Journal of Neuroscience Research 30:687498 (1991)

Rapid Communication Immunolocalization of Alzheimer p=Amyloid Peptide Precursor to Cellular Membranes in Baculovirus Expression System J.R. Currie, N. Ramakrishna, T.G. Burrage, M.-C. Hwang, A. Potempska, D.L. Miller, P.D. Mehta, K.S. Kim, and H.M. Wisniewski New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York

One characteristic of Alzheimer’s disease (AP disease) is the accumulation of amyloid deposits within the extracellular space of the brain and meninges. A 40 amino acid peptide called P-peptide or A4 protein is the subunit of the amyloid fibrils found in these deposits. The sequence of P-peptide is contained within those of a family of larger proteins called the Alzheimer P-amyloid peptide precursor (APP). These APPs contain, in addition to a signal sequence, a hydrophobic sequence that is believed to span cell membranes. Although biochemical studies indicate that some APPs have properties of integral membrane proteins, morphological confirmation of this has not been reported. We recently described an expression system in which human APP,,, cDNA was placed under the transcriptional regulation of the polyhedrin gene promoter in the baculovirus Autographica californica infecting a Spodoptera frugiperda cell line (Ramakrishna et al., Biochem Biophys Res Commun 174:983-989, 1991). As part of a larger biochemical and molecular biological study of APP, we have carried out an immunocytochemicalstudy using antibodies directed against several epitopes within APP to reveal, at both the light and the electron microscopic levels, the cellular localization of APP in the baculovirus expression system. These studies demonstrate that APP,,, is abundantly synthesized and inserted into certain of the membrane compartments of the cell. As early as 24 hr postinfection, APP75, is found associated with all membrane compartments excepting mitochondrial membranes. The patterns of immunolabeling are consistent with our biochemical findings that the protein is processed in these cells so as to release the extracellular domain and to retain a transmembrane and intracellular segment. These data provide the first morphological demonstration of the membrane location of APP,,,, its posttranslational processing to a secreted fragment, and its ex0 1991 Wiley-Liss, Inc.

clusion from the mitochondria1 membranes. This system is especially valuable for identifying conditions under which antibodies raised against APP or appropriate synthetic peptides will react with native APP. Key words: APP, AP, membrane protein, ultrastructure, immunocytochemistry INTRODUCTION In victims of Alzheimer’s disease, Down’s syndrome, and other AP diseases, the brain and meninges contain deposits of the 40 amino acid @/A4 peptide (Glenner and Wong, 1984; Masters et al., 1985). This peptide forms fibrils, which are concentrated in amyloid deposits in the extracellular space of the brain parenchyma and in the vascular elements of the brain and pia-arachnoid. Although the microglia/pericyte type of cell found in these locations has been implicated in the formation of these fibers (Terry and Wisniewski, 1970; Rohrer et al., 1988; Wisniewski et al., 1989, 1990), the precise cellular origin of the peptide has yet to be definitively shown. The P/A4 amino acid sequence is contained within a family of larger proteins, which are alternatively spliced transcripts of a single gene called the Alzheimer P-amyloid peptide precursor (APP) gene (Goldgaber et al., 1987; Kang et al., 1987; Robakis et al., 1987; Tanzi et al., 1987, 1988; Ponte et al., 1988; Kitaguchi et al., Received August 19, 1991; revised October 7, 1991; accepted October 9, 1991. Address reprint requests to Dr. Julia R . Cume, Departments of Pathological Neurobiology and Molecular Biology, Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314. T.G. Burrage’s present address is Plum Island Animal Disease Center, Greenport, NY 11944.

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1988; de Sauvage and Octave, 1989; Golde et al., 1990). TABLE I. Antibody Characteristics The accumulation of P/A4 peptide has been suggested to Antigen result from the faulty processing or degradation of one or Antibody APP domain APP amino acids" more of these presumed precursors (Goldgaber et al., C-terminal 672-695 1987; Kang et al., 1987; Robakis et al., 1987; Tanzi et R57 PIA4 R2 1 597-620 al., 1987; Kitaguchi et a]., 1988). Four of the five tran- 4G8 PlA4 597-620 scripts of the APP 'gene described so far (APP,,,, 6E 10 pJA4 597-620 Midregion 420-440 APP,,, , APP,,,, APP695) contain the P-peptide se- R12 N-terminal 42-62 quence and, thus, could .serve as a precursor. The C- R7 i Glial fibrillary acidic protein terminal 12-14 amino acids of P-peptide represent the GFAP AcNPV surface glycoprotein 64 external one-half of the putative transmembrane domain GP64 4D11 Cytomegalovirus phosphoprotein 65 of APP (Kang et al., 1987; Dyrks et al., 1988). Bio- NRS Pooled nonimmune rabbit IgG chemical evidence supports the membrane location of APPs (Dyrks et al., 1988; Selkoe et al., 1988; Weide- "Sequence numbered according to Kang et al. (1989). mann et al., 1989; Oltersdorfet al., 1990; Gardella et al., 1990); however, immunocytochemical studies, even those performed at the ultrastructural level (Card et a]., 1988; Benowitz et al., 1989; Klier et al., 1990), have These recombinant viruses containing the sebeen contradictory and inconclusive, indicating the pres- quences for APP,,, (AcNPV-APP,,,) and tRNA (Acence of APP immunoreactivity within lysosomes (gran- NPV-tRNA) or the wild-type virus were allowed to inular elements in the cell) or in the extracellular space. As fect cultures of Spodopteru frugiperdu cells (Sf9 strain, a part of our larger efforts to understand the synthesis and from American Type Culture Collection) at a multiplicity processing of APP and the biogenesis of the P/A4 pep- of infection of one plaque-forming unitkell. This retide in AD, this study uses an eukaryotic expression sys- sulted in a low level infection, which allows several cytem and light and electron microscopic immunocyto- cles of viral replication before cell lysis begins at about chemistry to demonstrate morphologically the membrane 72--96 hr postinfection (hpi). After 1 hpi, the inoculum insertion and compartmentalization of APP,,, . Two pre- was replaced with fresh Grace's medium (Gibco) supvious publications describe the molecular biology and plemented with 10% heat-inactivated fetal bovine serum biochemistry of this system (Ramakrishna et al., 1991) and the cultures were incubated at 27°C. In some experand the anti-APP antisera employed (Potempska et al., iments, uninfected cells were used as controls. At 24, 1991). 48, 52, 66 and 72 hpi, the cells were gently washed with serum-free medium or phosphate-buffered saline (PBS) and processed for microscopy. MATERIALS AND METHODS For light microscopic analysis, cells were briefly The APP cDNA construct used in this study has fixed (10-20 min) in either ice-cold 70% methanol or been described in detail (Ramakrishna et al., 1991). In room temperature 4% paraformaldehyde in phosphate brief, the entire coding sequence for human APP,,, was buffer (pH 7.4). The cells were fixed where they were inserted just downstream from the Autographica califor- grown on coverslips or in flaskettes or were fixed as nicu nuclear polyhedrosis virus (AcNPV) polyhedrin Cytospin preparations (at 40g, 10 min). Rabbit polyprotein promoter region; the APP protein is thus ex- clonal antibodies were affinity purified on APP synpressed while the polyhedrin protein is not. The polyhe- thetic-peptide columns as described in Potempska et al. drin protein is responsible for occluding enveloped virus (1991), and the optimum working concentrations were particles in the nucleus into crystalline structures called determined by serial dilution. The antibodies were dipolyhedra. The polyhedrin protein is usually expressed rected against epitopes found in the N-terminal (R71), late in infection (after about 10 hr) and at very high midextracellular (R12), P/A4 (R21), and C-terminal abundance for several days. By inserting the APP se- (RS7) domains of APP,,,. (See Table I for the descripquence behind the polyhedrin promoter, a very efficient, tions of the antibodies.) Protein A-purified normal high-level production of APP can be accomplished (Ra- pooled rabbit serum (Vector Laboratories) and antiglial makrishna et a]., I99 1). A similar construct containing a fibrillary acidic protein antibody raised in rabbit (DakotRNA coding sequence that also interrupts the polyhedrin patts) were used as rabbit IgG controls. Two mouse moncoding sequence (kindly supplied by Dr. P. Saikumar, oclonals (4G8 and 6E10) directed against the first 24 Wistar Institute, Philadelphia, PA) and wild-type Ac- amino acids of the @/A4 sequence (Kim et al., 1988; NPV (gift of Dr. M.D. Summers, Texas A&M Univer- 1990) were affinity purified before use, and a monosity, College Station, TX) were used as controls. clonal antibody against a cytomegalovirus 65 kD matrix

APP Localization to Membranes

phosphoprotein (Kim et al., 1983) was used as a mouse ascites control. The specificity of the affinity-purified APP antibodies has been tested by immunoblotting of human, rat, and recombinant APP (Potempska et al., 1991). To detect viral infection and to mark the membrane compartments of the cell, a monoclonal antibody (ascites fluid) directed against the AcNPV surface glycoprotein GP64 (kindly supplied by Dr. L.E. Volkman, University of California at Berkeley, CA) was used. The fixed cells were washed in PBS containing azide and blocked in 4% normal goat serum or 3% bovine serum albumin in PBS-azide for 2 hr at 4°C. Serial dilutions of several different antibodies in blocking solution were used for a 2 hr incubation at room temperature. (The cells fixed in paraformaldehyde were permeabilized by including 0.1% Triton-X 100 in all of the blocking, washing, and antibody solutions.) After a PBS wash, the cells were incubated either in goat antirabbit IgG conjugated to rhodamine or phycoerythrin (GARRh, GAR-PE) 1:200 or in goat antimouse IgG conjugated to fluorscein isothiocyanate (GAM-FITC) 1:200 in blocking solution for 1 hr. After final PBS and water washes, the cells were coverslipped with Mowiol (Calbiochem) as an antiquenching agent and examined under a Zeiss Axiophot fluorescence microscope. Photographs were taken with Kodak TMAX 400 or Kodak Ektachrome 800/1600 film. To compare the density of the label, the control cells were photographed at approximately the same exposure as that used for the labeled cells. For ultrastructural analysis, the infected and uninfected Sf9 cells (52 and 66 hpi) were fixed in 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 hr at 4°C. The cells were pelleted and processed either for routine epon embeddement, or they were dehydrated to 90% ethanol and embedded in Lowicryl. The Lowicryl blocks were polymerized with ultraviolet light at room temperature for 18 hr. Thin sections were cut and collected on uncoated or formvar-coated nickel grids. Immunoelectron microscopy was carried out with 1% bovine serum albumin (BSA) in PBS as the blocking solution followed by the monoclonal and polyclonal antibodies in blocking solution or with blocking solution alone. Some sections on uncoated grids were labeled on both sides with the same antibody, while other sections were labeled on only one side. To test the colocalization of some antibodies, grids were labeled on each side with a different antibody. After washing with 1% BSA, 0.05%Tween-20 in PBS, the distributions of the antibodies were revealed with a 1:50 dilution (in 1% BSA, 0.5 M NaCl, 0.05% Tween-20 in phosphate buffer) of 10 nm colloidal gold-covered goat anti-IgG against the appropriate species or of protein A-gold. In the double-sided experiments, the antibody

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binding on the second side was detected with 5 nm colloidal gold IgG. The thin sections were stained with uranyl acetate and lead citrate and were examined and photographed in a Hitachi 7000 electron microscope. Quantification of the APP molecules (as represented by gold particles) per micrometer of plasma membrane was carried out on APP-infected cells labeled with the anti-C-terminal antibody and the number compared with that present on the wild-type-infected cells. Micrographs of the different specimens were taken at X 20,000 and printed at X 60,000. Using the Jandel Sigma-scan program and tablet, measurements were taken of lengths of plasma membrane (in millimeters) and the number of gold particles found along the plasma membranes that had been measured on each micrograph. The total length of membrane measured, the mean number of gold particles per micrometer plasma membrane, and the standard error of the mean were then calculated for each preparation.

RESULTS The presence of human APP,,, in recombinant virus-infected Sf9 cells was determined with affinity-purified antibodies raised against synthetic peptides corresponding to distinct domains in APP. At 52 and 72 hpi, times at which the protein had been expressed for many hours, phycoerythrin-conjugated second antibody easily detected APP epitopes in alcohol-fixed cells. The APP C-terminal antibody intensely labeled the AcNPVAPP,,, infected cells, while the control, uninfected cells remained unlabeled (data not shown). The antibody labeled the plasma membrane, the nuclear membrane, and the cytoplasmic membrane compartments of the AcNPVAPP infected cells. However, the fluorescence was so bright that the resolution of different cytoplasmic membrane compartments was difficult. All cells incubated with the glial fibrillary acidic protein control antibody remained unlabeled (data not shown). Since the level of expression of APP was so high in the cell by 2 days postinfection, cells in which the protein had been expressed for only a few hours (at 24 hpi) were fixed briefly in paraformaldehyde to differentiate better the various membrane compartments. To control for the effects on cell metabolism of polyhedrin protein loss as a result of APP sequence insertion, cells were infected with an AcNPV construct containing a tRNA sequence similarly inserted into the polyhedrin coding sequence. Controls for nonspecific labeling were carried out by omitting the primary antibody (secondary antibody alone) or by using protein A-purified pooled normal rabbit IgG or anticytomegalovirus ascites fluid as non-APP primary polyclonal or monoclonal antibody, respectively. The results of this experiment are summa-

GAR-Rh

NRS-GAR-Rh

a-CMV-GAM-FITC

Fig. 1.

GAM-FITC

APP Localization to Membranes

rized in Figure 1. AcNPV-APP-infected cells remained unlabeled when incubated with the normal rabbit IgG (NRS), the anticytomegalovirus ascites, or the two secondary antibodies alone (Fig. 1A-D). (The original film for Figure 1D was exposed about 10 times longer than the frames for the other controls, so some autofluorescence is visible as a halo in the cells.) All three antiAPP antibodies labeled membrane compartments in the AcNPV-APP cells (Fig. 1E-G), whereas the AcNPVtRNA-infected cells remained unlabeled by these antibodies (Fig. 11-K). The anti-AcNPV surface glycoprotein antibody labeled both types of infected cells (Fig. lH,L). In the AcNPV-APP-infected cells, plasmalemma1 labeling is not as apparent with the antibodies directed against the extracellular APP epitopes (Fig. 1F,G) as with the antibody directed toward the cytoplasmic epitope (Fig. 1E). The distribution of APP revealed by the C-terminal APP antibody is illustrated at a higher magnification in Figure IM. Most of the label appears over globular compartments in the perinuclear zone of the cytoplasm. Some label is also present over the plasmalemma and the nuclear membrane. Figure 1N illustrates, at low power, the distribution of the AcNPV antibody fluorescence label at 24 hpi. It is obvious that, at the multiplicity of infection used, not every cell is infected or is expressing the AcNPV surface protein at a level that is detectable with immunofluorescence at this time. However, many cells do contain a level of infection high enough to produce readily discernible APP. By 72 hpi, virtually all of the cells have become infected and are expressing APP at

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a high level (Ramakrishna et al., 1991), so that, as was previously stated, an analysis of the fluorescence pattern is difficult (data not shown). Ultrastructural studies confirm and extend these observations. In the cytoplasm, the usual organelles are seen: rough and smooth endoplasmic reticulum, ribosomes, mitochondria, lysosomes, and vacuoles. The morphology of the Sf9 cells infected with the AcNPVAPP construct for 48 hr as revealed in epon-embedded specimens is illustrated in Figure 2. The cells are considerably swollen, primarily as a result of the enlargement of the nucleus due to viral replication. By this stage of infection, most of the viral particles are present in the nucleus rather than the cytoplasm, but viral inclusion into polyhedra is abortive. The cytoplasm is filled with swollen endoplasmic reticulum and other membranous structures. Small and large vacuoles filled with what appear to be empty viral envelopes and other tubulomembranous structures are found in the perinuclear zone. The mitochondria appear normal. Some compact fibrillar material is present in the cytoplasm, but this structure is also present in cells infected with other polyhedrin viruses (Summers and Arnott, 1969) and AcNPV constructs (data not shown; Hess et al., 1989; van Lent et al., 1990). Although the viral cytoplasmic fibrils have some physical characteristics of P-amyloid fibrils, i.e., a diameter between 5 and 10 nm and a relatively long, straight shape (Hess et al., 1989; Terry et al., 1964), recent studies have identified this fibrillar material as the viral p i 0 protein that is also involved in polyhedron formation (van der Wilk et al., 1987; Williams et al., 1989). Attempts to label the epon-embedded specimens with our antibody preparations were not successful, so a second method of embeddement was used. Fig. 1. Light micrographs of Sf9 cells 24 hpi with AcNPVThe morphology of the immunolabeled cells from APP,,, (rows 1, 2, and 4) or AcNPV-tRNA (row 3). Row 1: Lowicryl-embedded specimens can be assessed by inAntibody controls. A: GAR-Rh alone. B: Normal rabbit serum spection of Figure 3. While the resolution of cellular IgG (NRS) and GAR-Rh. C: Anti-CMV and GAM-FITC structures is not as clear as with epon embeddement, the (slightly overexposed). D: GAM-FITC alone. No specific lavarious normal and abnormal cellular elements illusbeling is seen with any of these control incubations. Rows 2 trated in the epon-embedded section are easily disand 3: Comparison of APP-infected cells (row 2) with tRNAcernible in the Lowicryl-embedded material. These infected cells (as controls; row 3) using anti-APP and antiphotographs were taken of sections from AcNPV-APPAcNPV antibodies; E, I: Anti-APP C-terminal antibody, R54. F, J: Anti-P-peptide antibody, R21. G, K: Anti-APP N-ter- infected cells incubated with the APP C-terminal antiminal antibody, R71. H, L: Anti-AcNPV GP64 monoclonal body and detected using goat antirabbit IgG complexed antibody. Membrane compartments in APP-infected cells in with colloidal gold. No etching of the sections was necrow 2 label with all four antibodies, while tRNA-infected cells essary. The gold particles heavily and specifically label in row 3 only label with the viral surface protein antibody. Row the membranous compartments of the cell, with the ex4: M: Higher magnification of AcNPV-APP infected cell la- ception of the mitochondral membranes. The fibrous inbeled with APP C-terminal antibody and GAR-Rh. Plasma clusions (p10 protein) in the cytoplasm are not labeled by membrane, nuclear membrane, and cytoplasmic membrane the APP antibody. compartments are labeled by the antibody. N: Lower magniA second example of the labelling pattern of the fication of AcNPV-APP infected cells labeled with antiC-terminal antibody over the APP-infected cells comAcNPV GP64 antibody and GAM-FITC. By 24 hpi many cells already show significant infection by and replication of the pared with control cells can be seen in Figure 4A. This specimen was labeled on both sides of the grid. The gold virus. Rows 1-3, X 650. M, X 1,550. N, X 150.

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Fig. 2 . Electron micrograph of epon-embedded Sf9 cell infected with AcNPV-APP,,, 48 hpi. Note the enlarged nucleus containing viral particles and an abortive polyhedron structure (asterisk). The perinuclear cytoplasm is filled with swollen

membranous compartments (arrowheads) and fibrillar accumulations (arrows). The mitochondria appear normal. Some vacuoles have ribosomes associated with their limiting membrane (inset).

label over the APP-expressing cell is dense and is specifically located over three membranous compartments: nuclear, plasmalemmal, and cytoplasmic (Fig. 4A). Only a very few scattered gold particles are seen over the control cells (Fig. 4B). Specimens used for quantitation of the plasmalemmal label were incubated on one side of

the grid to make the quantitation easier, since the unsupported double-sided labeled sections were extremely fragile (Fig. 5A,B). Fewer gold particles are present, but their distribution is the same. The results of the analysis are presented in Table 11. There were at least 52 times as many gold particles on the APP-infected cell plasma-.

APP Localization to Membranes

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Fig. 3. Electron micrograph of Lowicryl-embedded Sf9 cell infected with AcNPV-APP,,, 48 hpi and labelled on both sides of the grid with the APP C-terminal antibody detected with 10 nm colloidal gold conjugated to goat antirabbit IgG. The label

is concentrated over cytoplasmic membranous accumulations (arrowheads) and the plasmalemma (arrows). The fibrillar structures (asterisks) are essentially unlabeled.

lemma compared with the controls. We did not quantitate the gold particles over the intracellular membrane compartments in the APP-infected cells. It is obvious that the difference in labeling over many of these areas compared with the control cells is even more dramatic than that over the plasmalemma. Ultrastructural examination of APP-infected cells incubated with the N-terminal antibody (Fig. 6A) or the P-peptide antibody (Fig. 6B) revealed that they also labeled cellular membranes. Most of the label was located over aggregations of membranes in the cytoplasm. Comparatively little was found over the nuclear and plasma membranes, and none was found over the fibrous inclusions. When the sections from the APP-infected cells were double-labeled with the antibodies directed against the C-terminal domain (R57, 10 nm) and the midregion domain (R12, 5 nm), the compartment in which both gold particles are found corresponds to the membranous accumulations in the cytoplasm. The micrograph is reproduced at a higher magnification so that the 5 nm gold particles are discernible. The R 12 antibody demonstrates

a labeling pattern similar to the other antibodies recognizing APP epitopes N-terminal to the transmembrane region.

DISCUSSION The identity of the human APP,,, protein produced in the recombinant baculovirus-infected insect cell system has been confirmed through immune blotting and N-terminal sequencing (Ramakrishna et al., 1991). Immunofluorescence and immunogold analyses of the baculovirus expression system utilized in this study, employing antibodies directed against external and cytoplasmic domains of APP, indicate that human APP,,, is inserted in the correct transmembrane orientation and transported to the cell surface. The human APP apparently is not recognized as an abnormal, exogenous protein and thus escapes degradation within the rough endoplasmic reticulum (RER). The high level of production of APP,,, is not in itself lethal to the cells, nor does

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Fig. 4. Electron micrographs of Sf9 cells 48 hpi with AcNPV,,, (A) or wild-type AcNPV (B) embedded in Lowicryl, incubated on both sides of the grid with the APP Cterminal antibody, and detected using GAR-colloidal gold. A: APP,,,-infected cells were heavily labeled over all cell mem-

brane compartments with the exception of mitochondria: plasma membrane (pm), nuclear membrane (nm), and cytoplasmic membranes (cm). B: Cells infected with wild-type virus were only very lightly and nonspecifically labeled.

simple overproduction lead to 6-amyloid fiber formation. Although northern and western blots do not reveal expression of the APP mRNA or the protein by 24 hpi in cells infected at a multiplicity of infection (moi) of 1 (Ramakrishna, et al. 1991), some of the more highly infected cells are strongly and specifically immunoreactive with the APP antibodies at this early stage of infection. The polyhedrin protein normally is detectable on acrylamide gels by 8-10 hpi (Carstens et al., 1979), and polyhedron formation occurs in the nucleus by 18 hpi (Knudson and Harrap, 1976). Thus the presence of APP,,, in those cells with a high level of infection at 24 hpi is consistent with the expected activation of the polyhedrin promoter several hours before this time. That is, although only a few cells are infected with enough recombinant APP virus to be detectable at 24 hpi by immunofluorescence microscopy, the APP has been synthesized in the cells for at least 14 hr. The cell homogenates used for mRNA and gel electrophoresis studies would not allow the detection of a few highly infected cells in a larger population of cells with a lower level of expression. This study is the first to demonstrate definitively at

the ultrastructural level that APP,5, is, in fact, a membrane protein. Previous immunochemical studies at the electron microscopic level have produced a variety of results depending on the tissue or cell source, the antisera used, and the state of fixation. For example, APP has been localized, using anti-P-peptide antibodies, within the perikarya of neurons and glia in the rodent brain (Card et al., 1988). No assignment was made in that study to any structure or compartment in those cells. In another study, APP appeared to be restricted to secondary lysosomes in human cortical neurons when a C-terminal APP antiserum was used (Benowitz et al., 1989). The identity of the immunoreactive material in these studies with APP was not proved. In a third study, an antiserum directed against an extracellular epitope of APP was found associated primarily with the extracellular matrix of a neural cell line derived from the rat eye (Klier et al., 1990). Although other transmembrane proteins such as the influenza virus hemagglutinin (Possee, 1986; Kuroda et al., 1986) and the human epidermal growth factor receptor (Greenfield et al., 1988) have been studied in the baculovirus expression system, these investigations included immunolabeling only at the light microscopic

APP Localization to Membranes

Fig. 5. Micrographs of AcNPV-APP Sf9 cells 48 hpi labeled on a single side with the APP C-terminal antibody and used for morphometric analysis of the density of label on the plasma membrane. A: Even with the lower level of labeling, the spec-

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ificity of the label for the three cell membrane compartments is apparent in the AcNPV-APP-infected cells. B: No specific label is present in the wild-type cell.

TABLE 11. Morphometric Analysis of Gold ParticleslMicrometer of Plasmalemma

Cell type APP-infected Wild-type

Number of micrographs measured

Total length of plasmalemma

Total gold

Mean

(mm)

particles

goldlkm

SEM

25 25

6,266.44 6,871.79

737 16

6.956 0.134

3.093 0.175

level. By the use of colloidal gold immunolabeling at the ultrastructural level, we have definitively localized human APP,5, in a eukaryotic cell line to the membrane compartments: i.e., nuclear membrane, plasma membrane, and cytoplasmic membranes. This study demonstrates that APP is excluded from the mitochondria1 membranes, an observation not available from the biochemical analysis of APP synthesis. The cytoplasmic membrane compartments include the RER, aggregations of vesicles or tuboreticular structures, and sometimes membranes enclosed within a membrane, such as might be found in lysosomal elements. No label appeared over dense bodies that might be interpreted to be secondary lysosomes. The cytoplasmic membrane compartments containing APP,, are concentrated in the perinuclear region. No APP,5, epitopes were detected in the cytoplasmic fibrillar aggregations now known to be bundles of the viral protein p10 (van der Wilk et al., 1987). Antibodies directed against the extracellular epitopes of APP labeled the membranes much less intensely

than did the antibody to the intracellular (C-terminal) epitope. Although it is possible that this difference results from the differing accessibilities of the epitopes, we prefer another explanation. Our biochemical studies of APP-infected Sf9 cells demonstrate that the cells rapidly cleave the membrane-bound APP in a manner closely resembling the natural processing of APP in mammalian cells (Ramakrishna et al., 1991; Esch et al., 1990). Proteases in both systems cleave APP near its putative membrane-spanning sequence and release the extracellular form of the protein. We conclude that the cytoplasmic domain of APP turns over relatively slowly and accumulates in the cells to a much higher level than intact APP. It is interesting that the insect cells rapidly process APP in a manner similar to mammalian cells. Sf9 cells do not appear to have an endogenous protein similar enough to human APP to be detected by immunochemical or RNA-hybridization techniques (see also Ramakrishna et al., 1991). Drosophila contains a protein with some structural homology to APP (Rosen et al.,

Fig. 6 . Electron micrographs of Lowicryl-embedded Sf9 cells infected with AcNPV-APP,,, , fixed 48 hpi, and incubated with anti-APP antibodies. The N-terminal antibody R71 (A) and the P-peptide antibody 6E10 (B) heavily and specifically label certain aggregations of membranes in the cytoplasm, while the plasma membrane (top) and the nuclear membrane

(bottom) remain relatively unlabeled. C: In a high-magnification micrograph from a double-label study, the antibodies directed against the C-terminus (R57, 10 nm gold) and the midregion of the external domain (R12, 5 nm gold) are colocalized primarily in the cytoplasmic membranous aggregations.

APP Localization to Membranes

1989); however, this protein does not possess a sequence similar to the cleavage site of APP. Because the distribution of the APP in a recombinant cell line overexpressing the protein does not necessarily reflect the distributions found in more differentiated cells, such as glia, neurons, or smooth muscle cells, we have not attempted to explore further the finer points concerning the subcellular localization of APP in this system. Appropriate constructs are being investigated in cells more similar to those implicated in P-peptide formation in the brain. However, this baculovirus expression system has proven invaluable for the determination of the optimal processing of APP-containing cells, which results in the preservation both of the antigenicity of the various APP epitopes to which antibodies have been raised and of the ultrastructural integrity of the cells. The abundance of APP produced by these cells and its appropriate subcellular location in membranes, rather than in cytoplasmic accumulation as in Escherichia coli expression systems, make the characterization of the APP antibody binding patterns much more feasible than in tissues in which there is either little APP expressed or little or none preserved after tissue processing. The most basic questions about the function and turnover of APP have remained unanswered because APP has not been reliably localized in cells and tissues by the currently available antisera raised to synthetic peptides derived from the APP amino acid sequence. Antisera that identify APP on immunoblots often give equivocal results when applied to fixed cells or tissues. Fixation may block APP epitopes and create artificial epitopes. These problems most likely account for the diversity in the reported localization of APP (Benowitz et al., 1989; Bobin et al., 1987; Card et al., 1988; Kim et al., 1988; Selkoe et al., 1988; Shivers et al., 1988; Stern et al., 1989; Tate-Ostroff et al., 1989; Zimmerman et al., 1988). The system described here is, therefore, especially valuable for identifying antibodies that react with the native form of APP. The baculovirus expression system is proving to be a most useful system for studying the basic cell biology of proteins including ultrastructural localization, biosynthesis, posttranslational processing, and ligand binding and for providing large amounts of the native protein for such functional studies and for antibody production against full-length proteins.

ACKNOWLEDGMENTS This study was funded by NIH grants POI-AGO4220-06 and NS2523 I , by SENETEK PLC, and by the NYS OMRDD. The photographic assistance of Mary Ellen Nascimento, Dan Klitnik, and Richard Weed is gratefully acknowledged. Ms. A.T. Heaney provided valuable assistance with the morphometric analysis. The

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authors thank Louis Faso, head of the Electron Microscopy Core Facility, Institute for Basic Research, for his assistance. Robert Gould and David Soifer provided valuable suggestions concerning the manuscript.

REFERENCES Benowitz LI, Rodriguez W, Paskevich P, Mufson EJ, Schenk D, Neve RL (1989): The amyloid precursor protein is concentrated in neuronal lysosomes in normal and Alzheimer disease subjects. Exp Neurol 106:237-250. Bobin SA, Cume JR, Merz PA, Miller DL, Styles J, Walker WA, Wen GA, Wisniewski HM (1987): The comparative immunoreactivities of brain amyloids in Alzheimer’s disease and scrapie. Acta Neuropathol 74:313-323. Card JP, Meade RP, Davis LG (1988): Immunocytochemical localization of the precursor protein for P-amyloid in the rat nervous system. Neuron 1:835-846. Carstens EB, Tjia ST, Doerfler W (1979): Infection of Spodoprera frugiperdu cells with Autogrupha californica nuclear polyhedrosis virus. I. Synthesis of intracellular proteins after virus infection. Virol 99:386-398. de Sauvage F, Octave J-N (1989): A novel mRNA of the A4 amyloid precursor gene coding for a possibly secreted protein. Science 2451651-653. Dyrks T, Weidemann A, Multhaup G , Salbaum MJ, Lemaire H-G, Kang J, Muller-Hill B, Masters CL, Beyreuther K (1988): Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer’s disease. EMBO J 7:949957. Esch FS, Keim PS, Beattie EC, Blacker RW, Culwell AR, Oltersdorf T, McClure D, Ward P (1990): Cleavage of amyloid betapeptide during constitutive processing of its precursor. Science 248:1122-1124. Gardella JE, Ghiso J, Gorgone GA, Marratta D, Kaplan AP, Frangione B, Gorevic PD. (1990): Intact Alzheimer precursor protein (APP) is present in platelet membranes and is encoded by platelet mRNA. Biochem Biophys Res Commun 173:12921298. Glenner GG, Wong CW (1984): Alzheimer’s disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120:885890. Golde TE, Estus S , Usiak M, Younkin LH, Younkin SG (1990): Expression of beta amyloid protein precursor mRNAs: Recognition of a novel alternatively spliced form and quantitation of Alzheimer’s disease using PCR. Neuron 4:253-267. Goldgaber D, Lerman MI, McBridge OW, Saffiotti U, Gajdusek DC (1987): Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer’s disease. Science 2351877-880. Greenfield C, Patel G , Clark S, Jones N, Waterfield MD (1988): Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector. EMBO J 7:139-146. Hess RT, Goldsmith PA, Volkman LE (1989): Effect of cytochalasin D on cell morphology and AcNPV replication in a Spodoptera frugiperda cell line. J Invert Pathol 53:169-182. Kang J, Lemaire H-G, Unterbeck A, Salbaum JM, Masters CL, Grzeschik K-H, Multhaup G, Beyreuther K, Muller-Hill B (1987): The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell surface receptor. Nature 325:733-736. Kim KS, Sapienza VJ, Chen CM, Wisniewski K (1983): Production and characterization of monoclonal antibodies specific for a

698

Currie et al.

glycoprotein polypeptide of human cytomegalovirus. J Clin Microbiol 18:331-343. Kim KS, Miller DL, Sapienza VJ, Chen CJ, Bai C, Grundke-Iqbal I, Currie JR, Wisniewski HM (1988): Production and characterization of monoclonal antibodies reactive to synthetic cerebrovascular amyloid peptide. Neurosci Res Commun 2: 121-130. Kim KS, Wen G, Bancher C, Chen JC, Sapienza VJ, Hong H, Wisniewski HM ( 1990): Quantitation of amyloid P-protein with two monoclonal antibodies. Neurosci Res Commun 7: 113-122. Kitaguchi N, Takahashi Y, Tokushima Y, Shiojiri S , Ito H (1988): Novel precursor of Alzheimer disease amyloid protein shows protease inhibitory activity. Nature 331 530-532. Klier FG, Cole G, Stallcup W, Schubert D (1990): Amyloid P-protein precursor is associated with extracellular matrix. Brain Res 515:336-342. Knudson DL, Harrap KA (1976): Replication of a nuclear polyhedrosis virus in a continuous cell culture of Spodopterafrugiperda: Microscopy study of the sequence of events of the virus infection. J Virol 17:254-268. Kuroda K, Hauser C, Rott R, Klenk H-D, Doerfler W (1986): Expression of the influenza virus hemagglutinin in insect cells by a baculovirus vector. EMBO J 51359-1365. Masters CL, Simons G , Weinmann NA, Multhaup G, McDonald BL, Beyreuther K (1985): Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci USA 82:4245-4249. OltersdorfT, Ward PJ, Henriksson T, Beattie EC, Neve R, Lieberburg I, Fritz LC (1990): The Alzheimer amyloid precursor protein. Identification of a stable intermediate in the biosyntheticidegradative pathway. J Biol Chem 265:4492-4497. Ponte P, Gonzalez-DeWhitt P, Schilling J, Miller J , Hsu D, Greenberg B, Davis K , Wallace W, Lieberburg J, Fuller F, Cordell B (1988): A new A4 amyloid m RNA contains a domain homologous to serine proteinase inhibitor. Nature 33 1525-527. Possee RD (1986): Cell surface expression of influenza virus hemagglutinin in insect cells using a baculovirus vector. Virus Res 543-59. Potempska A, Styles J, Mehta P, Kim KS, Miller DL (1991): Purification and tissue level of the beta amyloid peptide precursor of rat brain. J Biol Chem 266:8464-8469. Ramakrishna N , Saikumar P, Potempksa A, Wisniewski HM, Miller DL (1991): Expression of human Alzheimer amyloid precursor protein in insect cells. Biochem Biophys Res Commun 174: 983-989. Robakis NK, Ramakrishna N, Wolfe G, Wisniewski HM (1987): Molecular cloning and characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid peptide. Proc Natl Acad Sci USA 84:4190-4194. Rohrer A, Gray EG, Paula-Barbosa M (1988): Alzheimer’s disease: Coated vesicles, coated pits and the amyloid-related cell. Proc R SOCLondon [Biol] 232:367-373. Rosen DR, Martin-Morris L, Luo L, White K (1989): A Drosophila gene encoding a protein resembling the human P-amyloid protein precursor. Proc Natl Acad Sci USA 86:2478-2482. Selkoe DJ, Podlisny MB, Joachim CL, Vickers EA, Lee G, Fritz LC, Oltersdorf T (1988): Beta-amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues. Proc Natl Acad Sci USA 85:7341-7345. Shivers BD, Hilbich C, Multhaup G, Salbaum M, Beyreuther K,

Seeburg PH (1988): Alzheimer’s disease amyloidogenic glycoprotein: Expression pattern in rat brain suggests a role in cell contact. EMBO J 7:1365-1370. Stern RA, Otvos L, Trojanowski JQ, Lee VM-Y (1989) Monoclonal antibodies to a synthetic peptide homologous with the first 28 amino acids of Alzheimer’s disease beta-protein recognize arnyloid and diverse glia and neuronal types in the central nervous system. Am J Path01 134:973-978. Summers MD, Arnott HJ (1969): Ultrastructural studies on inclusion formation and virus occlusion in nuclear polyhedosis and granulosis virus-infected cells of Tricoplusa ni (Hubner). J Ultrastruct Res 28:462-480. Tanzi RE, Gusella JF, Watkins PC, Bruns GAP, St. George-Hyslop PH, Vankeuren ML, Patterson D, Pagan S, Kurnit DM, Neve RL (1987): Amyloid beta protein gene: cDNA, mRNA distribution, and genetic linkage near Alzheimer locus. Science 235: 880-884. Tanzi RE, McClatchey AI, Lamperti ED, Villa-Komaroff L, Gusella JF, Neve RL (1988): Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer’s disease. Nature 33 1528-530. Tate-Ostroff B, Majocha RE, Marotta CA (1989): Identification of cellular and extracellular sites of amyloid precursor protein extracytoplasmic domain in normal and Alzheimer disease brains. Proc Natl Acad Sci USA 86:745-749. RD, Wisniewski HM (1970): The ultrastructure of the neurofibrillary tangle and the senile plaque. In Wolstenholme GEW, O’Conner M (eds): “Ciba Foundation Symposium on Alzheimer’s Disease and Related Conditions.” London: Churchill, pp 145-168. RD, Gonatas NK, Weiss M (1964): The ultrastructure of the cerebral cortex in Alzheimer’s disease. Trans Am Neurol Assoc 89:12. van der Wilk F, van Lent JWM, Vlak JM (1987): Immunogold detection of polyhedrin, p10 and virion antigens in Autographa californica nuclear polyhedrosis virus-infected S. frugiperda cells. J Gen Virol 68:2615-2623. van Lent JWM, Groenen JTM, Klinge-Roode EC, Rohrmann GF, Zuidema D, Vlak JM (1990): Localization of the 34 kDa polyhedron envelope protein in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus. Arch Virol I I1:103-114. Weidemann A, Konig G, Bunke D, Fisher P, Salbaum JM, Masters CL, Beyreuther K (1 989): Identification, biogenesis and localization of precursors of Alzheimer’s disease A4 amyloid protein. Cell 57:115-126. Williams GV, Rohel DZ, Kuzio J, Faulkner P (1989): A cytopathological investigation of Autographa californica nuclear polyhedrosis virus p10 gene function using insertion deletion mutants. J Gen Virol 70: 187-202. Wisniewski HM, Wegiel J, Wang KC, Kujawa M, Lach B (1989): Ultrastructural studies of the cells forming amyloid fibers in classical plaques. Can J Neurol Sci 16535-542. Wisniewski HM, Vorbrodt AW, Wegiel J , Morys J, Lossinsky AS (1990): Ultrastructure of the cells forming amyloid fibers in Alzheimer disease and scrapie. Am J Med Genet Suppl 7: 287-297. Zimmerman K, Herget T, Salbaum JM, Schubert W, Hilbich C, Cramer M, Masters CL, Multhaup G, Kang J, Lemaire H-G, Beyreuther K, Starzinski-Powitz A (1988): Localization of the putative precursor of Alzheimer’s disease-specific amyloid at nuclear envelopes of adult human muscle. EMBO J 7:367-372.