Identification of neuronal plasma membrane microdomains that colocalize β-amyloid and presenilin: implications for β-amyloid precursor protein processing

Neuroscience 120 (2003) 291–300

IDENTIFICATION OF NEURONAL PLASMA MEMBRANE MICRODOMAINS THAT COLOCALIZE ␤-AMYLOID AND PRESENILIN: IMPLICATIONS FOR ␤-AMYLOID PRECURSOR PROTEIN PROCESSING R. TORP,a* O. P. OTTERSEN,a C. W. COTMANb AND E. HEADb

1998; Naruse et al., 1998; Waragai et al., 1997; Xia et al., 1998). One hypothesis holds that PS1 may act as a ␥-secretase (Wolfe et al., 1999). A difficulty with this hypothesis is that PS lacks homology with known proteases. This suggests that PS may promote A␤ formation in an indirect manner, by interacting with APP (Waragai et al., 1997) and ␥-secretase so as to promote a direct contact between the two proteins. One of the arguments against a possible role for PS in cleaving APP is the apparent lack of spatial co-localisation (Annaert and De Strooper, 1999; Cupers et al., 2001; Perez et al., 1999). This has been termed the “spatial paradox” (Annaert and De Strooper, 1999; Annaert et al., 1999). However, in transfected DAMI and differentiated human NT2N neuronal cells, both APP and PS were co-localised on the plasma membrane (Dewji and Singer, 1997) or within axonal membrane components (Kamal et al., 2001). An alternative approach to determine the role of PS in processing APP is to study the spatial characteristics of PS and the final product of APP cleavage, A␤. It is critical to identify the nature and subcellular expression pattern of ␥-secretase because reducing the activity of this enzyme is a potential target for treatment or prevention of AD. If PS is involved in APP cleavage at the ␥-site then PS should be co-localised at the ultrastructural level with the cleavage product, A␤. The aim of this study was to explore this hypothesis by use of confocal and high-resolution immunogold electron microscopy.

a

Centre for Molecular Biology and Neuroscience and Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1105, Blindern, N-0317 Oslo, Norway b Institute for Brain Aging and Dementia, University of California, Irvine, CA 92697-4540, USA

Abstract—Alzheimer’s disease (AD) is associated with the accumulation of extracellular deposits of the ␤-amyloid protein (A␤). A␤ is a result of misprocessing of the ␤-amyloid precursor protein (APP). ␥-Secretase is involved in APP misprocessing and one hypothesis holds that this secretase is identical to PS1. We tested this hypothesis by determining whether PS is co-localised with A␤ in situ. Using confocal analyses and a sensitive immunogold procedure we show that PS and A␤ are co-localised within discrete microdomains of neuronal plasma membranes in AD patients and in aged dogs, an established model of human brain aging. Our data indicate that APP misprocessing occurs in discrete plasma membrane domains of neurons and provide evidence that PS1 is critically involved in A␤ formation. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: AD tissue, canine tissue, colocalization, amyloid, presenilin, lipid rafts.

Misprocessing of the amyloid precursor protein (APP) is a key event in the pathogenesis of Alzheimer’s disease (Selkoe, 1996). Thus, the sequential cleavage of APP by two proteases called ␤- and ␥-secretase leads to the formation of a 40 – 43 amino acid long amyloid peptide (A␤). The A␤ peptide is the main constituent of plaques, a hallmark of Alzheimer’s disease. ␤-Secretase was recently cloned and identified as an aspartate protease, named ␤ amyloid cleaving enyzme (Vassar et al., 1999). In contrast, the identity of ␥-secretase has not been determined. However, it has been suggested that presenilins (PS), a class of proteins that when mutated contributes to familial Alzheimer’s disease (AD; Hardy, 1997; Hutton and Hardy, 1997) are somehow involved in ␥-secretase-mediated cleavage of APP (Chen et al., 2000; De Strooper et al.,

EXPERIMENTAL PROCEDURES Tissue samples Dogs. Five beagle dogs were included in the study. Animals ranged in age from 12 to 148 months and were selected from a previous study describing membrane-associated A␤ (Torp et al., 2000). All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory Animals and all efforts were made to minimise animal suffering, to reduce the number of animals used, and to utilise alternatives to in vivo techniques, if available. Dogs were killed with an overdose of sodium pentobarbital, perfused transcardially with physiological saline followed by 4% paraformaldehyde. Tissue blocks were archived in phosphate-buffered saline, pH 7.5, with sodium azide at 4 °C prior to processing for electron microscopy. Multiple sections from two blocks of either the dorsolateral prefrontal (proreus) or posterior parietal cortex (area marginalis posterior; Kreiner, 1966), as listed in Table 1, were subsequently used for light and electron microscopic studies.

*Corresponding author. Tel: ⫹47-22-851-269; fax: ⫹47-22-851-278. E-mail address: [email protected] (R. Torp). Abbreviations: AD, Alzheimer’s disease; APP, amyloid precursor protein; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; PS, presenilin; TBNT, Tris buffer (5 nM) containing 0.01% Triton X-100 and 0.3% NaCl; TBST, Tris-buffered saline containing 0.05% Tween 20; TRIS, 0.1 M Tris buffer, 0.85% NaCl; TRIS A, 0.1 M Tris buffer, 0.85% NaCl with 0.1% Triton X-100; TRIS B, 0.1 M Tris buffer, 0.85% NaCl with 0.1% Triton X-100 and 2% bovine serum albumin.

Humans. Ten cases were selected and included three controls and seven AD cases and are described in Table 1. Tissue was obtained from the Human Brain Repository at the Institute for

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00320-8

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Table 1. Case demographics NPID

Case

Sex

Age (years)

ApoE

Final diagnosis

B&B stage

PMI (h)

Cause of death

AD AD AD AD AD AD AD Biopsy Normal Normal Normal

VI VI VI VI VI VI VI –

7.4 3.75 4.25 8.3 N/A 2.5 8.3 –

End stage AD End stage AD Pneumonia Pneumonia N/A End stage AD Pneumonia

II I

2.75 3.67

Cancer Lung cancer

07-01 07-98 33-99 14-99 7-99 1-01 14-99 4001

1 2 3 4 5 6 7 8

F M F F F M F F

65 70 86 78 88 81 78 81

N/A 3/4 3/4 3/4 N/A N/A 3/4 N/A

2-99 13-01

9 10

F M

74 95

N/A N/A

B&B, Braak & Braak; PMI, postmortem interval; N/A - not available. Table 1B. Canine brain samples Dog#

Case

Sex

Age (years)

2082 2083 2069 6443 2094

1 2 3 4 5

M F M F M

10 6 6 10.6 1

Brain Aging and Dementia at the University of California at Irvine. The tissue was immersion-fixed in 4% formaldehyde in 0.1 M Sorensen buffer pH 7.3 for 72 h. The tissue was archived in phosphate-buffered saline, pH 7.5 with sodium azide at 4 °C prior to processing for electron microscopy.

Post-embedding immunogold method After perfusion and fixation as described above, blocks from the frontal and parietal cortex were prepared for embedding in Lowicryl resin using the freeze substitution method as described previously. In brief, vibratome sections (200 ␮m) of cortex were cut and washed extensively in phosphate buffer. Deeper layers of cortex were dissected out to give blocks of approximately 5 mm. These were equilibrated in ascending concentrations of sucrose solution (10%, 20% and 30% in phosphate buffer) and then plunged into liquid propane (⫺170 °C) in a cryofixation unit KF 80; Reichert, Wien, Austria. The blocks were transferred to a cryosubstitution unit (AFS; Reichert) at ⫺90 °C, where freeze substitution followed. Slices were immersed in 0.5% uranyl acetate in methanol at ⫺90 °C and then the temperature was increased at 4 °C/h to ⫺45 °C and all the following steps were conducted at this temperature. The blocks were infiltrated with Lowicryl HM20 resin with a progressive increase in the ratio of resin to methanol. The slices were then placed in embedding capsules in fresh Lowicryl and polymerised under UV light for a minimum of 48 h.

Ultrathin sections were cut from the blocks and collected on coated single-slot grids in preparation for the subsequent postembedding immunohistochemistry. Sections were immunolabelled by the post-embedding method. The sections were treated with a saturated solution of NaOH in absolute ethanol (2–3 s) rinsed in phosphate buffer and incubated sequentially in the following solutions. (i) 0.1% sodium borohydride and 50 mM glycine in Tris buffer (5 nM) containing 0.01% Triton X-100 and 0.3% NaCl (TBNT; 10 min); (ii) 0.5% powdered milk in TBNT (10 min); (iii) followed by a 2 h incubation in primary antibody solutions at room temperature. Primary antibodies were diluted (anti A␤-42 or anti-PS [PS1 and PS2]) 1:300 and 1:500 respectively in TBNT containing 0.5% powdered milk. After several washes in TBS, the sections were incubated in the appropriate secondary antibodies conjugated to different-sized colloidal gold particles, 10 nm gold-conjugated goat anti-mouse IgG (British BioCell International, Cardiff, UK; 1:20) for PS or 10 nm gold-conjugated goat anti-rabbit IgG (British BioCell; 1:20) for A␤ 42 in TBNT containing 0.5% powdered milk supplemented with polyethylene glycol (5 mg/ml), for a minimum of 2 h at room temperature. Following washing, sections were incubated in 1% uranyl acetate for 15 min, washed in de-ionised water, stained with lead citrate for 2 min and examined in a Philips CM10 electron microscope.

Antibodies Table 2 provides a list of antibodies, dilutions used, and source. Two affinity-purified antibodies were used to visualise A␤. The first is a rabbit polyclonal (A␤42) raised against a synthetic A␤ peptide containing amino acids 1 through 42 (Cummings and Cotman, 1995). The second is a monoclonal antibody raised against A␤1–16 (6E10). Two monoclonal antibodies were used to label PS, one was specific for PS1 (APS11, anti-human PS1 N-terminal between 21 and 34 aa) and for PS2 (APS21, anti-human PS2 N-terminal; APS26, anti-human PS2 c-terminal loop epitope). The specificity of these antibodies has been described previously

Table 2. Antibodies Antibody

Used to detect

Dilution

Source

Anti-A␤42 6E10 APS11 APS21 A2B5 Flotillin

Senile Plaques Senile Plaques Presenilin-2 Presenilin-1 GM-1 Gangliosides Flotillin-1

1:300 1:8000 1:500 1:300 1:5000 1:100

Cummings and Cotman, 1995 Signet Laboratories, Inc., Dedham, MA, USA Diehlman et al., 1999 Diehlman et al., 1999 Chemicon International, Temecula, CA, USA BD Transduction Laboratories, Palo Alto, CA, USA

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Fig. 1. Co-localisation of PS and A␤. PS (red) and A␤ (green) are co-localised within diffuse plaques in the aged canine brain (A–C) and in punctate deposits on the neuronal membrane (arrows) (D–F).

(Diehlmann et al., 1999). Markers for membrane microdomains that are rich in cholesterol included anti-flotillin and anti-GM-1 gangliosides.

Enzyme-linked immunosorbent assay (ELISA) Flat bottom microtiter plates (Immunol 2; ISC BioExpress, Kaysville, UT, USA) were coated overnight at 4 °C with 100 ␮L of 2.5 ␮M of the desired peptide (soluble A␤1– 42 and A␤1– 40) in 50 mM carbonate-coating buffer, pH 9.6. Fibrillar A␤1– 42 was produced by incubating A␤1– 42 overnight at 37 °C and incubated in PBS buffer at pH of 7.5 at the same concentration as the other two peptides. Following three washes with Tris-buffered saline containing 0.05% Tween 20 (TBST), 100 ␮L of a blocking solution containing TBST with 3% nonfat milk was applied for 1 h at 37 °C. Serial dilutions of anti-PS1 and anti-PS2 (1:100 –1:400) were prepared in TBST with 0.3% nonfat milk (1:100 –1:400). An anti-A␤ antibody (6E10-Senetek) was used as a positive control and all antibody samples were incubated for 1 h at 37 °C. After washing the wells with TBST, horse radish peroxidase-conjugated horse anti-mouse IgG diluted (Vector Laboratories, Burlingame, CA, USA) to 1:5000 was added for 1 h at 37 °C. A positive reaction was detected using 3,3⬘,5,5⬘ tetramethyl benzidine substrate (BioRad, Hercules, CA, USA) and stopped by adding 1 N H2SO4. The resulting absorbence was monitored at A450 using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Double immunolabelling using the post-embedding procedure Double immunolabeling was carried out on the same ultrathin section using mixtures of primary antibodies. The following combinations were used: anti-A␤ and anti-PS (PS1 and PS2) antibodies (a mix of polyclonal A␤ and monoclonal PS1 or PS2). The same post-embedding procedure was followed as described above, except that the sections were incubated with drops of a cocktail of primary antibodies diluted to the same concentrations as used in the single labelling experiments. After several washes in TBS, the sections were incubated in a cocktail of 10 nm goldconjugated goat anti-mouse IgG (British BioCell; 1:20) and 20 nm gold-conjugated goat anti-rabbit IgG (British BioCell; 1:20) in 0.5% powdered milk in TBST supplemented with polyethylene glycol (5

mg/ml). Uranyl acetate and lead citrate was used for counterstaining. The specificity of the post-embedding techniques was established by the absence of labelling for the respective antigens when the secondary antibodies were omitted.

Immunofluorescence and confocal microscopy Tissue sections were washed in 0.1 M Tris buffer, 0.85% NaCl (pH 7.5; TRIS) and pretreated in 90% formic acid for 4 min to enhance A␤ immunostaining. Following a 5 min wash in TRIS and a 15 min wash in TRIS with 0.1% Triton X-100 (TRIS A), sections were blocked for 30 min in TRIS A with 2% bovine serum albumin (TRIS B). Sections were subsequently placed in anti-PS1 or anti-PS2 (1:1000) in TRIS B overnight at RT. Following washing, sections were incubated in biotinylated anti-mouse IgG with long arm spacers (Jackson ImmunoResearch, West Grove, PA, USA) at 1:200 for 1 h, washed, and then placed in streptavidin conjugated Cy-3 (Jackson ImmunoResearch; 1:500) for 1 h. Following several washes, sections were blocked for 30 min in TRIS B and then incubated overnight in anti-A␤1– 42 (1:500). The same procedure was subsequently followed except biotinylated anti-rabbit secondary antibody was used and streptavidin conjugated CY-5 (1:200). Serial sections were also used with the reverse order of antibodies being applied. Confocal images were collected on an Olympus IX70 inverted microscope using both a 20⫻ and 40⫻ objective for image analysis and barrier filters at either 605 or 700 nm. Each channel was collected separately to avoid the possibility of signal bleeding from one wavelength into the other. CY-3 fluorescence was captured at 568 nm and CY-5 at 647 nm. A z-series at 1 ␮m intervals was captured to determine the spatial co-localisation characteristics of A␤ and PS or GM1 ganglioside staining within individual neurons.

RESULTS Potential confounding binding interactions of PS and A␤ antibodies were studied using ELISA system. In contrast to the positive reaction obtained when using an anti-A␤ antibody (6E10), neither PS antibody bound to soluble or fibrillar A␤1– 42 or to A␤1– 40. In aged canine brains, A␤

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Fig. 2. Ultrastructural localisation of PS and A␤. Double immunogold labelling reveals co-localization of A␤ (large particles) and PS1 (small particles) in discrete patches of neuronal plasma membrane. The immunolabelled patch (framed) is enlarged in the inset. Asterisk indicates a stretch of unlabelled plasma membrane of the same neuron (N). Bars⫽0.3 ␮M, 0.2 ␮M.

and PS1 were co-localised within plaque regions when viewed with confocal microscopy (Fig. 1A–C). In addition, in areas without diffuse plaque formation, A␤ and PS were also observed along neuronal membranes of cells (Fig. 1D–F). Higher resolution electron microscopy further revealed that anti-PS1 produced distinct labelling in discrete patches along the neuronal plasma membrane in aged but not young canines suggesting that the antigen is restricted to specific membrane domains as previously reported for

A␤1– 42. Double label immunogold studies for PS1 (small particles) and A␤42 (large gold particles) show that these two proteins are co-localised in these discrete patches (Fig. 2). A␤42 was present in membrane domains as a fibrillar material (Fig. 3). Like A␤42, PS1 was detected along membranes of large and small dendrites (Fig. 3C, D). The distribution of PS1 (Fig. 4A) and PS2 (Fig. 4B) were similar. Although co-localisation was the rule, some membrane regions were labelled predominantly or exclusively with either A␤42 or PS1 (asterisk Fig. 4A). These

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Fig. 3. Plasma membrane domains shows an interaction between PS and A␤. Co-localisation of A␤ (large particles) and PS1 (small particles) occurs in plasma membranes of perikarya (A; framed area enlarged in B) and dendrites (C; framed area enlarged in D). Note that the double labelling coincides with apparent thickenings of the membrane, characterised by small fibrils. Very few particles are found in the cytoplasm. Asterisk indicates nuclear membrane; Cyt, cytoplasm. Bars⫽0.4 ␮M, 0.2 ␮M.

single labelled membranes were usually obliquely or tangentially cut, raising the possibility that the two antigens have a laminar organisation that cannot be easily identified in transversely cut membrane patches. Infrequent gold particles indicating PS1 were found at internal membranes, including the endoplasmic reticulum and Golgi cisternae (data not shown). Dendritic spine membranes were devoid of labelling, as were

axons and axon terminals. Glial plasma membranes remained relatively free of immunogold labelling for either A␤42 and PS antibodies. When glial and neuronal membranes were apposed, the particles were found to be associated with the latter (Fig. 4B). It is important to note that this was also evident at sites where neuronal and glial membranes were separated by intervening cell profiles. Thus the resolving power of the immunogold

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Fig. 4. PS1 and 2 show similar distribution patterns. PS1 (small particles in A) and PS2 (small particles in B) show the same distribution pattern with respect to A␤ (large particles). Double labelling is the rule except at sites where the plasma membrane has been cut obliquely (asterisk). Subtle differences in the distance between the respective epitopes and the plasma membrane may explain why such sites exhibit a single particle size (large or small). Where neuronal (N) and glial (G) membranes are apposed the particles representing PS are associated with the neuronal membrane. The large particles are spread more widely but the peak of the gold particle distribution coincides with the neuronal rather than the glial plasma membrane (see text). Arrowheads indicate neuronal plasma membranes. Bar⫽0.2 ␮M.

technique becomes a limiting factor when attempts are made to distinguish between contiguous membranes that are only 10 –20 nm apart. We then examined whether the pattern of labelling for PS and A␤42 in the human brain was consistent with that observed in aged canines. Although the ultrastructure in the human material was clearly inferior due to the inevitable postmortem delay, we could confirm that A␤42 immunogold labelling occurred on dendritic plasma membranes (identified as such by being postsynaptic to clearly identifiable axon terminals; Fig. 5). Double labelling revealed that A␤ co-localised with PS1 (Fig. 5, small particles). Sites of co-localisation were typically found at neuronal plasma

membranes that were obscured and partially disrupted by a fibrillar material most likely representing A␤. One possibility is that A␤ and PS co-localise within buoyant cholesterol-rich microdomains, characterised by an enrichment in GM-1 gangliosides. In both canine and human tissue used in the current study, GM-1 ganglioside immunoreactivity was observed as punctate deposits associated with neuronal membranes (Fig. 6). However, these deposits did not coincide with the accumulation of A␤ (Fig. 6). Antibodies to flotillin (another marker of putative cholesterol-rich domains) did not produce any labelling of neuronal membranes in the present material.

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Fig. 5. Plasma membrane accumulation of A␤ and PS in AD brain. Immunogold labelling indicates plasma membrane accumulation of A␤ (A) and A␤ co-localised with PS1 (B) and PS2 (inset in B) in the frontal cortex. The labelled profile in A is a postsynaptic dendrite (T, terminal forming an asymmetric synapse) while the labelled profiles in B and inset are perikaryal plasma membranes. Note that the neuronal plasma membranes are disrupted by a fibrillar material that is closely associated with clusters of large and small particles suggestive of A␤ and PS co-localisation. Arrowheads indicate plasma membrane. Bars⫽0.4 ␮M, 0.2 ␮M.

DISCUSSION PS is co-localised with A␤ on the plasma membrane A major finding of the present study of human and canine brain is that PS and A␤42 are co-localised in patches along the neuronal plasma membrane. Labelling of internal

membranes, including the rough endoplasmic reticulum (ER), was also observed, but the signal at these sites was much weaker than that at the neuronal surface. The expression of PS at the plasma membrane is consistent with three other reports in transfected Cos-7, DAMI and Jurkat cell lines, respectively (Dewji and Singer, 1997; Schwarz-

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Fig. 6. Segregation of A␤ and GM1 gangliosides. Z-scan at 1 ␮m increments obtained by confocal microscopy demonstrating a lack of association between A␤ and GM1 gangliosides. Membrane-associated A␤ (green) is present on neurons with GM1 ganglioside labelling (red, arrows point to the punctate staining of GM1) but the two proteins do not directly overlap. The asterisk indicates lipofuscin observed in both wavelengths. An overlay is shown in panel 3.

man et al., 1999; Takashima et al., 1996) but runs counter to other reports indicating a preferential association of PS with ER (Kim et al., 2000; Walter et al., 1996). In the latter studies the overexpression of PS may have led to an accumulation of PS within the ER. In transfected cell lines where both plasma and ER labelling are observed, the number of gold particles in ER is quite low relative to that seen on plasma membranes suggesting that PS is normally rapidly cleared from intracellular pathways (Takashima et al., 1996). The weak intracellular labelling for PS in the current study is not likely due to a masking effect. One of the advantages of the post-embedding immunogold technique is that all epitopes stand an equal chance of being labelled, provided that they are exposed at the surface of the ultrathin section (Takumi et al. 1999). Thus there should be no bias against the identification of intracellular epitopes. Clearly, the expression of PS and A␤42 in intact tissue fixed by immersion or perfusion should be more representative of the in vivo distribution of the respective proteins than the expression pattern ob-

served in transfected cell lines. The problem has been to obtain an immunocytochemical signal of sufficient strength. Here we have achieved this by use of a sensitive immunogold procedure (Matsubara et al., 1996) that allows co-localisation to be analysed at the nanometer level. Functional implications of PS/A␤42 colocalization in discrete membrane domains Co-localisation of PS and A␤ at plasma membranes was observed in aged brains only and was typically found in amyloid plaques. The accumulation of PS on plasma membranes was not observed in young dog brain and this could be due to a rapid turnover of PS and a lack of membraneassociated A␤. Thus the co-localisation of these molecules may identify the sites where APP misprocessing occurs. Our findings suggest that this key event in the pathogenesis of AD occurs in discrete patches of the neuronal membrane. Importantly, co-localisation also occurred at sites of fibrillar deposits, corroborating the idea that PS is directly or indirectly involved in plaque generation.

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Whether PS and A␤ are in direct physical contact cannot be resolved by means of the present immunogold technique. The discrete membrane domains that express PS and A␤42 may constitute the exit route of a functionally and anatomically distinct exocytotic pathway involved in APP processing. It is possible that the misprocessing of APP is initiated intracellularly (as could be surmised from transfection studies), but that the concentration of either protein remains below detection limits until they reach the neuronal surface where they aggregate. Do A␤ and PS containing membrane microdomains share features typical of lipid rafts? Punctate immunolabeling for PS and A␤ along plasma membranes suggests the presence of specialised microdomains where A␤ accumulates. We attempted to identify the characteristics of these microdomains through confocal and electron microscopic double label experiments. Biochemical studies have indicated that buoyant cholesterol-rich microdomains and possibly lipid rafts may be loci of A␤ production (Lee et al., 1998; Wahrle et al., 2002; Wolozin, 2001). However, we were unable to identify punctate deposits positive for flotillin, which is a protein found within detergent-insoluble glycolipid-enriched fractions associated with lipid rafts and observed in the AD brain (Bickel et al., 1997; Kokubo et al., 2000; Morishima-Kawashima and Ihara, 1998). Lack of immunoreactivity for flotillin may be a consequence of fixation or archive time. We used a second marker for lipid rafts, by immunolabeling for GM1 gangliosides that are enriched in these specialised membrane domains (Yanagisawa et al., 1995). Although one marker for GM-1 gangliosides exhibited punctate membrane labelling consistent with a previous report in AD brain (Emory et al., 1987), these microdomains were spatially distinct from those labelled with A␤. Thus the domains of A␤/PS co-localisation do not seem to coincide with cholesterol-rich rafts although the possibility remains that markers of cholesterol-rich microdomains are obscured by the A␤/PS deposits. The possibility that PS and A␤ interact within lipid rafts, characterised by a high cholesterol content is supported by epidemiological studies showing that cholesterol-lowering drugs (e.g. statins) significantly reduces the risk of AD (Jick et al., 2000; Wolozin et al., 2000). The present observations suggest that this interaction is indirect.

CONCLUSIONS Our immunocytochemical and morphological observations are consistent with the hypothesis that APP is processed in a PS-dependent manner at neuronal surfaces. These domains may generate, or release, A␤ fibrils (Fig. 3) bound to PS that later coalesce to form A␤ plaques, thus accounting for the observation that PS is associated with plaque formation (Busciglio et al., 1997; Diehlmann et al., 1999). Thus, A␤ and PS may become linked at the plasma membrane to form a complex that endures even after extracellular precipitates are formed. Our results suggest that pre-

299

vious studies in transfected cells have led to a focus on intracellular pathways for APP processing although the present data do not exclude the possibility that the APP misprocessing is initiated at intracellular membranes. Acknowledgements—The authors appreciate the technical assistance of Jorunn Knutsen, Mihaela Nistor and Floyd Sarsoza. The presenilin antibodies were kindly provided by Dr. Tobias Hartmann at the University Heidelberg, Heidelberg, Germany. Funded by National Institute on Aging grant AG12694. Supported by The Norwegian Research Council and Civitan Norway.

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(Accepted 6 April 2003)