from these E. coli cells were used in immunoblot to test the antibody reactivity against the pre-A4 amyloid proteins. The proteins present in the lysates were separated by 10% and 7.5% SDS polyacrylamide gel electrophoresis (PAGE) and electroblotted onto nitrocellulose filters. Nonspecific binding to the nitrocellulose was prevented by saturating the filters with 10% FCS overnight. The filters then were incubated with the pre-A4 amyloid protein antibodies for 1 hour at RT, followed by incubation with a secondary antibody conjugated to alkaline phosphatase for 40 minutes at RT. The disclosing buffer consisted of 0.33 mg/ml nitroblue tetrazolium and 0.165 mg/ml 5-bromo-4-chloro-3-indolylphosphate in AP buffer (100 mmol/l [millimolar] TRIS-HCI, pH 9.5, 100 mmol/l NaCI, 5 mmol/l MgCI2). The reaction was allowed to proceed for 5 minutes at RT.

Peptide Synthesis and Antibody Production The peptide ISEPRISYGNDALMPSLT, which spans from aa 506 to aa 523 of the pre-A4 amyloid 695, and ISQAVHAAHAEINEAGR, which spans from aa 323 to aa 339 of the chicken ovalbumin molecule, were synthesized by the solid-phase method of Merrifield with the modifications described,21 purified by gel-exclusion chromatography on a Bio-Gel P4 column, and coupled together with GA. The peptide belonging to the ovalbumin sequence was used as a source of foreign T cell epitope.2223 BALB-C mice were immunized intraperitoneally 3 times with 100 ,g of the complex formed by the two synthetic peptides in complete (for the first immunization) or incomplete Freund's adjuvant. Five days after the last immunization, the mouse spleen cells were fused with x63 Ag 8.653 myeloma cells24 and subsequently screened for antibody production. The supernatants from cultures of growing hybrids were tested in enzyme-linked immunosorbent assay (ELISA) against the synthetic peptide used for immunization (aa 506-523) coupled to a different carrier (bovine serum albumine). The supernatant from the culture 2-2 G8 contained an antibody (IgMk) that reacted in ELISA against the peptide and in immunoblot against the pre-A4 amyloid 695 (expressed in E. coli). The polyclonal guinea pig antiserum was produced by immunization with a synthetic peptide corresponding to the last 43 carboxyl-terminal residues of the pre-A4 amyloid proteins.14 This antiserum specifically recognizes the cytoplasmic domain and

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Localization of the Amyloid Precursor

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against the Fd fragment of the fusion protein; the antimouse secondary antibody alone gave the same result. Among the proteins of salivary gland and muscle homogenates, the MAb 2-2 G8 decorated bands of various molecular weight in immunoblotting (Figure 2a, b). The rounded shape of the bands of the salivary gland is due to the high content of proteins loaded on the gel. A band corresponding to the molecular weight described for the full-length glycosylated precursor"4 was not observed, although the antibody is able to detect such band among the proteins from a rat brain homogenate (Figure 2c). The bands of lower molecular weight found in salivary gland and muscle homogenates could represent tissue-specific processed forms of the A4 amyloid precursors. On incubation with the corresponding peptide, the antibody reactivity was abolished.

Immunolocalization of the A4 Amyloid Precursors

of mouise immunoglobulin (b and d) anid of a cell lysate from same E. coli strain not expressing the pre-A4 amyloid prothe

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AJPJuly 1990, Vol. 13 7, No. 1

mglmI).

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14 precursor molecules.

Because the sequences of the peptides used to raise the MAb 2-2 G8 and the guinea pig antiserum are identical both in humans and rats,25 all the experiments were carried out on rat tissue. Both antibodies were efficient when used in IF, whereas only the MAb 2-2 G8 was able to react with the tissues after the samples had been processed for IEM. Specificity controls were performed for each experiment by peptide inhibition of antibody binding or by using an irrelevant mouse monoclonal primary antibody.

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Results

b

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Antibody Specificity Among several monoclonal antibodies that were selected on the basis of their reactivity against the synthetic peptide (aa 506-523) used for immunization, 2-2 G8 specifically recognized the A4 amyloid precursors. When used in immunoblotting, the MAb 2-2 G8 decorated several bands among the proteins from a lysate of E. coli (Figure 1), which expressed the pre-A4 amyloid 695 fused to the Fd domain of the mouse immunoglobulins as described by Weidemann et al14 (Figure 1 a). The 1 24-kd band represents the full-length fusion protein, whereas the smaller bands are most likely degradation products, as no reaction was observed against a lysate of nontransfected E. coli (Figure 1 a). On previous incubation of the antibody with the peptide used for immunization, most of the antibody reactivity disappeared, although some bands are still weakly decorated (Figure 1 d). This could be due to the reactivity of the anti-mouse secondary antibody

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22 Catteruccia et al AIPJuly 1990, Vol. 137, No. 1

Figure 3. Immunizogold electron micrograph1s of submaxillary gland sbowing the specific decoration of the MAb 2-2 GI (2. 5 mng/ ml); (a), irrelevan t a ntibody (IgMk, N S. 4.14 mg/ml); (b), 2-2 GC8previously incubated u'ith the corresponding peplide,MA/I aa 506-523 (50 mg/ml), (c) and (d) and (e), differentfields ojfsecells rous incubated uith 2-2 G8I. a X 16,900: b X26,000: c X 14,500; d X3300: e X 7000.

Secretory Organs The adenohypophysis and the salivary glands (submaxillary) were processed for IEM to test whether the mor-

phologic data support the hypothesis that the A4 amyloid precursors are secreted. The electronmicrographs show that the MAb 2-2 G8 decorates the contents of the secretory granules both in the serous cells of the salivary glands

Localization of the Amyloid Precursor

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AJPJuly 1990, Vol. 137, No. 1

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and in some cell types of the adenohypophysis (Figures 3, 4). In the serous cells of the salivary glands, the granules (about 500 nm in diameter) are heavily decorated and the staining pattern indicates that most of the preA4 amyloid protein is associated with the content of the granules (Figure 3 c, d, e). In the adenohypophysis, the decoration of the granules was not so heavy or so uniform. A positive staining both of the endoplasmic reticulum and of the secretory granules was usually found in cells with small dense granules of about 100 nm (Figure 4b). In some cells, the secretory vesicles were negative, while endoplasmic reticulum was decorated by the antibody; in general this was the case for cells with larger granules. Incubation of the antibody with the corresponding peptide resulted in more than 80% inhibition of the staining as estimated by the number of gold particles still decorating the secretory vesicles of the serous cells of the salivary glands (Figure 3b).

Muscle In addition to secretory organs, the expression of the A4 amyloid precursors also was studied in skeletal muscle, where the presence of the corresponding mRNA was shown to be particularly abundant.15 17 Because of the length of the muscle cells, the same cells could be tested with both antibodies. Serial trans-

verse sections were analyzed using the MAb 2-2 G8 and the guinea pig antiserum by IF. The MAb 2-2 G8 and the polyclonal antibodies gave the same pattern of staining in IF. Sections cut along the longitudinal axis of the muscle showed a bright fluorescence along the edge of the myofibrils (Figure 5a, b). In transverse sections, a strong reactivity was observed in the space between the myofibrils (Figure 5c, d). The ultrastructural analysis of the muscle cells by IEM indicated that the A4 amyloid precursors are on or very closely associated with the sarcoplasmic reticulum (Figure 5e, f).

Discussion The ultrastructural localization of the A4 amyloid precursors is important to the understanding of the function of these proteins and the topology of the lesions in Alzheimer's disease. The distribution in tissues and organs and the function of the different A4 amyloid precursors have not yet been elucidated. The RNA expression data that indicate that the pre-A4 amyloid mRNA is ubiquitously expressed15 17 do not provide an explanation for the specific localization of the lesions in Alzheimer's disease. The structural analysis of the sequence of the A4 amyloid precursors6 and the evidence from in vitro translation experiments13 would predict that the proteins explicate their function on the cell membranes. Only biochemical

24 Catteruccia et al AJPu ulj 1990, Vol. 13 7, No. 1

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Figure 5. Transmission (phase contrast) and fluorescence microphotography of skeletal muscle cells, (a) and (b), longitudinal section decorated by the guinea pig antiserum; (b) and (c), transverse section decorated by the MAb 2-2 G8. IEM, (e) and (f) of muscle cells in longitudinal section decorated by the MAb 2-2 G8. a X2000; b X 1260; c X 700; d X 500; e X53, 000; f X 80, 000.

Localization of the Amyloid Precursor

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AJPJuly 1990, Vol. 137, No. I

but no morphologic evidence has so far been provided for the membrane localization of the precursors. Polypeptides thought to have originated from the A4 amyloid precursors have been found in the supernatant of cultured cells and in the CSF.'4"18,19 Although there are examples of active biologic peptides such as epidermal growth factor and transforming growth factor type a, generated from longer precursors that contain a transmembrane and a cytoplasmic domain,269 there is no direct evidence that the pre-A4 amyloid polypeptides found in supernatants and in the CSF are the result of active secretion. If this were the case, it has to be established -whether the generation of the secreted form is a general process that involves all cells or whether it reflects a cell-specific function. In this respect, HeLa and PC12 cells (which were used to detect the A4 amyloid precursors in culture supernatants) cannot be considered representative examples, as they originate from exocrine and endocrine tissues, respectively. For this reason, we decided to analyze the localization of the A4 amyloid precursors in skeletal muscle as well as in submaxillary glands and in the adenohypophysis by employing the MAb 2-2 G8 using IF and IEM. Submaxillary glands were chosen as an example of exocrine glands and because of their easy accessibility. The adenohypophysis was selected as an example of endocrine organ and because of the presence, within the gland, of several cell types that may differ either for the hormone produced or (and) for the processing pathways of the same precursor. The MAb used was shown to recognize the full-length pre-A4 amyloid 695. The MAb 2-2 G8 recognized a molecule of the expected molecular weight only in a lysate of E. coli expressing the pre-A4 amyloid 695 as a fusion protein. When tested by immunoblotting, the MAb 2-2 G8 decorated three bands from 93 to 130 kd in rat brain homogenate that also were recognized by other antibodies directed against the A4 amyloid precursors.14 In salivary gland and muscle homogenates, the antibody reacted with bands of lower molecular weight, which we interpreted as tissue-specific processed products, as this reaction could be blocked by preincubation of the MAb 2-2 G8 with the corresponding peptide. All the tissues processed for IEM were analyzed with the MAb 2-2 G8; for IF, a guinea pig antiserum raised against a synthetic peptide corresponding to the last 43 amino acid at the carboxyl terminus, common to all the known transmembrane forms of the A4 amyloid precursors, was also used. This antiserum in IEM did not give any positive staining, suggesting that the epitope did not survive the treatment used to process the tissues. The results of the immunolocalization of the A4 amyloid precursors in salivary glands and the adenohypophysis show that the protein is mainly localized in the secretory granules, thus indicating an in vivo secretion by both types of organs. The distribution of gold particles in the

secretory granules provides information on how the secreted form is generated. The fact that most of the reactivity is inside the granules, rather than on their surface, strongly suggests that a portion of the pre-A4 amyloid proteins is processed either in the Golgi or in the secretory vesicles before reaching the cell surface. Recently, a partial cDNA sequence for a minor transcript that does not include the transmembrane and cytoplasmic domain has been described.Y0 The reactivity found in the secretory granules cannot be attributed to the protein encoded by this transcript, as it lacks the sequence recognized by the MAb 2-2 G8. However, the possibility still exists that the pre-A4 amyloid protein detected in the secretory granules by the MAb 2-2 G8 originates from a not-yet-identified transcript encoding for a secretory form. The secretory granules of the different cell types show various intensities of staining. Granules of the serous cells of the salivary glands and the population of the small granules in the adenohypophysis are decorated, whereas the large granules of the cells of the adenohypophysis do not show any staining at all. Different levels of expression or different processing pathways, which either spare or destroy the epitope, could account for the differences in the level of staining in the secretory granules of the different cell types. The notion that all the pre-A4 amyloid proteins might be processed and secreted is not consistent with the finding that the putative A4 amyloid precursors in skeletal muscle cells are mainly associated with the sarcoplasmic membranes. The biochemical and morphologic data we provide suggest that the A4 amyloid precursors are processed in a tissue-specific manner. A specific membrane-sorting process involving one or more of the splicing products, each one with a different function and distribution in the tissues examined, could account for the different ultrastructural localization. In addition, tissue-specific factors such as proteases or protease inhibitors could differentially regulate the fate of the A4 amyloid precursors. Specific sorting or targeting of the A4 amyloid precursors in the brain may account for the tissue-specific accumulation of the A4 amyloid protein in Alzheimer's disease.

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Acknowledgments The authors thank Caroline Hilbich of the ZMBH for synthesizing the pre-A4 amyloid peptide, and Prof. Peter Traub for providing use of the electron microscopy facilities at the MPI Ladenburg. Special thanks is due to Dr. Siegfried Kuhn of MPI Ladenburg for his help and encouragement.