184

Brain Research, 503 (1991) 184-19,4 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993191/$03.50 A DONIS 000689939117122I)

BRES 17122

Localization of Alzheimer flA4 amyloid precursor protein at central and peripheral synaptic sites Walter Schubert 1'2, Reinhard Prior 1, Andreas Weidemann 1, Heinrich Dircksen 3, Gerd Multhaup 1, Colin L. Masters 4 and Konrad Beyreuther 1 ~Center for Molecular Biology, University of Heidelberg, Heidelberg (E R. G. ), 21nstitutefor Neuropathology and Jlnstitute of Zoophysiology, University of Bonn, Bonn (ER. G.) and 4Department of Pathology, University of Melbourne, Melbourne (Australia)

(Accepted 11 June 1991) Key words: Alzheimer's disease; flA4 Amyloid precursor; Synaptophysin; Laser scan microscopy; Immunoelectron microscopy

We have recently shown that the amyloid flA4 precursor protein (APP) is synthesized in neurons and undergoes fast axonal transport to synaptic sites [Koo et al., Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 1561-1565]. Using immunofluorescence, laser confocal microscopy and immunoelectron microscopy with simultaneous detection of APP and synaptophysin, we now report a preferential localization of APP at synaptic sits of human and rat brain and at neuromuscular junctions. APP is further found on vesicular elements of neuronal perikarya, dendrites and axons. The synaptic localization of APP implies (1) a role of APP in physiological synaptic activity and (2) a potential and early impairment of central synapses when synaptic APP is converted to flA4 amyloid during the pathological evolution of Alzheimer's disease and Down's syndrome. INTRODUCTION Alzheimer's disease (AD) is a progressive neurodegenerative disorder with intracerebral and cerebrovascular amyloid deposits among its main pathological features. Protein sequencing of the amyloid isolated from patients with A D and aged individuals with Down's syndrome (DS) revealed a 42-43 residue protein which is now termed flA4 because of its proposed r-pleated sheet structure and its molecular weight of 4 kDa 9A°'26'28. flA4 is part of a larger amyloid precursor protein (APP) which resembles a glycosylated cell surface receptor and is encoded by the A P P / P A D - g e n e (precursor of amyloid in A D and DS) located on chromosome 2115'38. Recently reported point mutations within the A P P / P A D - g e n e lead to single amino acid substitutions within the APP-sequence and are associated with an accelerated rate of flA4 formation in some familar cases of A D 1t'29 and Dutch-type cerebral hemorrhage 22. The molecular conformation of APP, its physiology, metabolism and the topography of A P P expression are therefore crucial for an understanding of the pathological mechanisms that lead to flA4 formation and dementia in A D . Various alternatively spliced A P P m R N A ' s have been identified, the main forms coding for A P P isoforms with 695, 751 and 770 amino acid residues which all contain the corn-

plete and partially membrane-inserted flA4-sequence TM 19,34,44 Proteolytic cleavage within the flA4-sequence gives rise to soluble forms of A P P s'42 which are found in cerebrospinal fluid 32'35'47 and blood 33'36. APP751 and APP770 contain a 56-amino acid insert with strong homology to the Kunitz type II family of serine protease inhibitors, their secretory forms are presumably identical to protease nexin-II 2°'31'46. Insert-containing peripheral A P P ' s appear to be involved in the regulation of proteolytic activation cascades such as blood coagulation where platelet-derived APP751/7702'45 show strong inhibition of blood coagulation factor Xla 43. Further, APP751/770 is secreted by stimulated peripheral blood lymphocytes suggesting a role of A P P during lymphocyte activation and immune response induction 27. Whereas APP751 and APP770 are expressed by peripheral organs, APP695 is produced mainly within the nervous system 12'a6. At the cellular level, neurons are the primary source of cerebral A P P 3'41. The physiological role of the neuronal APP695 is still obscure. The membrane-spanning localization of A P P suggests a role in cell-cell or cell-matrix interactions; there is some experimental evidence for the latter function t'38. Synapses and neuromuscular junctions represent types of specialized ceU-cell contacts specific for the nervous system and essential for its function. We have previously shown that APP under-

Correspondence: K. Beyreuther, Center for Molecular Biology, Laboratory for Molecular Neuropathology, Im Neuenheimer Feld 282, D-6900 Heidelberg, F.R.G. Fax: (49) (6221) 565891.

185 goes fast a x o n a l t r a n s p o r t to synaptic sites 21. H e r e w e p r e s e n t a d d i t i o n a l e v i d e n c e that A P P is p r e f e r e n t i a l l y l o c a l i z e d in synapses and n e u r o m u s c u l a r j u n c t i o n s . F r e s h f r o z e n h u m a n and rat s a m p l e s w e r e d o u b l e i m m u n o l a b e l l e d for A P P and s y n a p t o p h y s i n (SP) and a n a l y s e d by f l u o r e s c e n c e , laser c o n f o c a l m i c r o s c o p y and i m m u n o e l e c t r o n m i c r o s c o p y to d e m o n s t r a t e t h e synaptic distrib u t i o n o f A P P in the n e r v o u s system. MATERIALS AND METHODS

Antibodies Monoclonal mouse antibody (MAb) 22Cll and the polyclonal rabbit APP-antiserum 42.2 were raised against purified Fd-APP fusion protein containing APP695 and the Fd fragment ot the mufine IgM immunoglobufin heavy chain. Fd-APP and antibodies were prepared as described 47. MAb 22Cll recognizes an epitope within the extracellular domain of all APP isoforms. The polyclonal guinea pig APP-antiserum K5.1 was raised against a synthetic peptide corresponding to the C-terminal 43 residues of APP. The peptide was synthesized by the solid-phase method of Merrifield using an Applied Biosystem 430A peptide synthesizer. For the identification of synaptic sites in tissue sections, we used a commercially available monoclonal mouse antibody (SY38, Boehringer, ER.G.) and a polyclonal rabbit antiserum (ProGen, ER.G.), which both recognize synaptophysin, a 38 kDa integral membrane protein of presynaptic vesicles48. MAb 22Cll ascitic fluid and polyclonal antibody 42.2 were used at dilutions of 1:10,000 for immunoblotting and at 1:500 for immunocytochemistry. K5.1 was used at dilutions of 1:1000 and 1:50. HPLC-purified MAb SY38 was used at a concentration of 10/~g/ml, the polyclonal anti-SP serum at a dilution of I:100.

APP purification and immunoblotting Rat brain (0.5 g, adult Wistar rats) was homogenized with 10 vols. of homogenization buffer (0.02 M Tris/HC1 pH 6.8; 0.15 M NaCI). The 10,000 g supernatant was chromatographed on DEAEcellulose (Whatman DE-502, NaCI gradient from 0.15 M to 1.0 M). The DEAE-fraction eluting between 0.2 and to 0.25 M NaC1 was applied to heparin-sepharose 6B (Pharmacia, NaC1 gradient from 0.2 to 1.0 M), and the protein eluting at 0.6 M NaC1 was precipitated with chloroform-methanol. The precipitate was redissolved in 150/~1 Laemmli sample buffer containing 0.1% sodium dodecylsulfate (SDS). Human cortical brain tissue was collected at 10 h postmortem from a patient with histologically confirmed AD and homogenized in isotonic buffer (0.32 M sucrose; 0.01 M HEPESHCI; 0.005 M PMSF; pH 7.5). The homogenate was centrifuged at 1000 g for 25 min. The resulting supernatant was centrifugated at 25,000 g for 20 min and the remaining pellet was dissolved in 0.1% SDS Laemmli sample buffer containing 6 M urea. Aliquots corresponding to 500/~g of rat or human brain protein were applied to 8% SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were electroblotted onto nitrocellulose filter and, after blocking with phosphate-buffered saline (PBS) + 1% bovine serum albumin, incubated with the primary antibodies for 3 h at room temperature (RT). Bound antibody was visualized with the Protoblot alkaline phosphatase system (Promega, U.S.A.). The specificity of the immunolabelling was determined with antigen-absorbed antibodies. For MAb 22Cll and polyclonal 42.2, Fd-APP was used as absorbent. Antiserum K5.1 was absorbed with the synthetic C-terminal domain of APP.

Double immunofluorescence Brain tissue samples were collected from the parietal cortex of adult Sprague-Dawley rats and from fresh surgical human samples of the anterior temporal lobe and immediately snap frozen in liquid nitrogen. Diagnostic biopsies of biceps muscle were removed

from two patients after an area rich in neuromuscular junctions had been localized in the open muscle by electrical stimulation and stepwise reduction of the stimulation current. The biopsy specimens were prepared as described 4°. Frozen sections (5/~m) were placed on poly-L-lysine coated slides, fixed in ice-cold acetone for 10 min and air dried. After rehydration in PBS (pH 7.4), double immunofluorescence staining was performed following preincubation with normal goat serum. Rat brain sections were incubated with MAb 22Cll, followed by affinity purified biotinylated goat anti-mouse Ig (Dianova, ER.G., 1:100), then by Streptavidin-Texas red (SAV-TR, Amersham, ER.G., 1:100), then by polyclonal rabbit anti-SP followed by FITC-conjugated goat anti-rabbit Ig (Dianova, 1:100). Human brain and muscle sections were first incubated with MAb SY38, followed by the biotinylated goat anti-mouse Ig and by SAV-TR. APP was then labelled by polyclonal 42.2 and visualized with goat FITC-conjugated anti-rabbit Ig (brain sections) or by polyclonal K5.1 and visualized with goat fluorescein (FITC)-conjugated anti-guinea pig Ig (muscle sections). All incubation steps were performed for 20 rain at 37 °C. Specificity of the immunostaining was examined by preabsorption of the primary antibody with Fd-APP fusion protein or by use of control antibodies instead of monoclonal and polyclonal anti-SP. Sections were observed in a Zeiss Axiophot with filters BP485/ FT510/BP515-565 for FITC and BP530-580/FT600/LP615 for TR. Fluorescence patterns were documented on Kodak T max 400 or recorded on video tape with a video camera.

Laser scanning microscopy Sections of human brain, double labelled for APP and SP antibodies as described above, were analysed in a laser scan microscope (LSM, Zeiss, ER.G.) equipped with a confocal system 6. Different laser fines for the excitation of multiple dyes were used as described previously4°. The LSM studies were focused on individual nerve cell bodies to visualize the subcellular distribution of APP- and SP-antigens. Confocal fluorescence images of the double stained sections were generated using an argon ion laser (488 nm) for FITC and an HeNe laser (633 nm) for TR 39'40. Fluorescent perikarya were identified by conventional fluorescence optics and then scanned sequentially with the two lasers at zoom magnifications of 3414 to 5974. Emission filters used were BP485/FT510/ BP515-565 for FITC and LP660 for TR. Initial fluorescence signals were recorded on video tape. Sections with high and low intensities in the initial scanning procedure were bleached for 30-60 s by repeated scanning in a 2-s mode and recorded on tape. This procedure allowed a selective demonstration of remaining sites with high signals after complete bleaching of the weaker fluorescence. Overlapping fluorescences of FITC and TR, indicating presence of both antigens at the same sites, were identified by image overlays of the selectively filtered emission signals on the LSM monitor. Following fluorescence imaging in the LSM, the same sections were counterstained with hematoxylin-eosin (HE). Stained neurons which were recorded as fluorescent structures before HE staining, were then relocalized in the LSM at the same magnifications. By correlating the morphology with the double fluorescence staining pattern, we identified the subcellular sites of immunolabelled structures and compared them to adjacent cellular sites4°.

Immunoelectron microscopy In order to examine the subcellular location of APP at the ultrastructural level, we applied the Lowicryl K4M (Polysciences, ER.G.) embedding method 4. Adult Sprague-Dawley rats were anesthesized, thoracotomized and fixed by trans-aortic perfusion with 1% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.2 TM. Small pieces of brain tissue were removed from the parietal cortex and fixed for another 2 h at 4 °C in the perfusion solution. Tissue blocks were then washed in 0.1 M PB containing 0.1 M sucrose and kept in the same washing buffer overnight. The dehydration in ethanol series and ethanol/Lowicryl K4M mixtures was performed at decreasing temperatures (35-

186 0 °C). After overnight infiltration of the tissue with Lowicryl K4M, blocks were placed in gelatine capsules containing fresh resin and polymerized with ultraviolet light (360 nm) for 24 h at -35 °C. The polymerization was continued for 2 days at RT. Thin sections were cut with a glass knife on an Ultra-Cut E ultramicrotome (ReichertJung, ER.G.) and collected on Pioloform F-coated nickel grids. Immunogold labelling was carried out after preincubation with normal goat serum (1:30) for 45 min at RT. Grids were then transferred on drops containing the primary antibodies (polyclonal rabbit anti-APP 42.2 and anti-SP MAb SY38) and incubated overnight at 4 °C. For simultaneous localization of APP and SP, grids were placed for 30 min at RT on drops containing both 6 nm gold conjugated goat anit-rabbit Ig (1:40) and 15 nm gold conjugated goat anti-mouse Ig (1:40). Single labelling for APP was done with MAb 22Cll or polyelonal anti-APP 42.2 and visualized with the 15 nm

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In human brain homogenate, the APP C-terminalspecific antibody K5.1 recognizes 2 protein bands of 105 kDa and 115 kDa (Fig. la, lane 5), which correspond to the full-length forms of APP695 and APP751/770. MAb 22Cll (Fig. la, lane 1) appears to label the same bands,

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Fig. 1. Identification of APP in human and rat brain homogenate. The crude membrane fraction of brain homogenate from an Alzheimer's disease (AD) individual (a) reveals flA4 precursor protein (APP) isoforms with electrophoretic mobilities from 90 to 115 kDa. Full length forms that include the cytoplasmic domain are detected with the anti-C-terminal antiserum as immunolabelled 105 kDa and 115 kDa bands and correspond to the APP695 and APP751/770 isoforms (lane 5). Immunostaining with either MAb 22C11 (lane 1) or polyclonal rabbit anti-APP 42.2 (lane 3) reveals additional bands (90 kDa-98 kDa) that presumably correspond to secretory forms of APP695 and APP751/ 770. The latter bands do not react with the C-terminal antiserum (lane 5). Immunoblots of rat brain (b) show one major band of 105 kDa which is recognized by MAb 22Cll (lane 1), polyclonal 42.2 (lane 2) and polyclonal K5.1 (lane 3) antisera. This band corresponds to fulllength, membrane bound APP695. No bands are detected with antigen-absorbed antisera (a; lanes 2,4,6) or when the primary antibody is omitted (a, lane 7).

187 whereas the polyclonal antiserum 42.2 reacts preferentially with APP695 (Fig. l a , lane 3). A c c o r d i n g to the domain specificity of these antibodies both the ect o d o m a i n and the e n d o d o m a i n of A P P are present on the 105 k D a and 115 k D a proteins; the t r a n s m e m b r a n e

d o m a i n is therefore present in the forms recognized by all 3 antibodies. In rat brain, a major protein b a n d of 105 k D a is recognized by all 3 antibodies (Fig. l b ) . This band represents full-length, m e m b r a n e - b o u n d APP695 which is the major splice product in the brains of adult

@ Fig. 2. APP and synaptophysin (SP) at synaptic sites in rat brain and human skeletal muscle. Double immunofluorescence staining for APP (MAb 22Cll; a,c,e,g) and SP (polyclonal antiserum; b,d,f,h) of rat parietal cortex and white matter reveals strong overlapping punctuate signals in the neuropil (a-d). Perikarya are stained for APP (a,c, arrows; e) but unstained for SP (b,d, arrows; f). In the white matter (wm), APP is detected only in small blood vessels (a). Magnification of the perikaryon indicated by arrows in (c) and (d) shows positive immunostaining for APP in the perinuclear region (e, arrow), on or near the cell surface, and on punctuate structures at the cell periphery which are most likely axosomatic sites. Only the latter overlap with SP-staining (e,f, arrowheads). Higher magnification of neuropil staining for APP (g) and SP (h) reveal a remarkable overlap of the punctuate signals. Note APP positive/SP positive synapses (g,h, arrows without numbers), APP negative/SP positive synapses (h, arrowhead) and also individual APP positive/SP negative structures (g, arrow 1). Immunostaining of human skeletal muscle (i,k) reveals neuromuscular junctions that react with both the polyclonal anti-APP K5.1 antiserum (i) and with anti-SP MAb SY38 (k). Two neuromuscular junctions of neighboring cross sectioned muscle fibres are shown, one cross-sectioned (i,k, arrows) and one tangentially cut (i,k, arrowheads), a-d x346; e-h ×2079; i,k ×1406.

188 rats ~3'~6. The predominant form in rat brain thus appears to be transmembrane and not secreted APP. In human brain homogenates, there are additional faster migrating APP's (90 kDa-98 kDa) stained with MAb 22Cll and polyclonal antiserum 42.2 (Fig. lb, lanes 1,3), but not with polyclonal C-terminal directed guinea pig antiserum K5.1 (Fig. la, lane 5). These bands correspond to C-terminal, truncated forms. When the antibodies were preabsorbed or when the primary antibody was omitted, the respective protein bands were completely unstained (Fig. la; lanes 2,4,6,7).

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In the neuropil of rat cerebral cortices, the staining pattern for APP was punctuate (Fig. 2a,c). It is generally believed that such neuropil reactivity is characteristic for proteins associated with synapses 7. To investigate whether these punctuate APP positive sites correspond to synapses, immunolocalization was carried out by fluorescence double labelling for APP and SP, the latter being a general synaptic marker in brain and periphery48. As shown in Fig. 2 (a,b; c,d), the immunostaining for APP and SP had a very similar pattern. The cerebral white matter was largely unstained. As expected, neuronal cell bodies were not stained with the SP antibodies (Fig. 2b,d; arrows) but were intensely labelled with APP antibodies (Fig. 2a,c; arrows). Within the perikarya, the perinuclear immunoreactivity for APP (Fig. 2e, arrow) was found in the endoplasmic reticulum and Golgi apparatus. Part of the extended APP-immunoreactivity at the surface of the same cell (Fig. 2e, arrowhead) overlaps with the SP-reactivity (Fig. 2f, arrowhead). These latter sites would be consistent with axosomatic synapses. An even more complete overlap between staining for APP and SP is shown in Fig. 2g,h. Occasionally, some synapses were APP-negative (Fig. 2h, arrowhead). Also, some punctuate APP-positive and SP-negative structures were detected with low abundance (Fig. 2g, arrow 1). The synaptic nature of these latter structures seems likely due to the punctuate appearance and spatial relation with SP-positive sites. Figs. 2i,k show two neuromuscular junctions (NMJ) of adjacent muscle fibres, one cross-sectioned (arrows) and one cut tangentially (arrowheads). Immunoreactive APP (Fig. 2i) and SP (Fig. 2k) occurred in the same NMJ, but showed different spatial extensions with respect to

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Fig. 3. APP and SP at axosomatic synapses in human temporal cortex. Frozen sections were double labelled by polyclonal antiAPP 42.2 (a,b) and M A b SY38 (c,e). Fluorescence (a,b,c,e) or hematoxylin-eosin staining (d,f) was analysed by confocal laser scan microscopy. Note that bleaching removes the diffuse A P P surface fluorescence (a) and leaves 4 punctuate APP positive structures (b) which are located at axosomatic synapses as indicated by selective excitation of the SP-fluorescence (c). A n axon-like structure terminating in two of the APP-positive synaptic sites (a,b,e) is stained darkly by hematoxylin-eosin treatment of the same section (d,f, arrowheads). The weak fluorescence of the axon-like structure (a, arrowhead) indicates the presence of A P P at presynaptic terminals, but at a lower concentration than at synapses as shown by fluorescence bleaching (b). The schematic diagrams (right panels) relate the labelled structures to the perikaryon, a - d ×920; e,f × 1550.

Fig. 4. Ultrastructural localization of APP at presynaptic sites of rat cerebral cortex. Three different synapses are shown (a,c,e), discernible by presynaptic dense projections (dp), the postsynaptic density (double arrows in a; p in c,e) and the synaptic cleft. Immunogold labelling for APP (polyclonal 42.2 antiserum, 6 nm gold particles, arrows) and for SP (MAb SY38, 15 nm gold particles, arrowheads) localizes both antigens on different and presumably vesicular structures within the presynaptic area. Note negative staining of the pre- and postsynaptic membranes (d, arrows), m, mitochondria. Bars = 0.2 ~m (a,c,e) or 0.1 ~m (d,b).

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191 the plane of the section: in the cross-sectioned NMJ APP is represented by a relatively thin band on the muscle fibre surface (Fig. 2i, arrow) whereas the SP-reactivity is more extended (Fig. 2k, arrow) and corresponds to the axon terminal48. In the tangentially, less ideally sectioned NMJ, a more extended APP-positive site is seen (Fig. 2i, arrowhead) which cannot be clearly ascribed to the muscle fibre surface. In this latter NMJ only a small part of the axon terminal can be identified by its SP-reactivity (Fig. 2k, arrowhead).

Laser scanning microscopy Human temporal cortex was examined by confocal laser scanning microscopy after immunofluorescence double labelling for APP and SP (Fig. 3). The initial fluorescence signals obtained for APP are shown in Fig. 3a. Fluorescence bleaching removes the weaker APP surface fluorescence and leaves 4 punctuate APP-positive structures (Fig. 3b). The latter are located at axosomatic synapses, because their fluorescence signal overlaps with that of SP-immunoreactive sites visualized by excitation at 633 nm using the HeNe laser (Fig. 3c). HE-counterstaining of the same area shows the morphology of stained perikaryon (Fig. 3d). Transmitted light images using the argon ion laser (488 nm) revealed branching structures which appear to be afferent axons (Fig. 3d, arrowhead) because they terminate in two of the identified axosomatic synaptic sites. Higher magnifications of this surface area of the perikaryon are given in Fig. 3e,f. The corresponding fluorescence signal in Fig. 3a (arrowhead) suggests that APP is also present in axons near synapses.

Immunoelectron microscopy Relatively mild fixation conditions and Lowicryl K4M embedding of rat brain cortex were used to conserve the APP- and SP-epitopes for antibody binding. The ultrastructural preservation of the tissue was similar to that demonstrated previously18. Synapses, axonal and dendritic profiles as well as microtubules, mitochondria and perinuclear cisternae in perikarya (most likely corresponding to Golgi and ER structures) were discernible (Figs. 4 and 5). Membranes appeared as negatively contrasted lines (Fig. 4d, arrows). The vesicle morphology,

however, was less defined than in standard plastic embedded material. A distinction of the vesicle membrane and the vesicle lumen could be made in individual synaptic vesicles (Fig. 4a,b), but the majority of intraneuronal vesicles appeared as moderately electron dense ghosts or small empty spaces surrounded by darker material (Fig. 4c,e; d; arrows). Presynaptic dense projections, synaptic clefts and the postsynaptic densities were well defined (Fig. 4). Immunolabelling for APP revealed small clusters of gold particles located on vesicular structures at presynaptic sites (Fig. 4, arrows). Individual vesicles were loaded with gold particles at the vesicle surface suggesting the presence of APP in the vesicle membrane (Fig. 4a,b). SP-immunoreactivity was observed in the same location, but on distinct structures, most likely different types of vesicles (Fig. 4c,d; arrowheads). Analogous to our fluorescence results, synapses labelled by both APPand SP-antibodies were seen as well as synapses labelled by either antibody alone. Whereas SP-specific immunoreactivity was exclusively present at these synaptic sites, APP showed a more extended intraneuronal distribution and was detected within perinuclear cisternae (Fig. 5a, b), axoplasm of myelinated and unmyelinated nerve fibres (Fig. 5c,d) and dendrites. In dendrites the gold granules were frequently localized closely to, or directly associated with, microtubules (Fig. 5e,f). Less frequently we observed immunoreactivity for APP at the cell surface of neurons, suggesting the association of APP with the plasma membrane (Fig. 5e, open arrow). DISCUSSION

Several lines of evidence show that APP is present in different types of synapses. First, in the neuropil of the neocortex, most of the SP signals overlap with the APP signals. These sites in the neuropil can be assumed to represent the majority of cortical synapses, most of which are axo-axonal or axo-dendritic terminals 7,48. Second, SP-specific fluorescence signals at the surface of perikarya overlap with strong punctuate signals of APP. This suggests a high abundance of the latter antigen in afferent terminals of putative vesicle-containing axosomatic synapses. Third, colocalization of APP and SP in skeletal

Fig. 5. APP in different compartments of rat neurons. Immunogold labelling with MAb 22Cll (a,c,e) and polyclonal 42.2 (b,d,f) antiserum. APP is detected in perinuclear vacuoles most likely corresponding to ER structures of the nuclear envelope (a, arrows) and to Golgi cisternae (a,b, arrowheads), c: axon-profile of a myelinated nerve fibre; note the lamellae of the myelin sheath (arrow). d: unmyelinated nerve fibre containing vesicular APP-positive structures, with the limiting axon membrane outlined by arrowheads, e,f: dendritic profiles containing numerous microtubules (f, arrowheads) and some mitochondria. APP is found in close proximity to microtubules (arrows). The limiting membrane of the dendrite (e, arrowheads) is surrounded by adjacent glial cell processes (e, asterisks) and shows small clusters of APPreactive immunogold particles (e, open arrow). A, axon; AV, axonal vesicles; N, nucleus; D, dendrite, GCP, glial cell process; m, mitochondria. Bars = 0.2 am.

192 muscle is observed in neuromuscular junctions, the ellector sites of motor nerves. These results indicate a widespread distribution of APP at synaptic sites suggesting that APP might have a basic function in synapses. Immunoelectron microscopy revealed a majority of neurons labelled for APP; in addition, most synapses were positive for both APP and SP. The gold particle load was, however, generally lower than expected from the immunofluorescence intensities of the same antigens; plasma membranes, which are predicted to contain A P E were only occasionally decorated. Our immunogold procedure was optimized to preserve a maximum of immunoreactivity for APP and SP, even if low concentrations of glutaraldehyde had to be used to achieve an acceptable degree of ultrastructural preservation. In our experience APP is particularly sensitive to glutaraldehyde fixation, and similar observations have been reported for S P TM. Labelled structures in these sections therefore probably represent sites of high antigen concentration, whereas lower concentrations of the antigens might not have been detected. Using immunoelectron microscopy, synapses were identified by morphological criteria and by SP immunogold labelling. Interestingly, APP and SP were associated with different types of vesicles. SP is normally found in neurotransmitter-containing and electron-lucent small synaptic vesicles at presynaptic sites and neuromuscular junctions, whereas the neuropeptidecontaining dense core vesicles have much lower amounts of SP and are unlabelled with SP-antibodies 23'3°'48. The ultrastructural preservation of our sections does not permit the morphological differentiation between these vesicle types; however, APP bearing vesicular structures were constantly negative for SP and might correspond to residues of dense core vesicles. Dense core vesicles are part of the regulated secretory pathway and share functional properties with secretory vesicles of exocrine and endocrine organs (for review see ref. 17). We have recently described the subcellular localization of APP in secretory granules of salivary gland and anterior pituitary 5. Moreover, dense core vesicles, in contrast to small synaptic vesicles, are assembled in the perikaryal endoplasmic reticulum and Golgi compartments and then transported to synaptic sites. In accordance with our previously reported experimental evidence for the synthesis of APP in the neuronal cell bodies and its subsequent axonal transport to synaptic sites 2t, APP was now detected also within the perinuclear endoplasmic reticu-

REFERENCES 1 Breen, K.C., Bruce, M. and Anderton, B.H., Beta amyloid precursor protein mediates neuronal cell-cell and cell-surface adhesion, J. Neurosci. Res., 28 (1991) 90-100.

lum, the Golgi complex and on vesicular elements of myelinated and unmyelinated axons. The presence of APP in these axonal structures has also been found in monkey brain 24. The function of neuronal APP has yet to be established. Both immunofluorescence and immunogold techniques revealed a minor fraction of SP-positive synapses that did not react with APP-specific antibodies. APP therefore appears not to be an essential component of synapses, but required for hitherto unidentified synaptic states. We have observed in cultured cells that an abundance of APP correlates positively with increased surface activities such as membrane vesiculation and the formation of cellular extensions during growth (W. Schubert, in preparation). We suggest that APP is upregulated in synapses with increased membrane activity, e.g. in synapses subject to high transmission rates or during synaptogenesis which involves growth and remodelling of synaptic membranes. The view that APP is individually regulated at the cellular level in neurons is supported by our finding that the intensity and pattern of the subcellular staining in perikarya is not identical in different neurons. Strong pancellular APP reactivity is characteristic for the majority of perikarya. On the other hand, weak perinuclear and cell surface stainings are less frequent, but equally characteristic patterns of neuronal APP expression. In summary, we have shown that the Alzheimer flA4 amyloid precursor protein is located in neuronal perikarya and concentrated at axosomatic and other synaptic sites in brain and in muscle. Accordingly, APP expression and turnover might be linked to synaptic turnover and plasticity. The clinical symptomatology of AD is tightly linked to the development of synapse pathology and synapse l o s s 25 . If under pathological conditions flA4 is released from APP, synapses -- essential for the maintenance of intellectual brain function -- would be an early and critical site of flA4 amyloid formation. Acknowledgements. We thank Dr. G. Kersting, Dr. R, WtiUenweber and Dr. D.K. Btiker for access to their facilities and continuous support. The work was made possible by grants from the Thyssen Stiftung, the Deutsche Forschungsgemeinschaft through DFG (Schu 627/1-1) and SFB 317, the Bundesministerium ftir Forschung und Technologie, the Boehringer Ingelheim Fonds, The Cusanus Werk, the Fonds der Chemischen Industrie, the National Health and Medical Research Council of Australia and the Alzheimer's Disease and Related Disorders Association. R.P. was supported by DFG (Pr 319/1-1).

2 Bush, A.I., Martins, R.N., Rumble, B., Moir, R., Fullser, S., Milward, E., Currie, J., Ames, D., Weidemann, A., Fischer, P., Multhaup, G. and Beyreuther, K., The amyloid precursor protein of Alzheimer's disease is released by human platelets, J. Biol. Chem., 265 (1990) 15977-15983.

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